Review Reports - Keratinocytes with DNA Aberration Induced by UVB Become Susceptible to Ferroptosis

Round 1

Reviewer 1 Report

The authors investigate the effects of UVB irradiation on an immortalized human skin cell – keratinocyte – line focusing on events from control and ferroptosis-inducing treatments. From the analyses provided, the results appear definitive though limited in scope as to cellular responses in the “dual role” cost-benefit comparison and contrast they set out to report for ferroptosis affecting survival of DNA-mutated cells versus prevention of that damage. See also corrections / clarifications requested in Detailed Comments.

Journal                        Antioxidants (ISSN 2076-3921)
Manuscript ID antioxidants-4354632
Title                             Keratinocytes with DNA aberration induced by UVB …
Authors                       Smirnova et al

Line 88 – much of the experimental set up and data interpretation is in the context of cell replication. A tert – lg T-Ag transformed keratinocyte (KC) line is used and yet how this affects the cell population dynamics seems not to be considered. While Wagner et al (MS ref # 18 ) report SV40 lg T-Ag is down regulated in maturing, differentiating, suprabasal KCs, is it still expressed in the presumably basal (presumably low Ca++) cells used in these investigations? And, if so, what does it mean for any cell cycle and cell replication studies modeling response in normal untransformed cells?

Line 95 – volumes of CaCl2 added are meaningless as no final concentration is reported. If the authors are using Ca++ concentration as a means of enriching for basal phenotype replicating KCs (as opposed to mixed populations in “high” Ca++ concentrations), final Ca++ concentrations must be reported. Also, it would be beneficial to data interpretation if some independent marker of (e.g., population doubling time) cell cycle was reported to help characterize the proliferative status of the cells.

Supp Figure 1 – Documentation of the UV irradiation set-up and wavelength parameters is appreciated. It is unclear if for cells exposed in these conditions was media left on and what absorption that might cause. Also, for translational relevance, how do any of the delivered irradiation doses compare to that experienced at the basal layer of intact epidermis?

Line 101 – description of methodology seems conflicting “The plates were put into an ultrasonic “”ice water bath”” and sonicated at the lowest setting for 60 seconds at “”room temperature”” .

Line 112 – 116 – there are typos or missing words or parentheses in this section making it very unclear as to the experimental design and what is being measured and calculated as a LDH background / basal value. What is “sample m”? This seems partially corrected in Fig 1 legend, line 186. In both instances, is this really reporting “cell death rate” which might imply change over a given time period?

Line 184 – Figure 1 As to presenting intervention condition results to control, the fold changes seem biologically significant. However, given what should be very minimal values of LDH in controls, what do these fold changes mean for a cell population level? LDH seems to be equated to cell death, as opposed to transiently damaged membranes and cell death. A in dependent orthogonal measurement of cell death would strengthen interpretation and impact of the results.

Minor points: The different plot presentation in panels A and B is very distracting. The stacked brackets for significance results is very confusing. Compact Letter Display (CLD) labeling of the plot bars to (e.g., esp for Fig. 4) to indicate multiple pairwise comparison would streamline assessments for the reader. In Fig 1 and elsewhere, it is unclear what experimental repeats were done versus technical measurements and biological replicas in one experiment run.

Line 246 – for Fig 5 and elsewhere, what is the cell biological significance of doubling the UVB (5 – 10 kJ/m2) and instigating a 10-fold CPD induction difference? Was this address in the manuscript? (Are the 5 vs 10 numbers called out in line 274 correctly placed?)

Line 269 and Discussion in general – There is a great translational emphasis put on these results and yet the experimental system is of growth factor-stimulated, presumably basally-enriched cells (Ca++ concentration must be resolved). Does the high mitotic activity present in these cells over report effects of the UVB delivery compared to slower replication rate in intact epidermis?

Author Response

Major comments

Comment 1: The authors investigate the effects of UVB irradiation on an immortalized human skin cell – keratinocyte – line focusing on events from control and ferroptosis-inducing treatments. From the analyses provided, the results appear definitive

Response: We thank reviewer for the validation of our results.

though limited in scope as to cellular responses in the “dual role” cost-benefit comparison and contrast they set out to report for ferroptosis affecting survival of DNA-mutated cells versus prevention of that damage.

