Hy b1370

The following were the antibodies used: anti-mouse tubulin (Genetex, GTX27291), anti-rabbit EGFR (abcam, ab52894), anti-rabbit pEGFR (abcam, ab5644), anti-rabbit hsp70 (Genetex, GTX104126), anti-rabbit bag3 (Genetex, GTX102396), and anti-rabbit p62 (Genetex, GTX100685). The following were the reagents used: ver-155008 (Medchem Express, HY-10941), pif (Medchem Express, HY-10940), bafilomycin A1 (Medchem Express, HY-100558), hydroxychloroquine (Medchem Express, HY-B1370), ixazomib (Medchem Express, HY-10453), MG132 (Medchem Express, HY-13259), osi (Medchem Express, HY-15772), and gef (LC lab, G-4408). The synthesis of all curcumin derivatives has been reported in our earlier publication (26 (link)). The structures and synthetic procedures of MTH-3, 21b, 22a, 22b, 23a, 24b, 25, 33, 35a, 35d, 36, and 37 correspond to the synthetic methods employed for the synthesis of compounds 9a, 10a, 9b, 10b, 11, 14a, 14b, 15, 16a, 16d, 17, and 18, respectively, as depicted in Scheme 1A-C of the reference (26 (link)). Briefly, curcumin derivatives were synthesized from curcumin via a three-step sequence involving esterification of curcumin with 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid to afford an ester intermediate, which was in turn dialkylated with alkyl bromide and hydrolysis with HCl(aq).

Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of SLE patients or healthy controls using Ficoll-paque density gradient centrifugation. Purified CD4⁺ T cells were negatively isolated from PBMCs by CD4⁺ T-cell isolation kits (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol. CD4⁺ T cell purity was routinely > 90% as verified through flow cytometry. The cells were then cultured in Xvivo 15 medium (Lonza, Walkersville, MD, USA) supplemented with 10% human AB serum (Valley Biomedical, Winchester, VA, USA) at 37 °C with 5% CO₂. The treatments of the cells were: TNF-α (HY-P7058, MedChemExpress, NJ, USA), 10 ng/ml, 24 h; IL-6 (HY-P7044, MedChemExpress), 10 ng/ml, 24 h; 17β-estradiol (estradiol/E2) (HY-B0141, MedChemExpress), 100 nmol/L, 24 h; Lipopolysaccharides (LPS) (L8880, Solarbio, Beijing, China), 100 ng/ml, 24 h; ultraviolet B (UVB), 50 mJ/cm2 [10 (link)]; hydroxychloroquine sulfate (HCQ sulfate) (HY-B1370, MedChemExpress), 6 μg/ml, 24 h; 5-Azacytidine (5-aza C) (HY-10586, MedChemExpress), 1 mM, 24 h; prednisolone (HY-17463, MedChemExpress), 10 ng/ml, 24 h; AZD9496 (HY-12870, MedChemExpress), 5 nM, 24 h.