Journal: Research

Article Title: Proteogenomic Analysis Identifies Clinically Relevant Subgroups of Collecting Duct Carcinoma

doi: 10.34133/research.0859

Figure Lengend Snippet: RPF2 promotes progression of CDC through ribosome production. (A) IHC (top) and WB (bottom) results of RPF2 expression in tumors and NATs. (B) Transfection efficiencies of RPF2-siRNA and RPF2 overexpression in 786-O cells are detected by WB (left) and RT-qPCR (right). (C) CCK8 assays characterize the effects of RPF2-siRNA (top) and RPF2 overexpression (bottom) on the proliferation of 786-O cells. (D) Transwell assays detect the effects of RPF2-siRNA (top) and RPF2 overexpression (bottom) on the invasiveness of 786-O cells. (E) The knockdown or overexpression of RPF2 inhibits or promotes the expression of UBTF, but has no effect on SL1 in 786-O cells. (F) Nuclear localization detection of UBTF in 786-O cells. (G) ChIP results between UBTF and Rrn3. (H and I) CO-IP assays clarify the physical interaction between RPF2 and UBTF in 786-O cells (H) and tumor tissues (I). (J) The knockdown (left) or overexpression (right) of RPF2 inhibits or promotes the expression of POLR1A in 786-O cells. (K) RPF2 knockdown inhibits the transcription of 28s, 18s, and 5.8s rRNA. (L) RPF2 depletion disrupts the nucleolar localization of NPM1 (B23). ACHN cells were transfected with control vector or RPF2 siRNA, followed by IF staining. (M) The overexpression (left) or knockdown (right) of RPF2 reduces or increases the expression of p53 and its target genes. ACHN cells were transfected with control vector, RPF2 plasmids, or RPF2 siRNAs, followed by IB and RT-qPCR analyses. (N) RPL5 or RPL11 knockdown compromises the induction of p53 by RPF2 depletion. ACHN cells were transfected with control vector, RPF2 shRNA, RPL5 siRNA, and RPL11 siRNA as indicated. Cell lysates were subjected to IB analysis with indicated antibodies. (O) RPL5–MDM2 and RPL11–MDM2 interactions are increased by depletion of RPF2. ACHN cells were transfected with RPF2 shRNA, followed by CO-IP-IB assays using antibodies as indicated. The proteasome inhibitor MG132 was supplemented into medium for 5 h before cell harvest. (P) RPF2 knockdown extends the half-life of p53 protein. ACHN cells were transfected with control vector or RPF2 shRNA. Cycloheximide (CHX) (100 mg/ml) was supplemented into medium for the indicated time before cells were harvested for IB analysis. (Q) Top panel: RPF2 knockdown suppresses proliferation and migration and promotes apoptosis of RCC cells. ACHN cells were transfected with lentivirus of control vector or RPF2 shRNAs, followed by cell viability assay, apoptosis assay, and transwell cell migration assay. Bottom panel: RPF2 overexpression promotes proliferation and migration of RCC cells. ACHN cells were transfected by lentivirus of PCDH or PCDH-RPF2 for cell viability assay, apoptosis assay, and transwell cell migration assay. The asterisks represent the statistical P values (* P < 0.05, ** P < 0.01, *** P < 0.001). (R) A model depicting the regulation of RPF2.

Article Snippet: The primary antibodies used in this study were as follows: anti-UBTF antibody (Santa Cruz, sc-13125), anti-SL1 antibody (Santa Cruz, sc-393600), anti-POLR1A antibody (Proteintech, 20595-1-AP), anti-RPF2 antibody (Santa Cruz, sc-81060), anti-Flag antibody (ABclonal, AE005; Sigma-Aldrich, F1804), anti-Myc antibody (ABclonal, AE070), anti-GAPDH antibody (ABclonal, AC001; Proteintech, 60004-1-Ig), anti-p53 antibody (Santa Cruz, sc-126), anti-MDM2 antibody (Abcam, ab16895), anti-RPL5 antibody (Abcam, ab86863), anti-RPL11 antibody (Abcam, ab79352), anti-B23 antibody (Santa Cruz, sc-271737), anti-PUMA antibody (Cell Signaling Technology, 12450S), anti-β-actin antibody (Proteintech, 66009-1-Ig), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (BOSTER, BA1055; Proteintech, SA00001-2), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (BOSTER, BA1051; Proteintech, SA00001-1).

Techniques: Expressing, Transfection, Over Expression, Quantitative RT-PCR, Knockdown, Co-Immunoprecipitation Assay, Control, Plasmid Preparation, Staining, shRNA, Migration, Viability Assay, Apoptosis Assay, Cell Migration Assay