Anti-Inflammatory and Anti-Apoptotic Effects of Acer Palmatum Thumb. Extract, KIOM-2015EW, in a Hyperosmolar-Stress-Induced In Vitro Dry Eye Model

The aim of this study was to assess the anti-inflammatory and anti-apoptotic effects of KIOM-2015EW, the hot-water extract of maple leaves in hyperosmolar stress (HOS)-induced human corneal epithelial cells (HCECs). HCECs were exposed to hyperosmolar medium and exposed to KIOM-2015EW with or without the hyperosmolar media. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 production and apoptosis were observed, and the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal regulated kinase (ERK), p38 and c-JUN N-terminal kinase (JNK) signaling and nuclear factor (NF)-κB was confirmed. Compared to isomolar medium, the induction of cell cytotoxicity significantly increased in HCECs exposed to hyperosmolar medium in a time-dependent manner. KIOM-2015EW-treatment significantly reduced the mRNA and protein expression of pro-inflammatory mediators and apoptosis. KIOM-2015EW-treatment inhibited HOS-induced MAPK signaling activation. Additionally, the HOS-induced increase in NF-κB phosphorylation was attenuated by KIOM-2015EW. The results demonstrated that KIOM-2015EW protects the ocular surface by suppressing inflammation in dry eye disease, and suggest that KIOM-2015EW may be used to treat several ocular surface diseases where inflammation plays a key role.


Introduction
Dry eye syndrome (DES) is a common ocular surface disease attributable to disorders of the tear film and ocular surface [1].Inadequate tear secretion and increased tear evaporation are the two major causes of DES.The two common mechanisms underlying the pathogenesis of ocular surface injury in DES are ocular surface inflammation and increased tear hyperosmolarity [1][2][3][4][5][6].The following evidence has related ocular surface inflammation with DES: (1) elevated expression and production of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ) in both dry eye patients and experimental dry eye models [7][8][9][10][11]; and (2) effectiveness of anti-inflammatory therapy in treating DES.

Preparation of Herbal Extract, KIOM-2015EW
Acer palmatum Thumb.leaves purchased from Korea Medicine Herbs Association (Yeongcheon, Korea), were confirmed by Professor Ki Hwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and then stored at the herbal bank of KIOM.To prepare KIOM-2015EW, dried Acer palmatum Thumb.leaves (1700 g) were ground into a fine powder, KIOM-2015EW formula was soaked in 17 L distilled water and then heat-extracted in an extractor (Cosmos-600 Extractor, Gyeonseo Co., Inchon, Korea) for 3 h at 115 • C, filtered using standard testing sieves (150 µm, Retsch, Haan, Germany), and then concentrated to dryness in a lyophilizer.KIOM-2015EW powder (50 mg) dissolved in 1 mL distilled water was kept at −20 • C prior to use after filtration through a 0.22 µm disk filter.

Cell-Viability Assay
Cell viability was evaluated by the CCK-8 and MTT assays.Cells (7 × 10 3 /well) were inoculated in a 96-well plate and treated with KIOM-2015EW for 24 h or 450 mOsM media for the specified time.Cell viability assays were performed as described previously [45].After incubation, cell viability was determined using MTT colorimetric and CCK-8 assays.Color development was measured at 450 for CCK-8 or 560 nm for MTT using a microplate reader (SpectraMax i3, Molecular devices, CA, USA).

ELISA
HCECs were seeded into a 100-mm 2 dish (density, 1.5 × 10 6 cells).Following overnight incubation, cell lines were exposed to each of the conditions described in the following section, and incubated with KIOM-2015EW in serum-depleted DMEM/F-12.After 24 h, culture supernatants were collected, centrifuged to pellet any detached cells, and measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted using an RNA extraction solution (BioAssay Co., Daejeon, Korea) and reverse transcribed to cDNA using a 1st Strand cDNA synthesis kit (BioAssay Co., Daejeon, Korea), according to the manufacturer's protocol.cDNA aliquots were amplified by PCR using the specific primers in Table 1.PCR products were electrophoresed on 1% agarose gels and visualized by GreenLight (BioAssay Co., Daejeon, Korea).The following PCR conditions are indicated in Table 1.

