Journal: Redox Biology

Article Title: POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis

doi: 10.1016/j.redox.2025.103758

Figure Lengend Snippet: POLR1A is a key genetic dependency and a potential therapeutic target in PM. A, B. Enrichment scores of the 13 pathways significantly over-represented across the PM dependent genes (A) and gene set enrichment analysis (GSEA) of the 4 highlighted pathways (B). The analysis was based on the CRISPR dropout dataset from the DepMap ( https://depmap.org/portal/ ). C. mRNA expression and dependency scores of ribosome biogenesis genes (GO RIBOSOME BIOGENESIS gene set) across the indicated PM cell lines (n = 16). The analysis was based on the dependency and expression scores from the DepMap. D. mRNA expression values and dependency scores of the genes encoding RNA polymerase I subunits across the indicated PM cell lines (n = 16). The mRNA expression values and dependency scores are from Depmap. E. The dependency score of Pol I subunit genes (POLR1A, POLR1G) and the previously reported genes as therapeutic targets in PM. The analysis was based on the dependency score across 16 PM cell lines from the DepMap. F. The expression of the Pol I subunits POLR1A is significantly correlated with PM patient survival. Kaplan-Meier univariate survival analysis of the TCGA cohort of PM patients (n = 87) was conducted using the R ‘survival’ and ‘survminer’ packages. Patients are dichotomized by upper 50 % (POLR1A high ) and lower 50 % (POLR1A low ) of POLR1A mRNA expression. The p-value was calculated using the log-rank test. G. Correlation analysis of mRNA expression between POLR1A, POLR1F and POLR1G and the RiBi gene set. ssGSEA analysis was based on the TCGA-MESO dataset. Spearman correlation test is used to calculate the coefficients (Cor) and the p value. H. mmunoblot analysis of POLR1A knockdown in MESO1 and H2452 cells. MESO1 and H2452 cells expressing doxycycline (Dox)-inducible scramble control (shCTRL) or POLR1A-targeted shRNAs (shPOLR1A#1, shPOLR1A#2) were treated with 500 nM Dox for 48 h. I. Dual-luciferase reporter assay in which Firefly luciferase expression was driven by a ribosomal DNA (rDNA) promoter. After induction of shRNA targeting POLR1A (500 nM Dox for 48 h), Firefly and Renilla luciferase activities were measured, and the ratio of Firefly to Renilla luminescence was calculated to normalize for transfection efficiency. Data were presented as mean ± SD (n = 5), with ns (p ≥ 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test.

Article Snippet: The following antibodies were used: POLR1A (Proteintech, #20595-1-AP); β-actin (Cell Signaling Technology/CST, #3700S); TFAM (Proteintech, #22586-1-AP); PERK (CST, #3192S); p-PERK (CST, #3179S); ATF4 (CST, #11815S); eIF2α (CST, #9722S); p -eIF2α (CST, #3398S); PARK2 (Proteintech, #14060-1-AP); GPX4 (abcam, Cat# ab125066); TP53(CST, Cat#9282S); SLC7A11(Proteintech, #26864-1-AP); CYTB(Proteintech, #55090-1-AP); ATP6(Proteintech, #55313-1-AP); TOMM20(CST, #42406S); Phospho-Histone H2A.X (Ser139) (CST, Cat#9718S); FTL(proteintech,10727-1-AP); NCOA4(NBP3-18136); LC3A/B(CST, #4108S), IRDye 680RD-conjugated donkey anti-mouse IgG (Lot #D20503-05) and IRDye 800CW-conjugated donkey anti-rabbit IgG (Lot #D01216-10) from Li-COR Biosciences.

Techniques: CRISPR, Expressing, Biomarker Discovery, Knockdown, Control, Luciferase, Reporter Assay, shRNA, Transfection