Response: We fully agree with the reviewer that the scope of our study with respect to cellular responses is limited. Indeed, our work focuses on a relatively narrow range of biological endpoints, specifically ferroptosis and DNA damage, whereas other UVB-induced cellular responses, such as inflammation, cellular stress responses, and changes in gene expression, were not investigated. These additional responses may interact with the mechanisms examined in the present study and represent important directions for future research.

To address the reviewer's comment, we have added the following statement to the section “Limitations and Future Directions”:

This study focused on the interplay between induction of ferroptosis and DNA damage, whereas UVB irradiation also triggers numerous other cellular responses, including inflammatory signaling, stress responses, and changes in gene expression. The interaction between these processes and the mechanisms investigated here was not addressed in this study and warrants further studies.

Detailed comments

Comment 2: Line 88 – much of the experimental set up and data interpretation is in the context of cell replication. A tert – lg T-Ag transformed keratinocyte (KC) line is used and yet how this affects the cell population dynamics seems not to be considered. While Wagner et al (MS ref # 18 ) report SV40 lg T-Ag is down regulated in maturing, differentiating, suprabasal KCs, is it still expressed in the presumably basal (presumably low Ca++) cells used in these investigations? And, if so, what does it mean for any cell cycle and cell replication studies modeling response in normal untransformed cells?

Response: Thank you for this important question. Indeed, we have performed our experiments with the basal proliferating cells incubated with 0.1 mM Ca++ (see also below). We do no not address whether this mechanism occurs in untransformed and differentiated cells. This is a limitation of our study which we clearly indicate in the section “Limitations and Future Directions”.

A limitation of this study is that all experiments were performed using basal proliferating cells. Therefore, the findings cannot be directly extrapolated to untransformed or differentiated cells. Investigating whether these results also apply to those cell types will require further studies.

Comment 3: Line 95 – volumes of CaCl2 added are meaningless as no final concentration is reported. If the authors are using Ca++ concentration as a means of enriching for basal phenotype replicating KCs (as opposed to mixed populations in “high” Ca++ concentrations), final Ca++ concentrations must be reported.

Response: We thank reviewer very much for this comment. We apologize for this unclear description of preparation procedure. We added the following description to the section “Methods”:

New text: Cells were grown in KGM™-Gold BulletKit™ medium (Lonza; Lot No.: Basal Medium: 00195130; SingleQuots™: 00192152). 500 mL of medium was additionally supplemented with 50 µL of 1 M CaCl₂ and 500 µL of G418 instead of gentamicin. The final Ca²⁺ concentration was 0.1 mM, which supports keratinocyte proliferation.

Comment 4: Also, it would be beneficial to data interpretation if some independent marker of (e.g., population doubling time) cell cycle was reported to help characterize the proliferative status of the cells.

Response: To address this point, we added to methods section: The doubling time NHEK_SVTERT3 was established by Evercyte. They show that NHEK_SVTERT3 has a constant population doubling time of 48–60 hours (https://evercyte.com/wp-content/uploads/2021/05/NHEK_SVTERT3_5_leaflet.pdf).

We added the following description to the section “Methods”:

NHEK_SVTERT3 has a constant population doubling time of 48–60 hours (https://evercyte.com/wp-content/uploads/2021/05/NHEK_SVTERT3_5_leaflet.pdf).

If the reviewer considers it necessary, we would be willing to perform an additional experiment to determine exact doubling time in our experimental model.

Comment 5: Supp Figure 1 – Documentation of the UV irradiation set-up and wavelength parameters is appreciated. It is unclear if for cells exposed in these conditions was media left on and what absorption that might cause. Also, for translational relevance, how do any of the delivered irradiation doses compare to that experienced at the basal layer of intact epidermis?

Response: Thank you for this important question. We agree that the irradiation procedure was not described in sufficient detail, and we have therefore expanded the Methods section in the manuscript.

