Fig 2: Regulation of RGS22 expression by the binding of YY1 directly to the RGS22 promoter. (A-C) RGS22 expression in YY1-overexpressing PANC-1 cells or YY1-knockdown CFPAC-1 cells was measured by reverse transcription-quantitative PCR and western blotting. (D) Schematic diagram of the luciferase reporter construct containing the human RGS22 promoter and the mutant construct containing the RGS22 promoter in which the predicted YY1 binding site was mutated. (E) EMSA showing that YY1 binds to the RGS22 promoter. The wild-type probe was incubated without (lane 1) or with (lane 2) PANC-1-YY1 cell nuclear proteins in the absence or presence of unlabeled probes (lanes 3–6). Lanes 3 and 4 contain the wild-type probe, and lanes 5 and 6 contain the mutant probe, each at 50- and 100-fold molar excess. EMSA was performed using an anti-YY1 antibody (lane 7), and the IgG antibody was used as a negative control for the YY1 antibody (lane 8). (F) Luciferase assays demonstrated the luciferase activity of PANC-1 cells transfected with YY1-overexpression or control lentiviruses. Data are presented as the mean ± SD of three independent experiments.*P≤0.05, **P≤0.01, ***P≤0.001. EMSA, electrophoretic mobility shift assay; RGS22, Regulator of G-protein signaling 22; YY1, Yin Yang-1; OE, overexpression; NC, negative control; KD, knockdown; WT, wildtype; MT, mutant.

Fig 3: RGS22 as a functional target of YY1. (A) Cell Counting Kit-8, (B and C) colony formation, and (D and E) EdU assays were performed to analyze the proliferation of PANC-1-YY1 cells transfected with RGS22 siRNA. (F and G) Wound healing assays were performed to analyze the migration of PANC-1-YY1 cells transfected with RGS22 siRNA. (H-J) Cell migration and invasion assays were performed in PANC-1-YY1 cells transfected with RGS22 siRNA (magnification, ×100; scale bar, 100 µm). Data are presented as the mean ± SD value of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001. RGS22, Regulator of G-protein signaling 22; YY1, Yin Yang-1; siRNA, small interfering RNA; OD, optical density; OE, overexpression; NC, negative control; KD, knockdown.

Fig 4: RGS22 suppresses proliferation of pancreatic cancer cells. Relative (A) mRNA and (B) protein expression levels of RGS22 in RGS22-knockdown and RGS22-overexpressing pancreatic cancer cells. (C and D) Cell Counting Kit-8, (E and F) colony formation, and (G-J) EdU assays were performed to analyze the effects of RGS22 knockdown or overexpression on cell proliferation. Magnification, ×100; scale bar, 100 µm. Data are presented as the mean ± SD of three independent experiments. **P≤0.01. RGS22, Regulator of G-protein signaling 22; OD, optical density; OE, overexpression; NC, negative control; KD, knockdown.

Fig 5: Expression of RGS22 in PDAC. (A) Boxplot showing RGS22 RNA expression in 52 pairs of PDAC tissues and adjacent tissues, as determined by RT-qPCR. (B) A boxplot showing RGS22 protein expression in 52 pairs of PDAC tissues and adjacent tissues, as determined by IHC. (C) RT-qPCR analysis of RGS22 RNA expression in a panel of human PDAC cell lines and the HPNE cell line. (D) A boxplot showing RGS22 RNA expression in 179 PDAC tissues and 171 normal pancreatic tissues [data from TCGA and GTEx; unit of measurement: log2(TPM+1)]. (E) RGS22 expression was lower in the PDAC cells than in the adjacent tissues, as determined by IHC. Scale bar, 50 µm. (F) Western blot analysis of RGS22 protein expression in a panel of human PDAC cell lines and the HPNE cell line. (G) Kaplan-Meier curves for OS based on RGS22 expression in 52 cases of PDAC. (H) Kaplan-Meier curves for OS according to RGS22 expression in 174 cases of PDAC (data from TCGA and GTEx). *P≤0.05, **P≤0.01. RGS22, Regulator of G-protein signaling 22; PDAC, pancreatic ductal adenocarcinoma; RT-qPCR, reverse transcription-quantitative PCR; IHC, immunohistochemistry; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression; TPM, transcripts per million; OS, overall survival.