Journal: Redox Biology

Article Title: POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis

doi: 10.1016/j.redox.2025.103758

Figure Lengend Snippet: POLR1A controls TFAM expression through ATF4 A. Venn diagram of common differentially expressed genes (DEGs) in POLR1A high samples and POLR1A inhibitor-treated cell lines. DEGs were defined as those with a fold change (FC) > 1.5 and p < 0.05, determined using the limma package in R. The analysis incorporated four publicly available transcriptomic datasets: TCGA-MESO, EGAD00001001915, GSE145603 , and GSE204749 . Ten overlapping DEGs were identified across all datasets. B. Immunoblot analysis of MESO1 and H2452 cells expressing Dox-inducible shCTRL or shPOLR1A after 48h treatment with 500 nM Dox. Densitometric quantification of TFAM normalized to β-actin is shown beneath each band. C. Quantitative RT-PCR of MESO1 and H2452 cells expressing Dox-inducible shCTRL or shPOLR1A after 48h treatment with 500 nM Dox. Data are shown as mean ± SD (n = 4), with ns (p ≥ 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test. D, E, Genetic co-dependency analysis identifies POLR1A- and TFAM-relevant gene sets (PRGs; TRGs). Co-dependency analysis was performed using RNAi datasets from DepMap (Achilles + DRIVE + Marcotte, DEMETER2). The X-axis represents gene rank based on correlation with POLR1A (D) or TFAM (E), while the Y-axis shows Pearson correlation coefficients of gene effect scores. Genes above the dashed line exhibit significant positive correlations (p < 0.05). The top 100 positively correlated genes (rank <100) are highlighted in red (PRGs) and orange (TRGs) and were subjected to transcription factor (TF) enrichment analysis using ChEA3. F, G, TF enrichment analysis of PRGs and TRGs identifies ATF4 a key mediator linking POLR1A and TFAM. Venn diagram showing 11 TFs commonly enriched in both PRGs and TRGs, based on ChEA3 analysis (F). Odds ratio (OR)-based enrichment analysis identifies ATF4 as the top-ranked shared TF, suggesting it may function as a key upstream regulator of both gene sets (G). H. UMAP visualization of coordinated RiBi and ATF4 activity in single PM cells. UMAP projections of scRNA-seq data from treatment-naïve PM samples (n = 3), with each cell scored by ssGSEA for RiBi (red gradient; GO_Ribosome_Biogenesis) and ATF4 transcriptional activity (blue gradient; ATF4_Q2 gene set, https://www.gsea-msigdb.org/gsea/msigdb/cards /ATF4_Q2). The merged overlay (bright pink) highlights cells with high scores in both pathways. I. UMAP visualization of TFAM expression mirrors RiBi and ATF4 activity in PM single cells. Using the same scRNA-seq dataset as in (H), cells are colored by ssGSEA scores for TFAM mRNA levels, revealing a spatial distribution that parallels high RiBi and ATF4 activity. J, K. qRT-PCR analysis of MESO1 and H2452 cells after transiently transfected with control (siCTRL) or ATF4-targeted siRNA (siATF4) for 48 h (J), with empty vector (vector) or ATF4 overexpression plasmid (oe-ATF4) for 48 h (K). Data are shown as mean ± SD (n = 4), with ns (p ≥ 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test. L, M. Immunoblot analysis of POLR1A–ATF4–TFAM axis perturbations. MESO1 and H2452 cells were transiently transfected with siCTRL or siATF4 for 48 h (L), with or without ATF4 overexpression (oe-ATF4) (M). N, O. MESO1 and H2452 cells expressing Dox-inducible shCTRL or shPOLR1A after 48h treatment with 500 nM Dox.

Article Snippet: The following antibodies were used: POLR1A (Proteintech, #20595-1-AP); β-actin (Cell Signaling Technology/CST, #3700S); TFAM (Proteintech, #22586-1-AP); PERK (CST, #3192S); p-PERK (CST, #3179S); ATF4 (CST, #11815S); eIF2α (CST, #9722S); p -eIF2α (CST, #3398S); PARK2 (Proteintech, #14060-1-AP); GPX4 (abcam, Cat# ab125066); TP53(CST, Cat#9282S); SLC7A11(Proteintech, #26864-1-AP); CYTB(Proteintech, #55090-1-AP); ATP6(Proteintech, #55313-1-AP); TOMM20(CST, #42406S); Phospho-Histone H2A.X (Ser139) (CST, Cat#9718S); FTL(proteintech,10727-1-AP); NCOA4(NBP3-18136); LC3A/B(CST, #4108S), IRDye 680RD-conjugated donkey anti-mouse IgG (Lot #D20503-05) and IRDye 800CW-conjugated donkey anti-rabbit IgG (Lot #D01216-10) from Li-COR Biosciences.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Redox Biology