Immunofluorescence Staining
Harvested cells were fixed with 10% neutral-buffered formalin solution at room temperature for 10 min.Cells were permeabilized with 0.3% Triton X-100 for 15 min, and blocked overnight with 5% normal goat serum and bovine serum albumin (BSA) in TBS at 4 • C. Cells were then incubated with monoclonal or polyclonal antibodies against TNF-α, IL-6, and IL-1β (diluted 1:50-100) and further incubated with the appropriate Alexa Fluor 488-or 555-conjugated secondary antibody at room temperature for 1 h; nuclei were then counterstained with DAPI (1 mg/mL).Slides were mounted with Fluorescence mount, and images were captured using a Nikon fluorescence microscope NIS-Elements microscope imaging software (Nikon, Tokyo, Japan).

Flow Cytometry
An fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used to detect cell death.Briefly, after treatment with vehicle, HOS, or HOS + KIOM-2015EW for 24 h, cells were trypsinized and resuspended in binding buffer (0.1 M HEPES/NaOH pH 7.4, 1.4 M NaCl, and 25 mM CaCl 2 ).Annexin V-FITC (5 µL) and propidium iodide (PI; 5 µL) were added and incubated for 15 min at room temperature in the dark.Cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, CA, USA).

Cytoplasmic and Nuclear Protein Extraction
Proteins from the cytoplasm and nucleus were separated by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (#78835, Thermo scientific, Waltham, MA, USA).Briefly, HCECs were harvested with trypsin-ethylenediaminetetraacetic acid (EDTA) and washed twice with PBS.Then, cells were centrifuged at 16,000× g for 5 min, and the supernatants were removed.Ice-cold CER-I and -II solutions were added per the manufacturer's instructions to separate the cytoplasmic proteins from the nuclear-compartment proteins.Western blotting for β-actin and phospho-IκB-α was performed to ensure that there was no contamination.

Statistical Analysis
Analyses were performed using GraphPad PRISM ® (GraphPad PRISM software Inc., Version 5.02, San Diego, CA, USA) and SPSS version 23 software (IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY, USA).The nonparametric Mann-Whitney U test was performed to show the statistical significance (p < 0.05) between the controls (cells untreated with HOS) and the cells exposed to HOS or between the cells treated with/without KIOM-2015EW.Results are expressed as means ± standard error of the mean (SEM), and p < 0.05 was considered significant.
Nutrients 2018, 10, x FOR PEER REVIEW 6 of 18 inflammatory drugs (CsA or fluoremetholone (FML)) also reduced cytokine production, but their effects were not as marked as that of KIOM-2015EW.Immunofluorescence staining was performed to detect TNF-α, IL-1β, and IL-6 expression in HCECs.The immunoreactivity of control HCECs to these inflammatory markers was weak (Figure 2B).Positive-staining increased markedly when the cells were exposed to HOS for 24 h, and incubation with KIOM-2015EW attenuated the increased reactivity.
To investigate whether KIOM-2015EW affects pro-inflammatory cytokine expression, the mRNA levels of TNF-α, IL-1β, and IL-6 were determined in HOS-stimulated HCECs.Treatment with 450 mOsM medium increased TNF-α, IL-1β, and IL-6 mRNA expression by 6.41 ± 1.88, 1.36 ± 0.25, and 1.81 ± 0.38 fold, respectively, compared with the control (312 mOsM; Figure 2C).Cytokine expression decreased by 4.58 ± 1.29, 0.86 ± 0.71, and 1.07 ± 0.65 fold, respectively, in HCECs at 450 mOsM with the addition of 0.05 mg/mL KIOM-2015EW.Expression further decreased by 2.37 ± 1.48, 0.69 ± 0.20, and 0.80 ± 0.07 fold, respectively, upon treatment with 0.1 mg/mL KIOM-2015EW and by To evaluate the effect of HOS-induced cytotoxicity in HCECs, cells were seeded in 96-well plates (7 × 10 3 cells/well), cultured for 24 h, and then switched to hyperosmolar media for the specified times.Cell viability decreased in a time-dependent manner in cells exposed to 450 mOsM DMEM/F-12 (Figure 1B).Based on the results of these cytotoxicity studies, we subjected cells to 4 h and 24 h of HOS in subsequent biochemical assays.At these time points, a ~30% inhibition of cell viability was observed compared with the control.
Immunofluorescence staining was performed to detect TNF-α, IL-1β, and IL-6 expression in HCECs.The immunoreactivity of control HCECs to these inflammatory markers was weak (Figure 2B).Positive-staining increased markedly when the cells were exposed to HOS for 24 h, and incubation with KIOM-2015EW attenuated the increased reactivity.

