Fig. 5: P4HA3 interacts with ACLY to inhibit ACLY degradation.

A Schematic illustration of co-immunoprecipitation (Co-IP) and mass spectrometry analysis to identify P4HA3-associated proteins. B HeLa PR and SiHa PR cells were transfected with 0, 2, 4 µg of HA-ACLY in a 6 cm cell culture dish for 48 h. RT-qPCR and western blot analysis was performed to detect the mRNA and protein expression of ACLY and SLC7A11. C IF staining of P4HA3 and ACLY in HeLa and SiHa cells. D 293T cells were transfected with FLAG-P4HA3 and HA-ACLY for 48 h. The whole-cell lysates were subjected to immunoprecipitation using anti-FLAG beads and anti-HA beads, and subsequently immunoblotted with antibodies against FLAG and HA. E, F Co-IP assays were carried out to investigate the interaction between endogenous ACLY and P4HA3 in CCa cells. G Schematic diagram of ACLY truncation constructs. H 293T cells were transfected with HA-tagged constructs containing different domain of ACLY and FLAG-P4HA3. Cell lysates were immunoprecipitated with anti-FLAG beads and immunoblotted with anti-HA and anti-FLAG antibodies. FL full length. I HeLa PR and SiHa PR cells were transfected with 0, 1, 2, 4 µg of FLAG-P4HA3 in a 6 cm cell culture dish for 48 h. Western blot analysis was performed to detect the protein expression of P4HA3 and ACLY. J Western blot analysis of ACLY in HeLa LNM2 and SiHa LNM2 cells with P4HA3 knockdown. K Western blot analysis of ACLY in the vector and P4HA3-overexpressing cervical cancer cells with or without MG132 treatment (10 µM). L Western blot analysis of ACLY in the scramble and P4HA3-silencing cervical cancer cells with or without MG132 treatment (10 µM). M, N Assessment of SLC7A11 mRNA and protein levels in HeLa PR and SiHa PR cells transfected with Vector+shNC, P4HA3+shNC, and P4HA3+shACLY was performed using RT-qPCR and western blot. Error bars represent the means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.