Tepp 46

PLGA-COOH (50:50, 30kDa) including a premixed content of ~3.4kDa polyethylene glycol (PEG) was purchased from Xi’anRuiXi Co., Ltd (Xi’an, China). Polyvinyl alcohol (PVA, 26 kDa), 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 2- (N-morpholino) ethanesulfonic acid (MES monohydrate), N- (3-dimethyl-ami-nopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Solarbio Ltd (Beijing, China). Tepp-46 was obtained from MedChemExpress (New Jersey, USA). PD-1 antibody was purchased from Biolegend (114114, San Diego, USA). Human interphotoreceptor-retinoid-binding protein (IRBP651-670, LAQGAYRTAVDLESLASQLT) peptide was produced by Sangon Biochem Ltd (Shanghai, China). 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Boster Biological Technology Co., Ltd (California, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Kyushu, Japan).

Primary lung fibroblasts from a patient with IPF were gifted by W. Ning (Nankai University). All cell lines were obtained from the American Type Culture Collection. 293T and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and HFL1 cells were cultured in F12K, all supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C with 5% CO₂. Cells were transfected using Lipofectamine 3000 or RNAiMAX (Invitrogen) for plasmid or siRNA transfection, respectively. For scramble control, Flag-PKM2 was controlled by pCDNA3.1-Flag, Myc-Smads were controlled by pCDNA3.1-Myc, GFP-TβR1 was controlled by pEGFP, human siRNA was controlled by a scramble siRNA sequence 5′-UUCUCCGAACGUGUCACGU-3′, and mouse siRNA was controlled by scramble siRNA sequence 5′-CUUACGCUGAGU ACUUCGA-3′. The treatments used were as follows: TGF-β1 (5 ng/ml), MG132 (5 μM in DMSO; Sigma-Aldrich), cycloheximide (CHX in DMSO; 20 μg/ml; Sigma-Aldrich), TEPP-46 [10 μM in DMSO; MedChemExpress (MCE)], and compound 3k (1 μM in DMSO; MCE).

Eight‐week‐old male CD‐1 mice (Sankyo Lab Service, Tokyo, Japan) were used in our experiment. The mice received one intraperitoneal injection of streptozotocin (STZ) (200 mg/kg body weight) to induce diabetes. Blood glucose levels were measured 2 weeks later, and the establishment of diabetes was defined as a blood glucose level of > 220 mg/dL. One month after the diabetes induction, the mice received oral gavage with TEPP‐46 (30 mg/kg, MedChemExpress, USA) or vehicle for 2 weeks; at the same time, the mice received an intraperitoneal injection of bovine serum albumin (BSA) (0.3 g/30 g, Sigma‐Aldrich, USA). All mice were killed at 14 weeks. Before killing, blood pressure and blood glucose levels were measured, and all samples were stored at −80°C until use.
The animal experiment was approved by the IACUC of Kanazawa Medical University (protocol number 2019‐25).