The following text is added to method section “Methods”:

As the UV source we used a Dermfix 2000SX device equipped with TL/01-type 311 nm a narrowband UVB lamp. It was calibrated with Waldmann UV – meter, using UV21 calibration sensor. Although their emission spectrum of TL/01 is similar to that of Waldmann UV21 lamps, TL/01 lamps have a narrower bandwidth. Because of this the irradiance measurements were corrected for the TL/01 spectrum using the relative irradiance reported by Taylor et al. (PMID: 12000370) showing that the effective irradiance of the TL/01 lamps is approximately 30% higher than the UV21. Multiple components of cell culture medium, such as amino acids absorb the UVB light around 310 nm. With reported absorbance values of approximately 1 absorbance units (AU) (Ferencik et al. (PMID: 38790309). According to the Beer–Lambert law, an absorbance of 1.0 AU corresponds to approximately 10% transmission. To avoid such a high loss of irradiation we changed the medium for PBS for the time of irradiation.

We also added more detailed description of this procedure to the section “Methods”:

The experimental workflow was as follows. Cells were seeded into 12-well plates and cultured for 48 h. On day 3, the cells were treated with the indicated ferroptosis inducers and incubated for an additional 24 h. Before irradiation the culture medium was carefully removed and retained, and the cells were rinsed with PBS. Cells assigned to the irradiation group were exposed to UVB while covered with PBS. Immediately after irradiation, the PBS was removed and the original conditioned medium was returned to the corresponding wells. Control cells underwent the same procedure, except that they were incubated with PBS for 2–3 min without UVB exposure before the original medium was restored. Both the irradiated and control plates were then incubated for an additional 16 h, after which the samples were harvested for subsequent analyses. The data generated from each weekly run were therefore considered to represent an independent experiment.

With regard to translation relevance UVB irradiation used in our study we added the following text to the section “Discussion”:

It is widely accepted that UVB radiation reaches the basal layer of the intact epidermis at a depth of approximately 100 µm, although its intensity may decrease to only a few percent of the incident power (reviewed by Pustisek and Situm, 2011; PMID: 22220467). However, computer simulations based on Monte Carlo method and a skin tissue model suggest that UVB radiation can penetrate much deeper into the epidermis, reaching depths of up to 400 µm (PMID: 28900751). In contrast, UVA radiation penetrates the skin substantially deeper than UVB but is considerably less efficient at inducing CPDs. Specifically, exposure to the same radiant dose (1 J/m²) at 310 nm (UVB) produces approximately 100-fold more CPDs than exposure at 365 nm (UVA) (Cadet and Douki; PMID: 29405222).

Comment 6: Line 101 – description of methodology seems conflicting “The plates were put into an ultrasonic “”ice water bath”” and sonicated at the lowest setting for 60 seconds at “”room temperature”” .

Response: We apologize for the conflicting description. We have thoroughly revised the “Methods” section to improve its clarity and completeness by providing more detailed and specific information, making the methodology easier to understand and reproduce.

We have introduced “Sample collection” subsection to section “Methods”:

Sample collection

After the final incubation of an experiment, the medium containing released lactate dehydrogenase (LDH) and the live, adherent cells were separated and stored for further LDH-measurement and CPD-analysis. The medium was transferred to microcentrifuge tubes, and frozen with liquid nitrogen. The plates were put into an ultrasonic water bath and sonicated at the lowest setting for 60 seconds at room temperature to loosen the cells. The plate´s wells were scraped with the rubber tip of a syringe piston; the resulting cell suspension was transferred into microcentrifuge tubes and frozen with liquid nitrogen. Then they were kept for max. 2 weeks at -20C. One portion of the frozen cells was used to determine LDH levels in medium and cell lysate. The second portion of the samples was used to extract DNA from the entire population of cells (both floating in medium and adherent (pellet) cells) by pooling both frozen samples. In these samples we determined the quality of DNA using electrophoresis and analyzed CPDs in the extracted DNA. The third analytical setup used live cells, which were treated with BODIPY, a fluorescence reporter detecting oxidized and non-oxidized lipids separately and examined by laser scanning microscopy. The ratio of oxidized/non-oxidized lipids was used as the measure of lipid peroxidation.

