Fig 1: Influence of PIAS1 depletion on the AR cistrome. VCaP cells were treated as in Figure 7. (A) Venn diagram showing overlap of AR cistromes in siNON and siPIAS1 cells (upper panel). Non-overlapping sites were further analyzed using get-DifferentialPeak tool to achieve final categories. Heat map showing AR tag densities for siNON unique, siNON/siPIAS1-shared and siPIAS1 unique sites in a window ±2 kb (lower panel). (B) Comparison of AR and H3K4me2 average tag counts in ±500 bp from the centers of the shared and unique sites. (C) The four most enriched motifs of AR-binding sites in VCaP cells. Initial de novo motif discovery was performed on all AR-binding sites (siNON) on ±100 bp of the peak center. (D) Distribution of the most enriched de novo motifs in siNON unique, siNON/siPIAS1-shared and siPIAS1 unique sites. Statistical significances were calculated by ?2-test (***P < 0.001).
Fig 2: A schematic model of PIAS1 function on AR target genes regulating prostate cancer cell growth. Androgen-bound ARs recruit PIAS1 to chromatin enhancer sites that are neighbored by FOXA1 and surrounded by active chromatin H3 methyl marks. PIAS1 subsequently promotes SUMO2/3 (S2/3) modification of FOXA1 and/or AR and other factors, which also influences interactions with coregulator proteins (CoR). Pol II; RNA polymerase II and basal transcription machinery.
Fig 3: UBE2I accelerates PIAS1 ubiquitination for degradation. (A) The si-UBE2I was transfected into cells and the ubiquitination level of PIAS1 was detected by co-immunoprecipitation assay. (B) The pcDNA-UBE2I was transfected into cells and the ubiquitination level of PIAS1 was detected by co-immunoprecipitation assay. (C) UBE2I was overexpressed alone or simultaneously with NRIP1, and the ubiquitination level of PIAS1 was detected by co-immunoprecipitation assay. GAPDH was used as an internal reference. N = 6, *P < 0.01.
Fig 4: ChIP-seq track examples of AR-, PIAS1-, FOXA1- and SUMO2/3-binding events in growth-associated AR target loci in VCaP cells. The occupancy of AR (dark blue in siNON-treated cells and light blue in siPIAS1-treated cells) and H3K4me2 (violet) in the presence of androgen (+, R1881 2 h) and that of PIAS1 (green), FOXA1 (purple) and SUMO2/3 (red) in the absence (-) and presence (+) of androgen. Red bars below the tracks depict the positions of the identified peak sites and red arrows sites co-occupied by AR, PIAS1, FOXA1 and SUMO2/3. Except for IGFBP-1, the expression of all loci was affected by androgen exposure.
Fig 5: PIAS1 relies on its SUMO E3 ligase activity to suppress IAV transcription and replication.(A) siRNA knockdown of PIAS1 in HEK293T cells. HEK293T cells were transfected with PIAS1 siRNA or scrambled siRNA for 48 h. Knockdown of PIAS1 expression was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (B) Minigenome assay in siRNA-treated HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. HEK293T cells treated with siRNA as in (A) were transfected with plasmids for the expression of four viral RNP proteins (WSNPB2, WSNPB1, WSNPA, and WSNNP), together with pHH21-SC09NS F-Luc and pRL-TK. Thirty-six hours later, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the internal control, Renilla luciferase activity. ***, P < 0.001. (C) Establishment of a PIAS1_KO HEK293T cell line. The knockout of PIAS1 was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (D) Minigenome assay in PIAS1_KO HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (B). ***, P < 0.001. (E) RT-qPCR analysis to determine the effect of PIAS1 knockdown on the transcription and replication of viral RNAs. A549 cells were transfected with siRNA targeting PIAS1 or with scrambled siRNA for 48 h, followed by infection with WSN (H1N1) virus (MOI = 5). Total RNA was extracted at 6 h p.i. by using TRIzol reagent, and the levels of NP-specific vRNA, cRNA, and mRNA were analyzed by RT-qPCR and then normalized to GAPDH mRNA. The values shown are standardized to the corresponding RNA expression level in the scrambled siRNA-treated A549 cells (100%). **, P < 0.01, ***, P < 0.001. (F-I) Minigenome assay in HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. HEK293T cells were transfected with plasmids for the expression of four viral RNP proteins and increasing amounts of Myc-PIAS1 or Myc-PIAS1 mutant, together with pHH21-SC09NS F-Luc and pRL-TK. At 36 h post-transfection, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the Renilla luciferase activity. **, P < 0.01, ***, P < 0.001. (J-M) Minigenome assay in PIAS1_KO HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (F-I). *, P < 0.05, **, P < 0.01, ***, P < 0.001.
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