KIOM-2015EW Blocked Apoptosis Due to HOS-Induced Cytotoxicity in HCECs
Previous studies have indicated that HOS induces HCEC apoptosis through a cytochrome cdependent pathway, which may be mediated by MAPK signaling [12].Here, the HCEC survival index was evaluated by MTT assay after treatment of HOS-exposed HCECs with various concentrations of KIOM-2015EW for 24 h (Figure 3A).KIOM-2015EW (0.05-0.2 mg/mL) suppressed HOS-induced cytotoxicity in HCECs.KIOM-2015EW inhibited apoptosis more effectively than CsA and FML.
To quantify the number of apoptotic cells, we performed Annexin V and propidium iodide (PI) for flow cytometry on HOS-exposed KIOM-2015EW-treated HCECs.The percentage of late apoptotic and necrotic cells increased upon HOS-exposure, indicating that both apoptosis and necrosis are

KIOM-2015EW Blocked Apoptosis Due to HOS-Induced Cytotoxicity in HCECs
Previous studies have indicated that HOS induces HCEC apoptosis through a cytochrome c-dependent pathway, which may be mediated by MAPK signaling [12].Here, the HCEC survival index was evaluated by MTT assay after treatment of HOS-exposed HCECs with various concentrations of KIOM-2015EW for 24 h (Figure 3A).KIOM-2015EW (0.05-0.2 mg/mL) suppressed HOS-induced cytotoxicity in HCECs.KIOM-2015EW inhibited apoptosis more effectively than CsA and FML.
Caspase 3 and PARP cleavage, and Bcl-2 (anti-apoptotic factor) and Bax were detected with specific antibodies 24 h after hyperosmotic stimulation (Figure 3C).In the western blot analysis, high levels of cleaved caspase 3 and PARP were detected in HOS-exposed cells, whereas KIOM-2015EWtreatment decreased this cleavage in a dose-dependent manner.Bcl-2 expression increased with KIOM treatment, but Bax expression level was not different in HOS-and/or KIOM-2015EW-treated cells (Figure 3C).To quantify the number of apoptotic cells, we performed Annexin V and propidium iodide (PI) for flow cytometry on HOS-exposed KIOM-2015EW-treated HCECs.The percentage of late apoptotic and necrotic cells increased upon HOS-exposure, indicating that both apoptosis and necrosis are major events involved in HOS-induced cytotoxicity in HCECs (Figure 3B).The cell viability in HOS-exposed cells was 77%.In the presence of KIOM-2015EW (0.05-0.2 mg/mL), the cell viability recovered to 92%, 94%, and 95%, respectively.
HCECs were also subjected to apoptosis analysis in the presence/absence of KIOM-2015EW.Caspase 3 and PARP cleavage, and Bcl-2 (anti-apoptotic factor) and Bax were detected with specific antibodies 24 h after hyperosmotic stimulation (Figure 3C).In the western blot analysis, high levels of cleaved caspase 3 and PARP were detected in HOS-exposed cells, whereas KIOM-2015EW-treatment decreased this cleavage in a dose-dependent manner.Bcl-2 expression increased with KIOM treatment, but Bax expression level was not different in HOS-and/or KIOM-2015EW-treated cells (Figure 3C).
HPLC analysis of three compounds, including orientin, isoorientin, and vitexin.The HPLC chromatograms of the standard mixture and KIOM-2015EW extract are presented in Figure 7A,B.By comparing the retention times and ultraviolet (UV) spectral data with the standard compounds, the peaks 1, 2 and 3 of KIOM-2015EW were identified as orientin, isoorientin and vitexin, respectively.The mixed standards were indicated at the retention time of 18.21 min (1); 19.20 min (2) and 22.47 min (3) in the chromatogram.These compounds were identified in the KIOM-2015W at similar retention times (1, 18.19 min; 2, 19.19 min; 3, 22.45 min).Other major peaks of retention time were not able to be identified.Therefore, we are currently separating these peak for nuclear magnetic resonance (NMR) analysis.