For clarify we gave more details to the LDH analysis in the section “Methods”:

In both medium and cell homogenates, we determined the activity of LDH. The ratio of LDH determined in medium to total amount of LDH in the sample (medium plus cell homogenate) was considered as the relative portion of dead and dying cells. LDH activity measurements were performed as previously described by Weidinger et al [ PMID: 36933393] with modifications described by Smirnova et al. [ PMID: 40002376]. Briefly, a 25% sample volume of RIPA buffer was added to the cell suspension samples, mixed by pipetting up and down, and incubated on ice for 20 min. Then, 150 µl of RT-PBS was added. The samples were then pipetted into a 96-well plate.

Comment 7: In both instances, is this really reporting “cell death rate” which might imply change over a given time period?

Response: We fully appreciate this comment. As outlined in the comparative analysis below, the major methods used to assess cell death are unable to discriminate between dead and dying cells. Therefore, to more accurately reflect what was measured, we have replaced the term "cell death rate" with "relative amount of dead and dying cells" throughout the manuscript.

Comment 8: Line 184 – Figure 1 As to presenting intervention condition results to control, the fold changes seem biologically significant. However, given what should be very minimal values of LDH in controls, what do these fold changes mean for a cell population level? LDH seems to be equated to cell death, as opposed to transiently damaged membranes and cell death.

Response: Thank you for this excellent question. The LDH method that just determines relative amount of dead and dying cells, because it accounts the total population level measuring total amount of LDH in samples, not only released LDH.

Indeed, all widely used methods, such as LDH release, trypan blue (TB) staining, and propidium iodide (PI) staining, primarily detect compromised plasma membrane integrity. Therefore, strictly speaking, these assays identify dead or dying cells with damaged membranes. We have addressed this point in the manuscript accordingly saying that we identify dead and dying cells. Among these methods, we selected the LDH assay because TB and PI staining detect only cells that remain in the pellet and do not account for cells that have detached or disintegrated. In contrast, the LDH assay used in this study measures LDH released into the culture medium regardless of whether the releasing cells remain attached or have detached. Furthermore, it quantifies the total LDH content in the sample (medium plus cell lysate), which reflects the total number of cells present. Consequently, the ratio of extracellular LDH to total LDH (medium/[medium + pellet]) provides a measure of the cell death that is independent of the total cell number and is not affected by whether the cells remain attached or have detached.

We selected the LDH assay to evaluate the amount of dead and dying cells because a substantial body of evidence supports the validity of LDH release as a marker of cell death of keratinocytes.To address this point we added a text justifying the selection of the research methods to the section “Methods”:

Several studies have demonstrated a positive correlation between LDH release and other cell death assays, such trypan blue exclusion (PMID: 31710929; PMID: 24809892; PMID: 28077256). In addition, an inverse correlation between LDH release and cell viability assessed by the MTT assay has been reported in keratinocytes (PMID: 35444418; PMID: 38675427). Similar to trypan blue staining, the MTT assay evaluates only adherent, metabolically active cells and does not account for detached cells.

Furthermore, the LDH assay has been extensively and successfully employed to quantify UV-induced cell death in keratinocytes (PMID: 31710929; PMID: 15634218; PMID: 34751410; PMID: 34451678; PMID: 26933830; PMID: 22321694; PMID: 26498291; PMID: 19467030), further supporting its suitability for the present study.

With regard to the presentation of the data. The basal (control) level of cell death varied slightly between independent experimental sets (typically ranging from 1–4% and reaching approximately 8% in one experiment), That is the reason why we presented the results as fold change relative to the corresponding control to facilitate comparison between experiments. To improve clarity, we have now revised the manuscript by adding the absolute control values (percentage of dead and dying cells relative to the total cell number) to the legend of each relevant figure.

Comment 9: An independent orthogonal measurement of cell death would strengthen interpretation and impact of the results.

Response: We considered this point. As a second independent approach to assess ferroptotic cell death, we evaluated lipid peroxidation, a hallmark and essential prerequisite of ferroptosis. Lipid peroxidation was quantified by determining the ratio of oxidized to non-oxidized lipids using the fluorescent probe BODIPY™ 493/503.

We hope that the above explanation addresses the reviewer's concern and demonstrates that the combination of the LDH assay and lipid peroxidation (LPO) analysis provides a reliable assessment of ferroptosis in our experimental model.

If the reviewer considers it necessary, we would be willing to perform an additional experiment demonstrating the correlation between the LDH assay and another cell death assay, such as trypan blue staining, across increasing UVB doses.

