Acss2 inhibitor

Lipopolysaccharides (Escherichia coli O111:B4, L2630, for animal experiments; Escherichia coli O111:B4, L4391, for cell experiments) were purchased from Merck (New Jersey, USA). ACSS2 inhibitor (cat no: S8588) was purchased from Selleck (Houston, USA). ML264 (cat no: HY-19994) was purchased from MedChemExpress (New Jersey, USA). Lotus Tetragonolobus Lectin (LTL) (cat no: FL-1321-2) was purchased from Thermo Fisher (MA, USA).
The following antibodies were used. ACSS2 antibody (cat no: ab133664) from Abcam (Cambridge, UK); KIM-1 antibody (cat no: sc-518008) from Santa Cruz (California, USA); F4/80 antibody (cat no: 28463-1-AP) from Proteintech (Wuhan, China); Cleaved GSDMD (N Terminal) antibody (cat no: A22523) and NLRP3 antibody (cat no: A5652) from Abclonal (Wuhan, China); caspase-1 antibody (cat no: 24232), anti-IL-1β antibody (cat no: 12242) from Cell Signaling Technology (Massachusetts, USA); GSDMD antibody (cat no: AF4012), NF-κB p65 antibody (cat no: AF5006), phospho-NF-κB p65 (Ser536) antibody (cat no: AF2006), and KLF5 antibody (cat no: AF7542) from Affinity (Changzhou, China).

Immortalized mouse podocytes were a gift from Ai Hua Zhang from the Children’s Hospital of Nanjing Medical University (Nanjing, China). Mouse podocytes were cultured in RPMI 1640 medium (Wisent) with 10% fetal bovine serum and 10 U/mL recombinant mouse γ-interferon (γ-IFN; PeproTech) at 33°C for proliferation. Podocytes were then transferred to nonpermissive conditions at 37°C without γ-IFN for at least 7 days for differentiation (40 (link)). Podocytes were incubated in RPMI 1640 with 10% FBS in the presence of 30 mmol/L d-glucose (MilliporeSigma) as the final concentration. The cells were exposed to 11 mmol/L d-glucose as the control. According to the procedure recommendation, ACSS2 siRNA transfection was performed with Lipofectamine 2000 from Thermo Fisher Scientific. ACSS2 inhibitor (Selleck Chemicals, S8588) or DMSO as control was added to podocytes before HG treatment. The sequences of control siRNA and ACSS2 siRNA are listed in Supplemental Table 3; https://doi.org/10.1172/jci.insight.165817DS1

Immortalized mouse podocytes were a gift from Ai Hua Zhang from the Children’s Hospital of Nanjing Medical University (Nanjing, China). Mouse podocytes were cultured in RPMI 1640 medium (Wisent) with 10% fetal bovine serum and 10 U/mL recombinant mouse γ-interferon (γ-IFN; PeproTech) at 33°C for proliferation. Podocytes were then transferred to nonpermissive conditions at 37°C without γ-IFN for at least 7 days for differentiation (40 (link)). Podocytes were incubated in RPMI 1640 with 10% FBS in the presence of 30 mmol/L d-glucose (MilliporeSigma) as the final concentration. The cells were exposed to 11 mmol/L d-glucose as the control. According to the procedure recommendation, ACSS2 siRNA transfection was performed with Lipofectamine 2000 from Thermo Fisher Scientific. ACSS2 inhibitor (Selleck Chemicals, S8588) or DMSO as control was added to podocytes before HG treatment. The sequences of control siRNA and ACSS2 siRNA are listed in Supplemental Table 3; https://doi.org/10.1172/jci.insight.165817DS1