2.12 Reagents
Physalin B (purity ≥98%) were separated and purified in our laboratory; their structures, shown in Figure 1A, were confirmed by comparing the IR, 1H and 13C nuclear magnetic resonance and MS data with reported data (Zheng, Chen, Liu, Liang & Hong, 2016). Calyx Seu Fructus Physalis (3 kg) were extracted with 95% ethanol for three times. After solvent removal, the crude extractive was separated successively with ethanol (40%, 80%, 100%) on macroporous resin and obtained fraction B. The Fr.B was partitioned with H2O, ethyl acetate, methylene chloride and petroleum ether. Fractionation of the methylene chloride extract using silica gel column, eluting with CH2Cl2: CH3OH (100: 1-5: 1) to obtain subfraction B. Subfraction B were chromatographed over a MCI, eluting with gradient mixtures of CH3OH-H2O (from 30: 70 to 100: 0). Finally, compound PB was obtained by ODS and P-HPLC. Its structure was identified by comparing 1H, 13C -NMR data with those of literature (Zheng, Luan, Chen, Ren & Wu, 2012). The compound was more than 98% pure and dissolved in DMSO at a stock concentration of 5 mM.
An anti‐α‐smooth muscle actin antibody (α‐SMA; ab7817), anti‐COL1A1 (ab34710) and anti‐LAP2α (ab5162) antibodies were purchased from Abcam. Anti‐GLI1 (#2553), anti‐Acetylated-Lysine (#9441), anti‐Flag‐Tag (#14793) and anti‐Myc-Tag (#2276) antibodies and normal rabbit IgG (#2729) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti‐HA‐Tag (51064‐2‐AP and 66006‐ 1‐Ig), anti‐HDAC1 (66085-1-Ig) antibodies were purchased from Proteintech (Wuhan, China). Vorinostat (HY-10221), TGFβ1 (HY-P7118), GANT61(HY-13901) was acquired from MCE (Shanghai, China). GLI1 siRNA, LAP2α siRNA were acquired from RiboBio (Guangzhou, China). was obtained from MCE (Shanghai, China). Additional materials were obtained from commercial sources.