Article Title: POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis

doi: 10.1016/j.redox.2025.103758

Figure Lengend Snippet: The POLR1A/TFAM axis regulates mitophagy-dependent iron metabolism in PM cells. A. Immunoblot analysis of MESO1 and H2452 cells expressing Dox-inducible shCTRL or shPOLR1A after treated with 500 nM Dox for 48 h. B. The POLR1A–TFAM axis regulates mitochondrial membrane potential (Δψm). MESO1 and H2452 cells expressing Dox-inducible shCTRL, shPOLR1A, or shTFAM, with or without TFAM co-expression, were treated with 500 nM Dox for 48 h and stained with MitoTracker Red CMXRos to measure Δψm (left). Mean fluorescence intensity (MFI) was quantified as mean ± 95 % confidence interval (CI) from three independent experiments (right), with ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired two-tailed t -test. C. CX-5461 reduces oxygen consumption rate (OCR) in MESO1 and H2452 cells. Cells were treated with vehicle or 100 nM CX-5461 for 72 h. OCR was measured using a Seahorse analyzer. Data are shown as mean ± SD from at least three independent experiments (n > 3). D. Schematic of mt-Keima–based mitophagy reporter. The mt-Keima construct targets the pH-sensitive Keima fluorescent protein to the mitochondrial matrix. Upon delivery to acidic lysosomes, Keima's excitation shifts from 440 nm to 586 nm. The mitophagy index (%) is calculated as follows: (number of cells in the enhanced mitophagy gate/total cell number)∗100. E. Quantification of mitophagy using the mt-Keima reporter. MESO1 and H2452 cells expressing Dox–inducible constructs, with or without TFAM co-expression, were treated with 500 nM Dox for 48 h. Mitophagy index was determined by flow cytometry using the mt-Keima reporter. Data are shown as mean ± 95 % confidence interval (CI) from three independent experiments (n = 3), with ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired two-tailed t -test. F. Chemical structure and detection mechanism of RhoNox-1, a fluorescent probe that selectively reacts with labile Fe 2+ to generate an irreversible red-orange fluorescent product (Ex/Em: 540/575 nm). Upon binding Fe 2+ , the probe undergoes a chemical transformation that shifts its fluorescence emission, enabling sensitive and specific detection of intracellular ferrous iron levels. G, H. KD of POLR1A/TFAM axis increases labile Fe 2+ levels in PM cells. MESO1 (G) and H2452 (H) cells expressing shCTRL, shPOLR1A, or shTFAM, with or without TFAM co-expression, were treated with 500 nM Dox for 48 h, stained with RhoNox-1, and analyzed by flow cytometry. Mean fluorescence intensity (MFI) is presented as mean ± 95 % CI (n = 3), with ns (p ≥ 0.05), ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test. I, J. PARK2 silencing suppresses POLR1A- and TFAM deficiency-induced mitophagy and labile Fe 2+ accumulation. MESO1 and H2452 cells with Dox-inducible shPOLR1A or shTFAM were transfected with siCTRL or siPARK2#3 and cotreated with 500 nM Dox for 48 h. Mitophagy index was measured using the mt-Keima reporter by flow cytometry and labile Fe 2+ levels were quantified with RhoNox-1 staining. Data are shown as MFI ±95 % CI (n = 3), with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test.

Article Snippet: The following antibodies were used: POLR1A (Proteintech, #20595-1-AP); β-actin (Cell Signaling Technology/CST, #3700S); TFAM (Proteintech, #22586-1-AP); PERK (CST, #3192S); p-PERK (CST, #3179S); ATF4 (CST, #11815S); eIF2α (CST, #9722S); p -eIF2α (CST, #3398S); PARK2 (Proteintech, #14060-1-AP); GPX4 (abcam, Cat# ab125066); TP53(CST, Cat#9282S); SLC7A11(Proteintech, #26864-1-AP); CYTB(Proteintech, #55090-1-AP); ATP6(Proteintech, #55313-1-AP); TOMM20(CST, #42406S); Phospho-Histone H2A.X (Ser139) (CST, Cat#9718S); FTL(proteintech,10727-1-AP); NCOA4(NBP3-18136); LC3A/B(CST, #4108S), IRDye 680RD-conjugated donkey anti-mouse IgG (Lot #D20503-05) and IRDye 800CW-conjugated donkey anti-rabbit IgG (Lot #D01216-10) from Li-COR Biosciences.

Techniques: Western Blot, Expressing, Membrane, Staining, Fluorescence, Two Tailed Test, Construct, Flow Cytometry, Binding Assay, Transformation Assay, Transfection

