Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 alters cell sensitivity to largazole without comprising HDAC inhibition. (A) Largazole sensitivity assay in cells with PRDX6 knockdown and the indicated rescues. HCT116 cells with PRDX6 knockdown and PRDX6 knockdown rescued with WT, S32A, C47S, or S32A+C47S were treated with 100 nM largazole for 12 days, fixed with 4% formaldehyde in PBS, and stained with 0.05% crystal violet. A 3-day DMSO treatment of HCT116 parental cells served as a natural growth control. Images were inverted to grayscale to enhance visibility. (B) Quantification of panel A. Crystal violet stain was solubilized and absorbance measured at 570 nm. Data are mean ± SD. One way ANOVA with Tukey multiple comparisons test was used. **** p < 0.0001. (C) Western blot of pan H4ac levels in HCT116 parental, PRDX6 knockdown, and PRDX6 knockdown cells with the indicated rescues. Cells were treated with 100 nM largazole for 2 hours. GAPDH was used as a loading control. (D) Schematic of the combination treatment assay. 3×10 5 HCT116 parental cells were seeded per well in a 6 well plate. All wells were treated with 100 nM largazole for 9 days, then washed with PBS and replaced with 100 nM largazole, 10 µM MJ33, or 100 nM largazole plus 10 µM MJ33 for 3 additional days. (E-F) Results and quantification of panel D. 3-day DMSO and MJ33 treatments of HCT116 parental cells were used to indicate natural growth rate and any growth effect of MJ33 alone. Images were inverted to grayscale to enhance visibility. Crystal violet stain was solubilized and absorbance measured at 570 nm. Data are mean ± SD. One way ANOVA with Tukey multiple comparisons test was used. **** p < 0.0001. (G-H) IC 50 values for largazole and paragazole in HCT116 parental versus PRDX6 knockdown cells. HCT116 parental and PRDX6 knockdown cells were seeded into a 96 well plate in 4 lanes × 12 wells with 4,000 cells per well. Twelve hours after seeding, medium was replaced with fresh DMEM containing a serial dilution of largazole, and cells were treated for 3 days. Cells were then washed with PBS, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized, and absorbance was read at 530 nm. Wells without largazole were used as controls for normalization. Data are mean ± SD. A four-parameter logistic regression was used to calculate IC 50 . Student t -test was used for statistical analysis. **** p <0.0001.

Article Snippet: PRDX6 KD sensitizes the head and neck squamous carcinoma cell line FaDu to ferroptosis with largazole treatment. ( A and C ) After the indicated treatments, both FaDu shNT and PRDX6 KD cells were stained with 1LμM FerroOrange (Dojindo F374-10) or 1LμM LiperFluo (Dojindo L248-10) in complete FluoroBrite DMEM (10% FBS, 1% GlutaMAX, 1% pen/strep) and costained with 10Lμg/mL Hoechst 33342 for 30 minutes.

Techniques: Inhibition, Sensitive Assay, Knockdown, Staining, Control, Western Blot, Serial Dilution

