Serum Starvation Affects the Transcriptomic and Proliferative Response to ACTH in Primary Cultures of Rat Adrenocortical Cells
, Emilia Cicha 2,†, Marta Szyszka 1, Karol Jopek 1, Marianna Tyczewska 1,3, Izabela Pieścikowska 3, Ludwik K. Malendowicz 1,* and Marcin Rucinski 1,4Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have submitted a manuscript titled " Serum starvation affects the transcriptomic and proliferative response to ACTH in primary cultures of rat adrenocortical cells." This manuscript is a full-length article. In this manuscript, the authors aimed to evaluate the functional and transcriptomic effects of ACTH in primary rat adrenocortical cells cultured under both standard conditions and simulated serum (steroid hormone) starvation. To achieve their objective, the authors used primary rat adrenocortical cells incubated in four different conditions: Control, ACTH, Control(S), and ACTH(S).
This article is a revised version of a previous submission, for which some suggestions have been provided. This revised version represents a significant improvement, with additions that have overall increased the clarity of the experiments performed.
Here are the specific points you should convey to the authors regarding their revised submission:
- In this version, as in the previous one, the authors have not provided the raw transcriptomic data from microarray analysis. These data should be deposited in a publicly accessible database, such as GEO (Gene Expression Omnibus), in accordance with best practices for data sharing and transparency. If the data has already been deposited, please include the relevant accession numbers in the manuscript, along with a quick access link to the GEO database.
- The authors should confirm the transcriptomic data using real-time PCR (qPCR), selecting a few key targets from the most significant genes presented in the study. This validation is a critical step for microarray-based studies to ensure the reliability of the gene expression changes observed.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors|
Manuscript “Serum starvation affects the transcriptomic and proliferative response to ACTH in primary cultures of rat adrenocortical cells” presented by Blatkiewicz et al. describes investigation of extracellular media complexity reduction onto different parameters of cultured rat adrenocortical cells in presence and absence ACTH. Transcriptomes, extracellular corticosterone levels, cell viability and proliferation were evaluated. Significant differences between several parameters including transcriptome profiles and corticosterone levels were observed for all studied conditions. The manuscript contains most needed for understanding results and goals information. However, current requirements for research reproducibility and transparency dictate necessity for providing of actual data used in generation of final output. Therefore, supplemental information should contain files with appropriate data sets. Additionally, charcoal is not specific for steroids removal agent. It also can bind large number of other compounds. This along with significant rise in corticosterone levels in the stripped conditions makes interpretation of obtained results as well as stated goals less logically connected e.g. “However, the role of extracellular steroid availability in shaping ACTH responses is still not fully defined”, “absence of endogenous steroids sensitises cells”, “standard and steroid-depleted conditions”. Corticosterone is a steroid released in control conditions. The authors added a paragraph into Discussion section addressing this issue. However, no appropriate adjustments were made in other sections of the manuscript. The same is true for rising corticosterone levels. |
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Additional comments (Comments are made during continuous reading of the manuscript. Therefore, answers to some raised questions may occur later in the text.) |
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Experimental section 1) Describe how charcoal-stripped FBS (designated as “S” for serum starvation) was prepared (including final composition) and stored. Presence of charcoal in extracellular media during cell culturing has to be described. 2) Isolation of adrenal glands from 20 rats is mentioned. However, there is no details on number of biological and technical repeats made during the experiments. Such information should be added to appropriate locations in the manuscript e.g. “Control group achieved an NCI of 1.7487 ± 0.1643” n=?. Figure 1. Legend states study of individual samples with 2-3 data points visible on B panel. What these data points represent? |
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Results and discussion 1) Fig. 1. Explain what following means “different letters, with each letter representing statistically distinct groups”. What are α levels for each comparison? 2) Fig. 1. Why ACTH stimulation data without serum depletion has so low SD? Add number of biological repeats to figure legend. |
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors The authors addressed all my concerns raised in my previous revision process.Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors made important moditiions to the manuscript, which is sufficently clear now for publishing. Only a notesential comment related to clarity can be made to following sentence "This serum is prepared by the manufacturer through treatment with activated charcoal and dextran to reduce steroid hormones, growth factors, and other small molecules while preserving essential proteins." Term "essential proteins" is vague and may cover some growth factors. Also, charcoal is know for binding proteins e.g. https://doi.org/10.1021/la501005s .
