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.