Pai 1

The protein used for western blotting was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitors (Roche). The proteins were quantified using the BCA Protein Assay Kit (Pierce). The western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions.
Primary antibodies of NOD1 (ab170547; Abcam), p-IKK (#2697), t-IKK (#11930), p-IκBα (#2859), t-IκBα (#4814), p-NF-κB (#3033), t-NF-κB (#8242), p-Smad3 (#9520), t-Smad3 (#9523) and PAI-1 (#11907; Cell Signaling Technology) were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibodies were incubated with the membrane at 4 °C overnight, followed by washing and incubation with secondary antibody (1:5000, Abcam) marked by horseradish peroxidase for 1 h at room temperature.
After rinsing, the polyvinylidene difluoride (PVDF) membrane-carried blots and antibodies were transferred into the Bio-Rad ChemiDoc XRS system, and then Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab Software (Bio-Rad).

KYSE‐150 and EC9706 cells were lysed in RIPA buffer. Protein concentration was determined using BCA Protein Assay Kit (Beyotime Biotechnology). Thirty micrograms of protein from each group were electrophoresed on 10% SDS‐PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% nonfat milk (Beyotime Biotechnology) and then individually incubated with the listed primary antibodies (all in a 1:1000 dilution), E‐cadherin, β‐catenin, vimentin, Slug, ERK1/2, p‐ERK1/2, PAI‐1, H3, H3K9Ac, BRD4 (all from Cell Signaling Technology), and GAPDH (Boster). After washing in TBS‐T, the membranes were incubated with HRP‐conjugated secondary antibodies (1:8000, Dingguo) 1 h. After washing, the blots were processed with BeyoECL chemiluminescence kit (Beyotime Biotechnology) and imaged with an Amersham Imager 600 system (GE Healthcare Biosciences).

Recombinant human TNF-α was purchased from Pepro Tech EC (London, UK). The PAI-1 reporter plasmid (p800Luc) that contains 800 bp of the proximal promoter sequences of human PAI-1 gene was a generous gift of Professor Daniel Rifkin (New York University) [15 (link)]. Except for PAI-1 (BD Biosciences, San Jose, CA), other antibodies were purchased from Cell Signaling Technology (Beverly, MA). Doxycycline and all of the other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). CBHA, SB203580, SP600125, PD98059, LY294002, and parthenolide were obtained from Calbiochem (San Diego, CA).

Berberine (1065210) and Primary antibodies uPA (SAB1105036), tPA (HPA003412), TIMP-2 (WH0007077M1) were purchased from Sigma-Aldrich Company (Sigma-Aldrich, St. Louis, MO, USA). COX-2 (#4842S), PAI-1 (#11907S), p-p38 (#4511), p38 (#8690), p-Erk1/2 (#4370), Erk1/2 (#4695), p-JNK/SAPK (#9255), p-Src (#12432), Src (#2108), HMGB1 (#6893S), NF-κB (#3033, #8242), TIMP-1 (#8946), MMP-9 (#3852S), MMP-2 (#4022S) and actin (#3700) were purchased from Cell Signaling Company (Cell Signaling, Danvers, MA, USA). JNK/SAPK (EPR18841-95) was purchased from Abcam (Cambridge, MA, USA). Secondary antibodies were purchased from Beyotime Institute of Biotechnology (Beyotime Inst. Biotech., Shanghai, China).

Whole-cell lysates of M17 cells were prepared then loaded on SDS-PAGE gel and then the isolated proteins were subjected to Western blot analysis. The specific primary antibodies for SIRT1 (#9475, 1;1000), PAI-1 (#94,536, 1;1000), p21 (#2947, 1;1000) and β-actin (#3700, 1;10,000) and the corresponding secondary antibody (#7074 and #7076, 1;5000) were purchased from Cell Signaling Technology (MA, USA). The bands were visualized by incubating with the Chemiluminescent Substrate (Thermo Fisher Scientific) and the densitometry was imaged using Image J software.

All collected proteins were added to sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris (pH 6.7), 1.25% SDS, 12.5% glycerol, and 2.5% β-mercaptoethanol). Proteins and the prestained protein marker (10–315 kDa) (TD-PM10315, BIOTOOLS Co., Ltd., Taipei, Taiwan) were loaded into an SDS-PAGE gel. A PVDF membrane containing transferred proteins was incubated with 5% nonfat milk with primary antibodies anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) (Cell Signaling, Beverly, MA), collagen 1 (Proteintech, Rosemont IL), autophagy-related 5 (ATG5) (Proteintech), CTGF (Proteintech), PAI-1 (Cell Signaling), SIRT1 (ABclonal Inc., Woburn, MA), caspase-3 (ABclonal Inc.), Hsp27 (ABclonal Inc.), BAX (ABclonal Inc.), Klotho (Proteintech), p53 (Proteintech), p21(Proteintech), p16 (Proteintech), G6PD (Proteintech), and GAPDH (Proteintech). After the hybridization process with the abovementioned antibodies on the PVDF membrane, the membrane was rinsed with TBS-T for 15 min three times. Subsequently, the PVDF membrane was further treated with anti-mouse (Jackson) or anti-rabbit (Jackson) secondary antibody for 2 h and rinsed with TBS-T for 15 min over three times. The protein bands of the PVDF membrane were visible by performing an enhanced chemiluminescence system (Amersham, Little Chalfont, United Kingdom).