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 knockdown increases vulnerability to largazole induced lipid peroxidation stress. (A) Largazole induces lipid peroxidation in HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate at 10,000 cells per well. After 24 hours, medium was replaced with fresh DMEM containing the indicated treatments for the indicated durations. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde in PBS, and stained with 10 µM Image iT Lipid Peroxidation Sensor and 10 µg/mL Hoechst 33342 for 3 days at 4°C in the dark. After incubation, cells were washed three times with PBS and imaged using DAPI, FITC, and TRITC channels. Scale bar, 100 µm. (B) Quantification of panel A. Fifty cells per condition were randomly selected and the mean FITC and TRITC fluorescence intensities per cell were measured. The FITC/TRITC ratio was used as a readout of lipid peroxidation stress. Two-way ANOVA with Tukey multiple comparisons test was used. ** p < 0.01, **** p < 0.0001, n.s.= not significant. (C) PRDX6 knockdown induces iron accumulation in HCT116 cells upon largazole treatments. After indicated treatments, live cells were stained with 1LµM FerroOrange (Dojindo) and costained with 10Lµg/mL Hoechst 33342 for 30 minutes prior to imaging, without washout. Imaging was performed on the Revvity Opera Phenix Confocal System using a 20X water objective and standard settings. Nuclei were segmented using Hoechst signal, and perinuclear cytoplasmic regions were defined to quantify FerroOrange fluorescence on a single-cell basis. Border cells were excluded, and integrated fluorescence intensity per cell was calculated. “High” Fe²⁺ cells were defined using Otsu’s threshold from control wells, and the percentage of high-signal cells was quantified per well. Bars represent mean ± SEM from biological replicates. Statistical significance was determined by two-way ANOVA with Tukey’s post-hoc test. *** p < 0.001, **** p < 0.0001, n.s.= not significant. (D) Largazole increases GSSG in PRDX6 knockdown HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate at 10,000 cells per well. After 24 hours, medium was replaced with fresh DMEM containing 100 nM largazole for 18 hours. GSH and GSSG were measured using the GSH/GSSG Glo assay, with readings normalized using the CellTiter Glo luminescent viability assay. Two way ANOVA with Tukey multiple comparisons test was used. * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. = not significant. ( E-F ) Largazole shows increased synergy with erastin in PRDX6 knockdown HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate in 8 lanes × 8 wells at 8,000 cells per well. Twelve hours after seeding, medium was replaced with fresh DMEM containing serial dilutions of largazole and erastin2, and cells were treated for 3 days. Cells were then washed twice with PBS, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized, and absorbance was read at 530 nm. Wells without treatment served as normalization controls. Synergy scores were computed and visualized using SynergyFinder+.

Article Snippet: PRDX6 KD sensitizes the head and neck squamous carcinoma cell line FaDu to ferroptosis with largazole treatment. ( A and C ) After the indicated treatments, both FaDu shNT and PRDX6 KD cells were stained with 1LμM FerroOrange (Dojindo F374-10) or 1LμM LiperFluo (Dojindo L248-10) in complete FluoroBrite DMEM (10% FBS, 1% GlutaMAX, 1% pen/strep) and costained with 10Lμg/mL Hoechst 33342 for 30 minutes.

Techniques: Knockdown, Staining, Incubation, Fluorescence, Imaging, Control, Glo Assay, Viability Assay

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 is critical to maintain GPX4 level in head and neck squamous cell carcinoma. (A) Overall pan-cancer survival analysis stratified by PRDX6 expression. Patients were grouped by significantly upregulated versus significantly downregulated PRDX6 in tumor relative to matched normal tissue. Shaded bands indicate 95% confidence intervals. Log-rank test p -value < 0.05. (B) PRDX6 expression level is critical for survival probability for patients with head and neck squamous cell carcinoma (HNSCC). Five year-Kaplan-Meier overall survival from TCGA data stratified by median PRDX6 expression (High PRDX6, red; Low PRDX6, blue). Shaded bands represent 95% confidence intervals. Log-rank test p -value < 0.05. (C-D) PRDX6 knockdown slows proliferation in squamous carcinoma cell lines. For all indicated cell lines, 12 wells of 500 cells were seeded into 7 plates of 96-well plate (1 plate/day). After indicated duration of incubation, plates were fixed and crystal violet staining was performed. The plates were solubilized, and absorbance was measured at 570 nm. Data are mean± SD. (E-F) Largazole shows synergistic effect with RSL3 and with erastin2 in A223 and FaDu PRDX6 knockdown cells. For both FaDu and A223, shNT and PRDX6 knockdown cells were seeded into 8 lanes x 8 wells of 96-well plate with 8,000 cells in each well. 12 hours after seeding, DMEM was replaced by fresh DMEM with largazole and RSL3 or and erastin2 in serial-diluted manner, and cells were treated for 3 days. After 3 days treatment, cells were washed with PBS twice, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized and quantified by reading absorbance for each well at 570 nm. Wells with no treatment were used as control to normalize the data and synergy scores were calculated and visualized using SynergyFinder+. (G-H) Largazole downregulates PRDX6 and GPX4 mRNA in FaDu and A223 cells. FaDu and A223 cells with either shNT or PRDX6 knockdown were treated with 100LnM largazole for the indicated times. Total RNA was extracted at each time point and subjected to RT-qPCR to assess PRDX6 and GPX4 mRNA expression. β-actin was used as the housekeeping gene for normalization. mRNA levels were normalized to the respective 0Lhr WT control group for both cell types. Data represent the relative expression of PRDX6 and GPX4 over time in response to largazole treatment. A regular one-way ANOVA was performed for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. = not significant. (I) PRDX6 regulates GPX4 protein level. Western blot for GPX4 expression levels in A223 or FaDu WT, PRDX6 KD and PRDX6 overexpression (OE) cell lines. Actin was used as a loading control. (J) PRDX6 and GPX4 co-expression and survival in HNSCC. Five year Kaplan Meier overall survival from TCGA stratified by combined PRDX6 and GPX4 expression (Both high, red; Both low, blue) using median cutoffs. Shaded bands indicate 95 percent confidence intervals. Log rank test p < 0.05.

