Figure 3.

PARG inhibition (PARGi) decreases basal and IL-6-induced phosphorylated STAT3 (pSTAT3) in immune cells and stimulates immune activation in vitro. (A) Splenocytes from tumor-free mice were isolated and cultured in the presence of vehicle (DMSO), 10 µM olaparib (PARPi), or 10 µM COH34 (PARGi) overnight. The levels of pSTAT3 and total STAT3 were analyzed by immunoblotting. (B) Immunoblot comparing pSTAT3 in healthy donor-derived PBMCs and ovarian cancer patient ascites-derived CD3⁺ T cells after overnight incubation with either DMSO or 5 µM PARGi. (C) Immunoblotting of pSTAT3 in mouse naïve CD19⁺ B cells, CD4⁺ and CD8⁺ T cells pretreated with vehicle control or PARGi (5 µM, overnight) and stimulated with 20 ng/mL IL-6 for 30 min. (D) Analysis of IL-6-induced pSTAT3 similar to (C) in human immune cells from (B) after overnight pretreatment with either DMSO or 5 µM PARGi. (E) Real-time PCR of Ifng gene expression in mouse splenocytes cultured in the presence or absence of PARGi for 24 hours. Gene expression data are shown after normalization to Actb expression and are presented as the mean-fold induction (mean±SD) relative to unstimulated samples. (F) ELISA measuring IFN-γ levels in the supernatants from cocultures of mouse CD8⁺ T cells with murine ID8 tumor cells in the presence of vehicle or 10 µM PARGi for 24 hours. Data are presented as the mean-fold induction (mean±SD) relative to vehicle-treated samples. (G) Levels of IFN-γ and granzyme B in the supernatants from ovarian cancer patient ascites-derived CD3⁺ T cells after treatment with either 5 µM PARGi or DMSO for 24 hours as determined by ELISA. The protein levels of pSTAT3 shown in the immunoblotting images were quantified by band intensity using ImageJ software and normalized with the levels of GAPDH. DMSO, Dimethyl sulfoxide; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PARG, poly(ADP-ribose) glycohydrolase; PBMCs, peripheral blood mononuclear cells; STAT3, signal transducer and activator of transcription 3.