Identification of the Main Components in KIOM-2015EW Using HPLC
According to the maximum absorption of the standards, a UV detector was set at 280 nm for HPLC analysis of three compounds, including orientin, isoorientin, and vitexin.The HPLC chromatograms of the standard mixture and KIOM-2015EW extract are presented in Figure 7A,B.By comparing the retention times and ultraviolet (UV) spectral data with the standard compounds, the peaks 1, 2 and 3 of KIOM-2015EW were identified as orientin, isoorientin and vitexin, respectively.The mixed standards were indicated at the retention time of 18.21 min (1); 19.20 min (2) and 22.47 min (3) in the chromatogram.These compounds were identified in the KIOM-2015W at similar retention times (1, 18.19 min; 2, 19.19 min; 3, 22.45 min).Other major peaks of retention time were not able to be identified.Therefore, we are currently separating these peak for nuclear magnetic resonance (NMR) analysis.
Nutrients 2018, 10, x FOR PEER REVIEW 13 of 18 immunofluorescence staining.Scale bar, 100 μm.The full size blot is shown in Supplementary Figure S4.

Orientin, Isoorientin and Vitexin Reduced Level of HOS-Induced Pro-Inflammatory Cytokines
To examine the anti-inflammatory effects of three major compounds, we identified levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in HOS-treated HCECs (Figure 7).The levels TNFα, IL-1β and IL-6 were significantly reduced by orientin, isoorientin and vitexin (1, 5 and 25 μM) treatment in HOS-induced cytokines.The anti-inflammatory effects of these compounds were confirmed to be in a dose dependent manner (Figure 7B-D).

Discussion
DES is caused partly by increased osmolarity of the tear film, resulting in inflammation and subsequent cell damage [1,50].In both animal-and cell-based studies, HOS induced the production and expression of pro-inflammatory cytokines in the ocular surface cells [13,14,19,20].
Increasing evidence suggests that the expression of inflammatory mediators (IL-6, IL-1β, and TNF-α) on the ocular surface may play a role in DES pathogenesis [8,51,52].Changes in tear

Orientin, Isoorientin and Vitexin Reduced Level of HOS-Induced Pro-Inflammatory Cytokines
To examine the anti-inflammatory effects of three major compounds, we identified levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in HOS-treated HCECs (Figure 7).The levels TNF-α, IL-1β and IL-6 were significantly reduced by orientin, isoorientin and vitexin (1, 5 and 25 µM) treatment in HOS-induced cytokines.The anti-inflammatory effects of these compounds were confirmed to be in a dose dependent manner (Figure 7B-D).