Minor points:

Comment 10: The different plot presentation in panels A and B is very distracting. The stacked brackets for significance results is very confusing. Compact Letter Display (CLD) labeling of the plot bars to (e.g., esp for Fig. 4) to indicate multiple pairwise comparison would streamline assessments for the reader. In Fig 1 and elsewhere, it is unclear what experimental repeats were done versus technical measurements and biological replicas in one experiment run.

Response: Thank you for this great tip we have now replaced the brackets with Compact Letter Display (CLD) labelling. This makes easier to understand the displayed results.

With regard to technical replicates. Given the week-long duration and the requirement for parallel treatment of irradiated and non-irradiated plates, experimental throughput was limited; therefore, reproducibility was assessed using independent biological experiments rather than technical replicates. We have added the information replicates to graphs and figure legends.

Comment 11: Line 246 – for Fig 5 and elsewhere, what is the cell biological significance of doubling the UVB (5 – 10 kJ/m2) and instigating a 10-fold CPD induction difference? Was this address in the manuscript? (Are the 5 vs 10 numbers called out in line 274 correctly placed?

Response: To address this important question of the reviewer we added the following text to the section “Discussion”:

The irradiation doses used in this study (5 or 10 kJ/m²) are fairly comparable with natural irradiation. It has been reported that on a clear summer day in a north European country, approximately 1 kJ/m2 UVB can be received during 15–30 min of solar noon exposure, while substantially higher doses may occur in tropical regions (Kolari at al., PMID: 3588354).

Also we corrected the statement regarding CPD induction in the section “Discussion”:

Our data suggest that cells susceptible to ferroptotic death harbor higher levels of UVB induced CPDs than the average keratinocyte. Consequently, the selective elimination of these cells through mild induction of ferroptosis substantially reduces the overall CPD burden in the remaining keratinocyte population.

Comment 12: Line 269 and Discussion in general – There is a great translational emphasis put on these results and yet the experimental system is of growth factor-stimulated, presumably basally-enriched cells (Ca++ concentration must be resolved). Does the high mitotic activity present in these cells over report effects of the UVB delivery compared to slower replication rate in intact epidermis?

Response: We have addressed the issue regarding Ca²⁺ above. With respect to its application, we acknowledge the following limitation in the section “Limitations and Future Directions”:

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents an interesting observation: "Keratinocytes with DNA aberration induced by UVB become susceptible to ferroptosis" below is my concern

The study relies on: RSL3, Erastin and Lipid peroxidation measurements but lacks definitive ferroptosis validation. The gold-standard controls would include: Ferrostatin-1, Liproxstatin-1, GPX4 analysis, ACSL4 expression, Iron chelation experiments Without these, cell death cannot be unequivocally attributed to ferroptosis.
"Cells were irradiated using a Dermfix lamp and Waldmann UV meter. But the Missing information like UVB spectrum,Irradiance, Exposure time, Calibration procedure are essential for reproducibility.
Cell death is assessed only through LDH release. Additional assays like Annexin V/PI staining, Caspase activity, Live/dead assays, Morphological analysis is recommended.
Figure 4 shows Cell death increases dramatically after combined UVB + RSL3 treatment. However: Y-axis scales differ between panels. Error bars are difficult to interpret. Sample size is not shown. Improve the caption with maximum information.
The CPD data represent the key finding. However: Raw CPD values should be reported alongside fold changes. Normalization procedure should be clarified., DNA loading controls should be described.

Expand the caption of figures with sufficient details including Exact n values and Biological replicate numbers
Introduction Current: "a beneficial by preventing damage to keratinocytes" Should be: "a beneficial role by preventing the survival of damaged keratinocytes"

Cell Culture Section Current: "which specifically belong to the cell type" Suggested: "which are human epidermal keratinocytes

Author Response

Major comments

Comment 1: The manuscript presents an interesting observation: "Keratinocytes with DNA aberration induced by UVB become susceptible to ferroptosis"

Response: Thank you for this positive feedback.

below is my concern

Comment 2: The study relies on: RSL3, Erastin and Lipid peroxidation measurements but lacks definitive ferroptosis validation. The gold-standard controls would include: Ferrostatin-1, Liproxstatin-1, GPX4 analysis, ACSL4 expression, Iron chelation experiments Without these, cell death cannot be unequivocally attributed to ferroptosis.