Journal: Redox Biology

Article Title: POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis

doi: 10.1016/j.redox.2025.103758

Figure Lengend Snippet: The POLR1A/TFAM promotes ferroptosis resistance in PM cells A, B. Cell viability assay of MESO1 and H2452 cells expressing Dox–inducible shCTRL, shPOLR1A, or shTFAM, with or without TFAM overexpression (oeTFAM), after treated with 500 nM Dox and increasing concentrations of RSL3 for 48h, in the presence or absence of Fer-1 (5 μM). Data are presented as mean ± SD (n = 6). C. Cell death assay of MESO1 and H2452 cells expressing Dox–inducible shCTRL, shPOLR1A, or shTFAM, with or without TFAM overexpression (oeTFAM), after treated with 500 nM Dox and 200 nM RSL3 for 48h, in the presence or absence of Fer-1 (5 μM). Data are presented as mean ± SD (n = 5), with ns (p ≥ 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test. D. Clonogenic assay of MESO1 and H2452 cells with Dox-inducible shCTRL, shPOLR1A, or shTFAM, with or without TFAM overexpression (oeTFAM), after treated with Dox (500 nM) and increasing concentrations of RSL3 for 48h, in the presence or absence of 5 μM Fer-1. E. MESO1 and H2452 cells expressing Dox–inducible shCTRL, shPOLR1A, or shTFAM were treated with Dox (500 nM) and RSL3 (100 nM) for 48 h, with or without TFAM overexpression (oeTFAM) and in the presence or absence of deferoxamine (DFO; 5 μM) and Fer-1 (5 μM). Lipid peroxidation was quantified by C11-BODIPY staining and flow cytometry. Data are presented as MFI ±95 % CI (n = 3), with ns (p ≥ 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001 by unpaired t -test. F, PARK2 silencing suppresses POLR1A- and TFAM deficiency-induced lipid peroxidation. MESO1 and H2452 cells with Dox-inducible shPOLR1A or shTFAM were treated with 500 nM Dox for 48 h, in the presence or absence of siPARK2. Lipid peroxidation was quantified by C11-BODIPY staining and flow cytometry. Data are shown as MFI ±95 % CI (n = 3), with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by unpaired t -test.

Article Snippet: The following antibodies were used: POLR1A (Proteintech, #20595-1-AP); β-actin (Cell Signaling Technology/CST, #3700S); TFAM (Proteintech, #22586-1-AP); PERK (CST, #3192S); p-PERK (CST, #3179S); ATF4 (CST, #11815S); eIF2α (CST, #9722S); p -eIF2α (CST, #3398S); PARK2 (Proteintech, #14060-1-AP); GPX4 (abcam, Cat# ab125066); TP53(CST, Cat#9282S); SLC7A11(Proteintech, #26864-1-AP); CYTB(Proteintech, #55090-1-AP); ATP6(Proteintech, #55313-1-AP); TOMM20(CST, #42406S); Phospho-Histone H2A.X (Ser139) (CST, Cat#9718S); FTL(proteintech,10727-1-AP); NCOA4(NBP3-18136); LC3A/B(CST, #4108S), IRDye 680RD-conjugated donkey anti-mouse IgG (Lot #D20503-05) and IRDye 800CW-conjugated donkey anti-rabbit IgG (Lot #D01216-10) from Li-COR Biosciences.

Techniques: Viability Assay, Expressing, Over Expression, Clonogenic Assay, Staining, Flow Cytometry

Journal: Redox Biology

Article Title: POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis

doi: 10.1016/j.redox.2025.103758

Figure Lengend Snippet: Working model of the POLR1A–ATF4–TFAM axis in ferroptosis defense. Under homeostatic conditions, POLR1A promotes ATF4–mediated transcription of TFAM, ensuring proper mitochondrial gene expression and function. TFAM supports mitochondrial integrity, limiting mitophagy and the consequent release of labile Fe 2+ , thereby preventing iron-driven lipid peroxidation and ferroptotic cell death. Inhibition of POLR1A or disruption of ATF4–TFAM signaling triggers excessive mitophagy, elevates cytosolic Fe 2+ , and sensitizes cancer cells to GPX4 inhibitors (e.g., RSL3), leading to hyperactivation of ferroptosis.

Article Snippet: The following antibodies were used: POLR1A (Proteintech, #20595-1-AP); β-actin (Cell Signaling Technology/CST, #3700S); TFAM (Proteintech, #22586-1-AP); PERK (CST, #3192S); p-PERK (CST, #3179S); ATF4 (CST, #11815S); eIF2α (CST, #9722S); p -eIF2α (CST, #3398S); PARK2 (Proteintech, #14060-1-AP); GPX4 (abcam, Cat# ab125066); TP53(CST, Cat#9282S); SLC7A11(Proteintech, #26864-1-AP); CYTB(Proteintech, #55090-1-AP); ATP6(Proteintech, #55313-1-AP); TOMM20(CST, #42406S); Phospho-Histone H2A.X (Ser139) (CST, Cat#9718S); FTL(proteintech,10727-1-AP); NCOA4(NBP3-18136); LC3A/B(CST, #4108S), IRDye 680RD-conjugated donkey anti-mouse IgG (Lot #D20503-05) and IRDye 800CW-conjugated donkey anti-rabbit IgG (Lot #D01216-10) from Li-COR Biosciences.

Techniques: Gene Expression, Inhibition, Disruption

Journal: eLife

Article Title: A unified view of low complexity regions (LCRs) across species

doi: 10.7554/eLife.77058

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-POLR1A (rabbit polyclonal) , Novus Biologicals , NBP2-56122 , (1:100).

Techniques: Recombinant, Expressing, Plasmid Preparation, Software