Journal: Science signaling

Article Title: PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer.

doi: 10.1126/scisignal.aag3326

Figure Lengend Snippet: Fig. 4. Manipulation of PSMA in both mouse TRAMP-C1 and human 22RV1 and PC-3 PCa cell lines mimic pathway switch. (A) CRISPR knockdown of PSMA in both the mouse TRAMP-C1 cell line (TRAMP-PSMAKO) and the human 22Rv1 cell line (22RV1-PSMAKO). Western blot analysis of both CRISPR cell lines to exam- ine changes in PDK-Ser241, IGF-1R, survivin, and pERK1/2 compared to controls (Scr). (B) TRAMP-C1 cells transiently transfected with peptides blocking the PSMA NH2-terminal cytoplasmic tail. Western blot at 24 hours for pERK1/2, cleaved PARP Asp214, and survivin at 24 hours compared to the control (Scr). (C) Western blot analysis of PC-3 cells expressing human PSMA (hPSMA) and empty vector (EV) control cell lysates to investigate changes in the previously identified signaling pathways (PSMA, IGF-1R, AKT-Ser380, AKT-Thr308, PDK1-Ser241, pERK1/2, survivin, and b-actin). All data are representative images from the mean ± SE of n = 3 for each experimental condition and three experimental replicates normalized to b-actin and presented as fold change, where WT is equal to 1. *P < 0.05, paired Student’s t test.

Article Snippet: 10 of 13 SC I ENCE S I GNAL ING | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 CRISPR knockout of PSMA in PCa cell lines TRAMP-C1 and 22RV1 PSMA CRISPR/Cas9 constructs were created in both TRAMP-C1 and 22RV1 cell lines using the lentiCRISPRv2 plasmid #52961 (Addgene) (85, 86).

Techniques: CRISPR, Knockdown, Western Blot, Transfection, Blocking Assay, Control, Expressing, Plasmid Preparation, Protein-Protein interactions

Journal: Science signaling

Article Title: PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer.

doi: 10.1126/scisignal.aag3326

Figure Lengend Snippet: Fig. 5. PSMA expression markedly alters FAK phosphorylation, and PSMA is in a complex with RACK1 and IGF-1R. (A) Western analysis for FAK-Tyr397 and FAK-Tyr925 in tumor lysates from 18-week-old WT and KO mice. (B) Western blot analysis for FAK-Tyr397 in parental WT, control scramble 22Rv1 cells (Scr), and 22Rv1-PSMAKO cells. (C) Immu- noprecipitation (IP) of PSMA in WT 22Rv1 cells using PSMA monoclonal antibody or rabbit immunoglobulin G (IgG) control and Western blot for IGF-1R (arrow indicates band). Input refers to unbound fraction. (D) Immuno- precipitation of PSMA in WT 22Rv1 cells using PSMA rabbit monoclonal antibody or rabbit IgG control and Western blot for RACK1. (E) Immunoprecipitation of RACK1 in both 22Rv1-PSMAScr and 22Rv-PSMAKO cells and Western blot analysis for b1 integrin. (F and G) Both 22Rv1-PSMAScr and 22Rv-PSMAKO cells were put either not in suspension (adherent) or in suspension (nonadherent) for 2 hours and assayed by Western blot for the direct activation of FAK- Tyr925 (F) and ERK (G) by addition of extracellular matrix (ECM) for 30 min. All data are representative images from the means ± SE of n = 3 for each experimental conditions and three experimental replicates normalized to b-actin and presented as fold change, where WT is equal to 1. *P < 0.05, paired Student’s t test.

Article Snippet: 10 of 13 SC I ENCE S I GNAL ING | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 CRISPR knockout of PSMA in PCa cell lines TRAMP-C1 and 22RV1 PSMA CRISPR/Cas9 constructs were created in both TRAMP-C1 and 22RV1 cell lines using the lentiCRISPRv2 plasmid #52961 (Addgene) (85, 86).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Immunoprecipitation, Suspension, Activation Assay