Response: We thank the reviewer for raising this important point. We have carefully considered this issue. Indeed, recent evidence indicates that RSL3 can also induce pyroptosis in cancer cells. Notably, both ferroptosis and pyroptosis are driven by lipid peroxidation, and both pathways can be inhibited by Ferrostatin-1 (Fer-1). However, the majority of published studies using erastin and RSL3 as ferroptosis inducers have not reported activation of alternative cell death pathways and therefore did not include Fer-1 as a control (PMID: 42260608, PMID: 42402515, PMID: 39240413, PMID: 41744671).

Considering the available evidence and the technical constraints of maintaining identical irradiation conditions across all experimental groups, we prioritized the use of two mechanistically distinct ferroptosis inducers, erastin and RSL3, rather than including an additional Fer-1 treatment group. The concordant effects observed with these two independent inducers strengthen the conclusion that the observed phenotype is associated with ferroptosis induction.

We also appreciate the reviewer's suggestion to assess additional ferroptosis markers, such as GPX4, glutathione (GSH), and ACSL4. These molecules are important upstream regulators of ferroptosis; however, their effects ultimately converge on the modulation of lipid peroxidation. Changes in GPX4 or ACSL4 expression or activity are not, by themselves, sufficient evidence of ferroptosis unless they result in increased lipid peroxidation. For this reason, we chose to quantify lipid peroxidation, which represents the key functional hallmark of ferroptosis and integrates the net effects of these regulatory pathways.

Nevertheless, we agree that the reviewer's suggestion would further strengthen the mechanistic interpretation of our findings. Regardless of whether Fer-1 inhibits ferroptosis alone or additional lipid peroxidation-dependent forms of cell death, its inclusion would help determine whether the reduction in UVB-induced DNA aberrations mediated by erastin and RSL3 depends on lipid peroxidation-driven cell death. We therefore acknowledge this as a limitation of the present study and have added the following statement to the revised manuscript to the section “Limitations and Future Directions”.

Further studies are required to determine whether RSL3 selectively eliminates cells carrying UVB-induced DNA aberrations through ferroptosis or other LPO-dependent cell death pathways. Future experiments should include Ferrostatin-1 or other selective ferroptosis inhibitors to establish whether inhibition of ferroptosis reverses the protective effects of erastin and RSL3 on UVB-induced DNA aberrations in both in vitro and in vivo models.

Comment 3: "Cells were irradiated using a Dermfix lamp and Waldmann UV meter. But the Missing information like UVB spectrum, Irradiance, Exposure time, Calibration procedure are essential for reproducibility.

Response: Thank you for this comment. We apologize for the inconsistency in the description of the Methods section. We have fixed this issue by adding the following text to the section “Methods”:

As the UV source we used a Dermfix 2000SX device equipped with TL/01-type 311 nm a narrowband UVB lamp. It was calibrated with Waldmann UV – meter, using UV21 calibration sensor. Although their emission spectrum of TL/01 is similar to that of Waldmann UV21 lamps, TL/01 lamps have a narrower bandwidth. Because of this the irradiance measurements were corrected for the TL/01 spectrum using the relative irradiance reported by Taylor et al. (PMID: 12000370) showing that the effective irradiance of the TL/01 lamps is approximately 30% higher than the UV21. -Multiple components of cell culture medium, such as amino acids absorb the UVB light around 310 nm. With reported absorbance values of approximately 1 absorbance units (AU) (Ferencik et al. (PMID: 38790309). According to the Beer–Lambert law, an absorbance of 1.0 AU corresponds to approximately 10% transmission. To avoid such a high lost of irradiation we changed the medium for PBS for the time of irradiation.

We also added more detailed description of this procedure to section “Methods”:

Comment 4: Cell death is assessed only through LDH release. Additional assays like Annexin V/PI staining, Caspase activity, Live/dead assays, Morphological analysis is recommended.