Discussion
DES is caused partly by increased osmolarity of the tear film, resulting in inflammation and subsequent cell damage [1,50].In both animal-and cell-based studies, HOS induced the production and expression of pro-inflammatory cytokines in the ocular surface cells [13,14,19,20].
Increasing evidence suggests that the expression of inflammatory mediators (IL-6, IL-1β, and TNF-α) on the ocular surface may play a role in DES pathogenesis [8,51,52].Changes in tear composition, including increased cytokine, chemokine, metalloproteinase, and T cell numbers in the conjunctiva are reported in dry eye patients and animal models.This inflammation is partly responsible for eye irritation, ocular surface epithelial disease, and altered corneal epithelial barrier function in dry eye [53].There are several anti-inflammatory therapies for dry eye that target one or more inflammatory mediators/pathways (corticosteroids, cyclosporine, tetracyclines and their derivatives, and essential fatty acids) [53].
To date, the only Food and Drug Administration (FDA)-approved drug for DES treatment in the United States is CsA (Restasis ® , Allergan, Irvine, CA, USA), which is purported to act partly by inhibiting T-cell-stimulated cytokines by binding to the nuclear proteins required for T-cell activation [54,55].Topical corticosteroids can be used to ameliorate DES, but their long-term use is limited owing to side effects such as increased intraocular pressure and cataract formation [54,55].
Recent studies have reported that many natural products (blueberry, green tea, curcumin, quercetin, and resveratrol) have anti-inflammatory effects on DES or ocular disease [56][57][58][59].In oriental medicine, herbal roots, bark and branches of the genus Acer (maple) have been used as a medicine for their antioxidant, antitumor, and anti-inflammatory activity [57].Over the years, medicinal plants of the Acer genus have been shown to treat rheumatism, bruises, eye disease, and pain, in addition to detoxification [57].However, the traditional uses of these plants have been recorded primarily in local herbal books or have been passed down orally from one generation to another [57].The medicinal use of this genus, compared to its ornamental and food uses, must be investigated further as it is widespread and of known therapeutic efficacy [57].Little is known about the bioactivity and potential clinical implications of KIOM-2015EW, a natural-substance-derived herbal product, on the health of the human eye.Here, we present evidence that KIOM-2015EW may have beneficial effects on eye and ocular surface diseases.We explored the beneficial effects of KIOM-2015EW using an in vitro HCEC culture model of HOS-induced dry eye.Our findings demonstrate that HOS induces inflammation and that KIOM-2015EW inhibits HOS-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in HCECs, with comparable efficacy and potency as corticosteroids and CsA.
Several studies have reported that MAPK signaling is activated by HOS [12,13,38].HOS induces HCEC apoptosis through a cytochrome c-mediated death pathway, which may be mediated by JNK and ERK1/2; JNK and ERK mediate IL-1β, TNF-α, and IL-8 induction in human limbal epithelialcells [12].The MAPK cascades are serine/threonine-specific kinases that play key roles in regulating proinflammatory cytokine production [60].Hyperosmolarity-induced cytokine release in HCECs is mediated via multiple MAPKs, including ERK, JNK, and p38 [13,14].We demonstrated that these cytokines are stimulated by HOS and inhibited by KIOM-2015EW, similar to the effects of CsA and FML.
NF-κB activation by HOS has been reported previously.HOS induces nuclear translocation of NF-κB in HCECs and cultured cardiomyocytes with dichotomic actions on caspase activation and cell death [49,61].Moreover, the transcription factors NF-κB and AP-1 play a major role in the induction-inflammation-related genes (cytokines, cytokine receptors, chemotactic proteins, and adhesion molecules) [62].Here, we demonstrated that KIOM-2015EW inhibits HOS-induced NF-κB transcriptional activity, indicating that the anti-inflammatory effects of KIOM-2015EW are mediated via this signaling pathway.
These findings suggest that the dry eye treatment strategy should include a combination of agents that protect the eye from inflammatory injuries, in addition to simple tear-related interventions.The development of a natural product for dry eye prevention and treatment would benefit many patients with eye disorders.
In summary, the current study demonstrated that the natural substance, KIOM-2015EW, effectively protects HCECs from HOS-induced inflammation by reducing inflammatory cytokine production through the regulation of MAPK signalling and NF-κB translocation to the nucleus.Our findings for the first time suggest the potential benefits of a natural plant extract on ocular surface disorders, such as DES.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/10/3/282/s1, Figure S1: KIOM-2015EW regulates mRNA of proinflammatory cytokines in HOS-induced HCECs.This is a full length image of the cropped blots presented in the Figure 2C, Figure S2: KIOM-2015EW reduces HOS-induced apoptotic cell death in HCECs.This is a full length image of the cropped blots presented in the Figure 3C

Figure 4 .
Figure 4. Regulation of Mitogen-activated protein kinase (MAPK) activation by KIOM-2015EW in HOS-induced MAPK phosphorylation.(A) HCECs were exposed to HOS for 15, 30, 60, 120, and 180 min, and the expression levels of MAPK proteins, including p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), phospho-p38 (pp38), phospho-ERK (pERK), and phospho-JNK (pJNK), were determined by western blot analysis; (B) HOS-exposed HCECs were treated with KIOM-2015EW for 30 min, and the expression levels of pp38, pERK, and pJNK were verified by western blot analysis.CsA and FML were used as positive anti-inflammatory drugs.β-Actin was used as the loading control.Data are presented as the mean ± SD of three independent experiments.The full size blot is shown in Supplementary Figure S3.

