Fig. 6: RORC acetylation promoted NEDD4L transcription. | Cell Death & Disease

Fig. 6: RORC acetylation promoted NEDD4L transcription.

From: Intratumoral Brevibacillus parabrevis enhances antitumor immunity by inhibiting NK cell ferroptosis in hepatocellular carcinoma

Fig. 6

A DNA pull-down and mass spectrum assays showing protein on the NEDD4L promoter in NK-92 cells. B DNA pull-down assay confirming that RORC bound to the NEDD4L promoter in NK-92 cells (n = 3). C Docking model between RORC and NEDD4L promoter and surface map of their interface residues. Purple, RORC; green, DNA; rod-shaped structures, interacting amino acid residues; dark green dashed lines, hydrogen bonds. D DNA pull-down and immunoblotting assays analyzing the binding of RORC to the WT or mutant sequences of NEDD4L promoter in NK-92 cells (n = 3). RT-qPCR (E) and immunoblotting (F) showing the transcription and protein expression of NEDD4L, respectively (n = 3). NK-92 cells were co-transfected with the WT or mutant sequences of NEDD4L promoter. G DNA pull-down assay analyzing the effect of B. parabrevis transplantation on the binding of RORC to the NEDD4L promoter in NK cells (n = 3). H, I Impact of overexpressing the WT NEDD4L promoter sequence in NK-92 cells, pre-stimulation of NK-92 cells transfected with the WT promoter sequences using the ferroptosis inducer FIN56, or transfection with NEDD4L promoter sequences containing an AAAGGGGTCT deletion, on orthotopic HCC growth in Hu-SRC mice (n = 6). NK-92 cells were infused into mice with orthotopic HCC. H Representative. I Survival curve. J, K Effect of overexpressing the WT NEDD4L promoter sequence in NK-92 cells, pre-stimulation of NK-92 cells transfected with the WT promoter sequences using FIN56, or transfection with NEDD4L promoter sequences containing an AAAGGGGTCT deletion, on subcutaneous tumor growth in Hu-SRC mice (n = 6). J Representative. K Tumor mass weight and growth curve. L Flow cytometry evaluating the impact of overexpressing the WT NEDD4L promoter sequence in NK-92 cells, pre-stimulation of NK-92 cells transfected with the WT promoter sequences using FIN56, or transfection with NEDD4L promoter sequences containing an AAAGGGGTCT deletion, on the expression of effector genes in tumor-infiltrating NK cells in Hu-SRC mice (n = 6). M Effects of B. parabrevis transplantation on lipid or glucose metabolism of NK cells (n = 6). Immunoblotting showing the effects of acetyl-CoA (N) or knockdown of RORC (O) on NEDD4L protein expression in NK-92 cells (n = 3). P NK-92 cell lysate was incubated with anti-IgG control or -RORC antibodies (n = 3). 5% of lysate was used as input control. Q RORC acetylation was detected after B. parabrevis transplantation or stimulation with a CPT1/2 inhibitor (Perhexiline maleate) in NK cells (n = 3). R Evaluation of acetylation of five Myc-tagged RORC lysine mutants in NK-92 cells (n = 3). S Sequence alignment of RORC from different species showing the conservation of the K120 residue. T Immunoblotting showing the effect of acetyl-CoA on RORC acetylation in NK-92 cells (n = 3). U DNA pull-down assay demonstrating the binding of RORC lysine mutants to the NEDD4L promoter in NK-92 cells (n = 3). V, W Investigation of the impact of WT, K120R, K120Q of RORC, and FIN56-pretreatment in NK cells on orthotopic HCC growth in Hu-SRC mice (n = 6). (V) Representative. (W) Survival curve. X, Y Analysis of the influence of WT, K120R, K120Q of RORC, and FIN56-pretreatment in NK cells on the growth of subcutaneous tumors in Hu-SRC mice (n = 6). X Representative. Y Tumor mass weight and growth curve. E, F, K-O, Y represented mean ± SD analyzed by unpaired t test, I, W were analyzed by Log-rank test. *P < 0.05, ** P < 0.01. WT wild-type.

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