rt qpcr analyses  (Bio-Rad)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Paroxetine repurposing enhances antitumor immunity via SPOP-mediated PD-L1 ubiquitination and proteasomal degradation

doi: 10.1186/s13046-026-03648-z

Figure Lengend Snippet: PAR targeting SPOP enhances PD-L1 degradation. A Protein expression of SPOP and PD-L1 in PAR-treated RKO cells was evaluated via Western blotting after treatment with siRNA targeting SPOP or a negative control (siRNA-NC). B RT-qPCR was conducted to measure PD-L1 RNA levels in RKO cells treated with various PAR concentrations for 24 h or with a fixed PAR concentration over different time periods. C and D The total protein profile of SPOP was examined via Western blotting after treating RKO cells with different PAR concentrations for 24 h ( C ) or with 10 µM PAR for various durations ( D ). E and F 293T cells were treated with or without PAR (10 µM) for 12 h and MG132 (10 µM) for 6 h. Exogenous SPOP ( E ) or PD-L1 ( F ) was pulled down with PD-L1 antibody ( E ) or SPOP antibody ( F ), respectively, and then PD-L1 and SPOP were detected by western blotting. G The thermal stability of SPOP was assessed via the CETSA method at temperatures of 24 °C, 27 °C, 30 °C, 33 °C, 36 °C, 39 °C, and 42 °C when interacting with PAR. H and I The effects of different PAR concentrations on SPOP protein stability at 42 °C were analyzed ( H ), as was the stability of SPOP after treatment with varying PAR concentrations at a 1:300 pronase-to-protein ratio ( I ). J and K Molecular docking of PAR with SPOP is illustrated ( J ), along with potential binding sites ( K ). L - Q The binding of PAR to wild-type SPOP ( L ) and mutant forms at Agr70A ( M ), Tyr87A ( N ), Lys129A ( O ), Trp131A ( P ), and Asp130A ( Q ) was detected via microcalorimetric thermophoresis. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Dunnett’s test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; N.S., not significant)

Article Snippet: RT-qPCR analyses were conducted on a LightCycler ® 96 system (Roche, Basel, Switzerland).

Techniques: Expressing, Western Blot, Negative Control, Quantitative RT-PCR, Concentration Assay, Binding Assay, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Paroxetine repurposing enhances antitumor immunity via SPOP-mediated PD-L1 ubiquitination and proteasomal degradation

doi: 10.1186/s13046-026-03648-z

Figure Lengend Snippet: PAR enhances T cell killing in vitro by targeting SPOP to promote PD-L1 degradation. A and B RKO cells were treated with SPOP siRNA ( A ) or SPOP plasmid ( B ), followed by 12 h of PAR (10 µM) treatment. The interaction between PD-L1 and SPOP was then assessed using immunofluorescence. C - H Tumor cells that survived after being cocultured with PAR and Jurkat cells for a period of time after being subjected to siRNA for PD-L1 ( C ) or SPOP ( F ) were observed via crystal violet staining. ( D) and ( G ) Knockdown efficiencies of PD-L1 and SPOP assessed by RT-qPCR, respectively. ( E) and ( H ) Statistical quantitative plots of surviving tumor cells in ( C ) and ( F ), respectively. I - K Tumor cells were subjected to crystal violet staining of RKO-overexpressing SPOP ( I ) treated with PAR for 12 h and then were cocultured with Jurkat cells for a period of time. ( J) Overexpression efficiency of SPOP in RKO cells detected via Western blotting. ( K) Statistical analysis of the number of surviving tumor cells in ( I ). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; N.S., not significant)

Article Snippet: RT-qPCR analyses were conducted on a LightCycler ® 96 system (Roche, Basel, Switzerland).

Techniques: In Vitro, Plasmid Preparation, Immunofluorescence, Staining, Knockdown, Quantitative RT-PCR, Over Expression, Western Blot