Fig 2: Model for XAB2 in gene expression and cellular senescence.

Fig 3: Translation and mRNA surveillance pathway were required for POLR2A RNA downregulation induced by XAB2 knockdown. (A) Translation inhibition by CHX rescued POLR2A at RNA level after XAB2 depletion. Cells were treated with 50 ug/ml CHX for 24 h after XAB2 depletion. (B) Quantitation of relative mRNA expression compared to control sample in A (n = 3, n.s.: no significance, *P < 0.05, **P < 0.01). (C) Translation inhibition by emetine rescued POLR2A at RNA level after XAB2 depletion. Cells were treated with 10 ug/ml emetine for 24 h after XAB2 depletion. (D) Quantitation of relative mRNA expression compared to control sample in C (n = 3, n.s.: no significance, *P < 0.05, **P < 0.01). (E) KEGG analysis based on TMT proteome results showing gene functions with significant upregulation after XAB2 knockdown. (F) Differential expression of genes (>1.2-fold, P < 0.05) related to mRNA surveillance pathway after XAB2 knockdown. (G) Depletion of Dom34 after XAB2 knockdown partially rescued POLR2A at RNA level. Cells were treated with siRNAs specific for XAB2 and Dom34 for 48 h. (H) Quantitation of relative mRNA expression compared to control sample in G (n = 3, *P < 0.05). (I) Depletion of Dom34 after XAB2 knockdown partially rescued POLR2A at protein level. Cells were treated with siRNAs specific for XAB2 and Dom34 for 48 h. (J) Quantitation of relative protein expression compared to control sample in I (n = 3, n.s.: no significance, *P < 0.05, ***P < 0.001). (K) Depletion of Dom34 increased the stability of POLR2A mRNA. (L) Quantitation of relative mRNA expression in K (n = 3).

Fig 4: Cell senescence induced by XAB2 depletion was mediated by POLR2A. (A) SA-ß-gal staining showed that depletion of XAB2 in HFF1 cells led to severe senescence phenotype. SA-ß-gal staining was performed at 96 hours after siRNA treatment. (B) Significant increase of SA-ß-gal positive cells after XAB2 depletion. More than 200 randomly selected cells were counted (n = 3, ***P < 0.001). (C) XAB2 depletion led to elevated expression of p53 and p21. (D) Quantitation of relative protein expression compared to control sample in C (n = 3, **P < 0.01, ***P < 0.001). (E) Depletion of POLR2A in HFF1 cells resulted in senescence phenotype. SA-ß-gal staining was performed at 96 hours after siRNA treatment. (F) POLR2A depletion led to significant increase of SA-ß-gal positive cells. More than 200 randomly selected cells were counted (n = 3, ***P < 0.001). (G) Depletion of POLR2A caused enhanced expression of p53 and p21. (H) Quantitation of relative protein expression compared to control sample in G (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (I) Re-expression of POLR2A in XAB2 depleted HFF1 cells alleviated the senescence phenotype. SA-ß-gal staining was performed at 96 hours after siRNA treatment (60 h) and POLR2A transfection (36 h). (J) Re-expression of POLR2A in XAB2 depleted HFF1 cells resulted in substantial reduction of SA-ß-gal positive cells. More than 200 randomly selected cells were counted (n = 3, n.s.: no significance, ***P < 0.001). (K) Western blot to show that re-expression of POLR2A after XAB2 depletion led to moderate decrease of elevated p53 and p21 level. (L) Quantitation of relative protein expression compared to control sample in K (n = 3, n.s.: no significance, *P < 0.05, **P < 0.01)

Fig 5: XAB2 depletion led to substantial loss of POLR2A and reduction of newly transcribed RNA. (A) RT-PCR showing decreased expression of POLR2A mRNA in HeLa cells treated with XAB2 siRNA. Cells were harvested after 48 hours of siRNA treatment. (B) Quantitation of relative mRNA expression compared to control sample in A (n = 3, ***P < 0.001). (C) RT-PCR showing decreased expression of POLR2A mRNA in HeLa cells expressing XAB2 shRNA. Cells were harvested after 72 hours of Dox induction. (D) Quantitation of relative mRNA expression compared to control sample in C (n = 3, ***P < 0.001). (E) Western blot showing reduction of POLR2A protein in HeLa cells treated with XAB2 siRNA. Cells were harvested after 48 h of siRNA treatment. (F). Quantitation of relative protein expression compared to control sample in E (n = 3, **P < 0.01, ***P < 0.001). (G) Western blot showing reduction of POLR2A protein in HeLa cells expressing XAB2 shRNA. Cells were harvested after 72 h of Dox induction. (H) Quantitation of relative protein expression compared to control sample in G (n = 3, **P < 0.01, ***P < 0.001). (I) Immunofluorescence (IF) staining to show substantial loss of POLR2A and altered SC35 pattern after XAB2 depletion. IF staining of POLR2A was performed after 48 h of siRNA treatment. (J) EU incorporation assay showing significant decrease of newly transcribed RNA after XAB2 depletion. Assay was performed after 48 h of siRNA treatment.