Fig. 5: Tumor-derived mutant p53 inhibits glycolysis and promotes apoptosis under metabolic stress in Jurkat T lymphocytes.
From: Regulated secretion of mutant p53 negatively affects T lymphocytes in the tumor microenvironment
A Dynamic monitoring of extracellular acidification rate (ECAR), as the parameters of glycolytic flux, were measured by the XF Glycolysis Stress Test Kit, after the Jurkat cells pretreatment with HT-29 NC or HT-29 shp53 cells derived exosomes for 18 h (4 μg/ml). B Seahorse tracing curves for ECAR in Jurkat cells that were treated with exosomes (4 μg/ml) from Con or p53R273H OE H1299 cells for 18 h. C Seahorse tracing curves for ECAR in Con or p53R273H OE Jurkat cells. D Analysis of glucose consumption and lactate production in Jurkat Con or R273H cells according to the instructions. E A co-culture system was used to detect the uptake of H1299-secreted GFP-p53R273H (lower layer) into the Jurkat cells (upper layer) over 24 h of incubation. The internalization of GFP-p53 was detected by immunoprecipitation of recipient cell lysates using anti-GFP antibodies, followed by WB analysis. F WB analysis of glycolysis markers (p-PKM2 (Y105), PKM2, HK I, PFKP) in the recipient Jurkat cells after co-cultured with Con or p53R273H OE H1299 cells by co-culture chamber system for 24 h. G WB analysis of glycolysis change (p-PKM2 (Y105), PKM2, HK-I, PFKP) in the recipient Jurkat cells after co-cultured with HT-29 NC or HT-29 shp53 cells by co-culture chamber system for 24 h. H The glycolysis change of Jurkat cells, detected by the p-PKM2 (Y105), PKM2, HK I, PFKP, after co-cultured with H1299 empty vector (EV), Flag-p53R273H or p53R273H LL25AA for 24 h. I The glycolysis markers of Jurkat cells, detected by the p-PKM2 (Y105), PKM2, HK I, PFKP, after co-cultured with H1299 donor cells for 24 h. The H1299 donor cells were divided into 3 group: empty vector with SiRNA control, GFP-R273H with SiRNA control and GFP-R273H with SiAP1B1. J Jurkat con and expressing-p53R273H cells in normal Glucose media (NG, 2000 mg/L) or in low glucose media (LG, 40 mg/L) for 24 h. The statistical diagram of apoptosis was shown as mean ± s.e.m, compared to Con in LG group, according to the flow cytometer analysis of apoptosis data. K WB detection of apoptosis markers, cleavage-PARP and cleavage-Caspase3, in Jurkat con and expressing-p53R273H cells in normal Glucose (NG, 2000 mg/L) or in low glucose (LG, 40 mg/L) for 24 h.
