U73122

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

AG1478 (EGFR inhibitor), MK2206 (Akt inhibitor), U0126 (MEK inhibitor) and U73122 (PLC-y1 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). EGF and TRIzol reagent were purchased from Invitrogen Corp. (Carlsbad, CA, USA). Prolactin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine™ 2000 and RNAiMAX were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Unless otherwise specified, all of the chemicals were purchased from China.

For the surface markers and intracellular cytokine detection, CD8⁺ and Vδ2⁺ T cells were treated with 10 mM sodium acetate (AA; Sigma, S2889), 1 mM sodium propionate (PA; Sigma, P1880) or 0.5 mM sodium butyrate (BA; MCE, HY-B0350A) for two times at 2-day intervals. In some experiments, SCFAs pretreated-CD8⁺ and Vδ2⁺ T cells were activated with 5 μg/mL anti-human CD3 and 1 μg/mL anti-human CD28 for 4 h, followed by flow cytometry analysis. For histone acetyltransferase inhibition, CD8⁺ and Vδ2⁺ T cells were treated with control, 0.5 mM BA, 1 μM A-485 (Selleck, S8740), or BA+A-485 for 48 h, and then the expression of PD-1 and CD28 on CD8⁺ and Vδ2⁺ T cells was measured by flow cytometry. Similarly, the levels of histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 27 acetylation (H3K27ac) in protein lysates were detected by immunoblotting. For the p-PLCγ1 detection, vehicle or 0.5 mM BA pretreated CD8⁺ and Vδ2⁺ T cells were treated with or without 2 μM U73122 (Selleck, S8011) under the condition of 5 μg/mL anti-human CD3 and 1 μg/mL anti-human CD28 activation for 1 hour, followed by immunoblotting. Unless mentioned otherwise, SCFAs pretreated T cells (CD8⁺ and Vδ2⁺) used in all the experiments were treated by 10 mM AA, 1 mM PA or 0.5 mM BA for two times at 2-day intervals in vitro.

Immortalized mouse podocytes were a generous gift of Professor Ai Hua Zhang from Children's Hospital of Nanjing Medical University; the cells were cultured in RPMI 1640 medium with 10% foetal bovine serum (FBS) at 33 °C as described previously 24 (link). Podocytes were thermo-shifted to 37 °C for 10 days before they were used in the experiments. Chemical agents, GLPG0974 (10 μmol/L) (R&D Systems, USA), AICAR (10 μmol/L), U73122 (10 μmol/L), and Go 6983 (10 μmol/L) (Selleck, USA), were acquired for the experiments.