Response: Thank you for this point. We carefully evaluated these experimental options during the study design phase. Because the number of samples that could be simultaneously exposed to equal UV radiation was limited, we prioritized experiments that directly addressed the primary objectives of the study, induction of ferroptosis. Consequently, we did not include apoptosis-specific analyses or the corresponding assays. Morphological assessment is well established and reliable for tissue specimens, whereas many apoptosis assays are optimized for cell culture systems and are less straightforward to apply to our experimental model. Regarding the live/dead assay, we agree that it could provide valuable complementary information. However, our decision not to include it was based on the following considerations. All widely used methods, such as LDH release, trypan blue (TB) staining, and propidium iodide (PI) staining, primarily detect compromised plasma membrane integrity. Therefore, strictly speaking, these assays identify dead or dying cells with damaged membranes. We have addressed this point in the manuscript accordingly saying that we identify dead and dying cells. Among these methods, we selected the LDH assay because TB and PI staining detect only cells that remain in the pellet and do not account for cells that have detached or disintegrated. In contrast, the LDH assay used in this study measures LDH released into the culture medium regardless of whether the releasing cells remain attached or have detached. Furthermore, it quantifies the total LDH content in the sample (medium plus cell lysate), which reflects the total number of cells present. Consequently, the ratio of extracellular LDH to total LDH (medium/[medium + pellet]) provides a measure of the cell death that is independent of the total cell number and is not affected by whether the cells remain attached or have detached.

We selected the LDH assay to evaluate the amount of dead and dying cells because a substantial body of evidence supports the validity of LDH release as a marker of cell death of keratinocytes. To address this point, we added a text justifying the selection of the research methods to the section “Methods”:

As a second independent approach to assess ferroptotic cell death, we evaluated lipid peroxidation, a hallmark and essential prerequisite of ferroptosis. Lipid peroxidation was quantified by determining the ratio of oxidized to non-oxidized lipids using the fluorescent probe BODIPY™ 493/503.

Comment 5: Figure 4 shows Cell death increases dramatically after combined UVB + RSL3 treatment. However: Y-axis scales differ between panels. Error bars are difficult to interpret. Sample size is not shown. Improve the caption with maximum information.

Response: Thank you for this important comment. We have revised all figure legends throughout the manuscript. Specifically, we (i) replaced the significance labeling with a compact letter display (CLD), which facilitates clearer visualization of differences among groups; (ii) added the number of replicates above each bar; (iii) we added absolute values of dead and dying cells in basal (control) samples, which we used to calculate the fold -increase, (iiii)updated the figure legends accordingly.

Comment 6: The CPD data represent the key finding. However: Raw CPD values should be reported alongside fold changes. Normalization procedure should be clarified., DNA loading controls should be described.

Response: Thank you very much for this helpful comment. The manuscript has been revised accordingly. We present quantitative CPD values along with the corresponding fold changes in the Results section. In the Methods section, we added information about the data normalization used to quantify CPD. We also clarified that DNA concentrations were determined prior to the assay, and identical amounts of DNA were loaded into each well to ensure equal DNA input.

The section “CPD analysis” is modified as following:

CPDs were measured in duplicate and in a random order using an OxiSelect Cellular UV-Induced DNA Damage ELISA Kit (CPD) (Cell Biolabs, Lot-No.: 3221514), according to the manufacturer’s instructions. Briefly, equal amounts of DNA (4 µg/ml) were added into each ELISA well according to the manufacturer's protocol, DNA was denatured and then immobilized on the plate. The wells were treated with an СPD-specific antibody and incubated. Several washing cycles were performed. The HRP conjugate was added and incubated, then the wells were thoroughly washed. Then the substrate was added and the absorbance was measured at 450 nm. OD450 within each plate were normalized to the average values of cells treated with only 5 kJ/m2, this value was taken as 100 %.

Detailed comments

Comment 7: Expand the caption of figures with sufficient details including Exact n values and Biological replicate numbers

Response: Thank you, we have done so (see our response to your question # 4).

Comment 8. Introduction Current: "a beneficial by preventing damage to keratinocytes" Should be: "a beneficial role by preventing the survival of damaged keratinocytes"

Response: Thank you we have changed this statement accordingly.

Comment 9:Cell Culture Section Current: "which specifically belong to the cell type" Suggested: "which are human epidermal keratinocytes

Response: Thank you we have changed this statement accordingly.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I appreciate the authors’ detailed and organized responses and changes.