Figure 4 .
Figure 4. Regulation of Mitogen-activated protein kinase (MAPK) activation by KIOM-2015EW in HOS-induced MAPK phosphorylation.(A) HCECs were exposed to HOS for 15, 30, 60, 120, and 180 min, and the expression levels of MAPK proteins, including p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), phospho-p38 (pp38), phospho-ERK (pERK), and phospho-JNK (pJNK), were determined by western blot analysis; (B) HOS-exposed HCECs were treated with KIOM-2015EW for 30 min, and the expression levels of pp38, pERK, and pJNK were verified by western blot analysis.CsA and FML were used as positive anti-inflammatory drugs.β-Actin was used as the loading control.Data are presented as the mean ± SD of three independent experiments.The full size blot is shown in Supplementary Figure S3.

Figure 5 .
Figure 5.Effect of KIOM-2015EW on MAPK blockade on HOS-induced apoptosis.HOS-exposed HCECs were treated with 20 μM MAPK inhibitors, including SB203580, PD98059, and SP600125, for 24 h.CsA and FML were used as positive anti-inflammatory drugs.(A) Cell viability was measured by MTT assay; (B) After incubation with MAPK inhibitors, apoptotic HCECs were stained with Annexin V and PI, and analyzed by flow cytometry.The data are presented as the mean ± SD of three independent experiments * p < 0.05 vs. control, # p < 0.05 vs. HOS.

Figure 5 .
Figure 5.Effect of KIOM-2015EW on MAPK blockade on HOS-induced apoptosis.HOS-exposed HCECs were treated with 20 µM MAPK inhibitors, including SB203580, PD98059, and SP600125, for 24 h.CsA and FML were used as positive anti-inflammatory drugs.(A) Cell viability was measured by MTT assay; (B) After incubation with MAPK inhibitors, apoptotic HCECs were stained with Annexin V and PI, and analyzed by flow cytometry.The data are presented as the mean ± SD of three independent experiments * p < 0.05 vs. control, # p < 0.05 vs. HOS.

Figure 6 .
Figure 6.Effects of KIOM-2015EW on the nuclear translocation of NF-κB and phosphorylation of IκBα in HOS-induced HCECs.(A) HCECs were simultaneously exposed to HOS and treated with 0.2 mg/mL KIOM-2015EW for 5 min.Cytosolic proteins were subjected to western blot analysis with the indicated antibodies.β-Actin was used as the internal control for the cytosolic fraction.The numbers represent the average densitometric values relative to β-actin.Data are summarized as mean ± SD from three independent experiments; (B) NF-κB p65 activation was examined by

Figure 6 .
Figure 6.Effects of KIOM-2015EW on the nuclear translocation of NF-κB and phosphorylation of IκB-α in HOS-induced HCECs.(A) HCECs were simultaneously exposed to HOS and treated with 0.2 mg/mL KIOM-2015EW for 5 min.Cytosolic proteins were subjected to western blot analysis with the indicated antibodies.β-Actin was used as the internal control for the cytosolic fraction.The numbers represent the average densitometric values relative to β-actin.Data are summarized as mean ± SD from three independent experiments; (B) NF-κB p65 activation was examined by immunofluorescence staining.Scale bar, 100 µm.The full size blot is shown in Supplementary Figure S4.
, Figure S3: KIOM-2015EW regulates HOS-induced MAPK phosphorylation.(A) is an uncropped image for Figure 4A.(B) is an uncropped image for Figure 4B, Figure S4: KIOM-2015EW regulates the phosphorylation and nuclear translocation of NF-κB in HOS-induced HCECs.This is a full length image of the cropped blots presented in the Figure 6A.

Table 1 .
Primer sequences, annealing temperature, and cycles used for reverse transcription polymerase chain reaction (RT-PCR).