Article Snippet: PRDX6 KD sensitizes the head and neck squamous carcinoma cell line FaDu to ferroptosis with largazole treatment. ( A and C ) After the indicated treatments, both FaDu shNT and PRDX6 KD cells were stained with 1LμM FerroOrange (Dojindo F374-10) or 1LμM LiperFluo (Dojindo L248-10) in complete FluoroBrite DMEM (10% FBS, 1% GlutaMAX, 1% pen/strep) and costained with 10Lμg/mL Hoechst 33342 for 30 minutes.

Techniques: Expressing, Knockdown, Incubation, Staining, Control, Quantitative RT-PCR, Western Blot, Over Expression

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 restrains largazole induced inflammatory and ferroptotic responses in squamous carcinoma cells. (A) GSEA shows that PRDX6 knockdown in A223 cells enhances largazole induced inflammatory responses. Bubble plot summarizing GSEA results of Hallmark gene sets enrichment across three conditions: KD_DMSO vs WT_DMSO, WT_LAR vs WT_DMSO and KD_LAR vs KD_DMSO. Each point represents a significantly enriched pathway (FDR < 0.25). Color indicates the normalized enrichment score (NES), reflecting the direction and magnitude of enrichment. Bubble size reflects the negative log10 of the adjusted p -value. (B-C) Largazole induces inflammatory signaling and ferroptosis in a PRDX6 knockdown dependent manner. Heatmaps display expression of genes in inflammation and ferroptosis pathways across the indicated conditions. Gene values were taken from DESeq2 normalized counts, log2 transformed, and row scaled (z score). The color scale represents relative expression (blue, low; red, high). Only genes detected in the RNA seq dataset and present in the normalized matrix are shown. (D) Cytokine array reveals elevated inflammatory responses induced by largazole treatment in A223 PRDX6 KD cells. A223 WT and PRDX6 KD cells were seeded into a 6-well plate with 2×10 6 cells in each well. After 24 hours, medium was replaced with 1 mL fresh DMEM containing DMSO or 100 nM largazole for 18 hours. Cells were collected and cytokine levels were measured using the Proteome Profiler Array Mouse XL Cytokine kit. (E) Quantification of panel D. Spot intensities were measured and analyzed by two way ANOVA with Tukey multiple comparisons. * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. = not significant.

Article Snippet: PRDX6 KD sensitizes the head and neck squamous carcinoma cell line FaDu to ferroptosis with largazole treatment. ( A and C ) After the indicated treatments, both FaDu shNT and PRDX6 KD cells were stained with 1LμM FerroOrange (Dojindo F374-10) or 1LμM LiperFluo (Dojindo L248-10) in complete FluoroBrite DMEM (10% FBS, 1% GlutaMAX, 1% pen/strep) and costained with 10Lμg/mL Hoechst 33342 for 30 minutes.

Techniques: Knockdown, Expressing, Transformation Assay, RNA Sequencing