Sc 130335

Four-octyl itaconate (4-OI) was synthesized by Ruilu Chemicals (Shanghai, China). H₂O₂ was provided by Sigma Aldrich Chemicals (St Louis, Mo). Antibodies for HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), Tubulin (#2125), and Lamin B1 (#13435), as well as cleaved-poly (ADP-ribose) polymerase (PARP, #5625), cleaved-caspase-9 (#20750), and immunoresponsive gene 1 (IRG1, #77510) were obtained from Cell Signaling Tech China (Shanghai, China). The anti-GCLC antibody (ab55435) and the anti-adenine nucleotide translocase-1 (ANT1) antibody (ab102032) were purchased from Abcam China (Shanghai, China). Antibodies of PGK1 (sc-130335) and cyclophilin D (CyPD, sc-137136) were provided by Santa Cruz Biotech (Santa Cruz, CA). RNA assay reagents were obtained from Thermo-Fisher Invitrogen (Suzhou, China). Cell Counting Kit-8 (CCK-8) was provided by Dojindo Laboratories (Kumamoto, Japan).

Immunoprecipitation (IP) and western blot were used to semi-quantify PGK1 acetylation level in NF-PitNETs compared to controls. Three NF-PitNET tissue samples were equally mixed as the NF-PitNET sample, and five control protein samples were equally mixed as the control sample (Supplemental Table 1), which were used to extract protein samples, respectively. An amount (1 mg) of each protein sample (NF-PitNETs; controls) was incubated with the specific antibody against PGK1 (6 μg; sc-130335, Santa Cruz Technology) to immunoprecipitate PGK1 from total proteins. The negative control IP experiment was performed with the use of the normal mouse IgG antibody (6 μg; B900620, Proteintech) to replace the anti-PGK1 antibody, which tested the specificity of anti-PGK1 antibody. The IP products (PGK1 product; IgG product), anti-PGK1 antibody (2 μg), and total protein samples (NF-PitNETs: 60μg; Controls: 60μg) were simultaneously immunoblotted with anti-acetyl-lysine antibody (1:1000; A2391, ABclonal).

Immunohistochemistry (IHC) staining and evaluation was carried out as we previously described [23 (link)]. The primary anti-PGK1 antibody (sc-130335, Santa Cruz) and anti-Ki67 antibody (ET1609–34, Hangzhou Hua-An Biotechnology) were diluted at 1:200 to perform IHC. Cells were plated and grown on coverslips overnight for cell immunofluorescence staining following reported approach [20 (link)].