ha tagged atf4  (Addgene inc)

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: Human overexpression plasmids for ATF4 (Addgene #115969) and TFAM (pLV [Exp]-Bsd-CMV > hTFAM [ NM_003201.3 ]) were obtained from Addgene and VectorBuilder, separately.

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 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: Human overexpression plasmids for ATF4 (Addgene #115969) and TFAM (pLV [Exp]-Bsd-CMV > hTFAM [ NM_003201.3 ]) were obtained from Addgene and VectorBuilder, separately.

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: Human overexpression plasmids for ATF4 (Addgene #115969) and TFAM (pLV [Exp]-Bsd-CMV > hTFAM [ NM_003201.3 ]) were obtained from Addgene and VectorBuilder, separately.

Techniques: Gene Expression, Inhibition, Disruption