P stat1

Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18 (link)]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).

Immunoblots were prepared as described^{41 (link)} using Abs for STAT3 (79D7, #4904), pSTAT3 (Y705, #9145), STAT5 (#9363), pSTAT5 (Y694, 9351), pSTAT1 (Y701, #9171), β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA), or STAT1 (#610185) (BD Transduction Labs, San Jose, CA, USA). Following incubation with horseradish peroxidase-conjugated secondary Ab, immune complexes were detected via SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Western blot assay was performed as described previously 20 (link). Briefly, total protein from cells was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). The extracted protein was separated on SDS polyacrylamide gels and transferred to the PVDF membranes. Western blotting was performed using antibodies against human COL6A1 (Abcam, Milton, Cambridge, UK), N-cadherin, E-cadherin, Vimentin, FAK, Src, p-FAK, p-Src, p-STAT1 (Tyr701), p-STAT1 (Ser727), p-STAT3 (Tyr705), STAT1, STAT3, CD133, ABC2G, SOX2, Nanog, CD63, CD9, TSG101, SOCS5, Flag, GFP, HA, Ubiqution (Ub), GAPDH, p300, c-Jun were purchased from Cell Signaling Technology (CST, USA).

One snap-frozen proliferating and one involuted IH tissue samples from the original cohort of patients used for DAB IHC staining were used for Western blotting (WB). These tissue samples were processed as previously described (3 (link)) and transferred to nitrocellulose membranes (Thermo Scientific) using an iBlot 2 (Thermo Scientific). The membranes were blocked for 90 min at 4°C in 1x iBind™ Flex Solution (Thermo Scientific) and probed using an iBind™ Flex device (Thermo Scientific) with the following primary antibodies: pSTAT1 (1:1,000; cat# 9167, Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (1:1,000; cat# 9145, Cell Signaling Technology), pSTAT5 (1:1000; cat# 9314, Cell Signaling Technology) and β-actin (1:2,000; cat# ab8226, Thermo Scientific). Detection and imaging of the blots were undertaken as routinely done in our laboratory (3 (link)). Human tonsil, mouse lung, and human liver total protein extracts were used as the positive controls for pSTAT1, pSTAT3, and pSTAT5, respectively.

Cells were seeded in six-well plates at a concentration of 5 × 10⁵ per well in 1 mL of medium and transfected with miRNA mimics or miRNA inhibitors. The cells were stimulated with type I IFN (1000 U/mL) 24 h after transfection and the cells were lysed at the indicated time points. The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively). The secondary antibodies used were obtained from Cell Signaling: horseradish-peroxidase (HRP)-linked anti-rabbit IgG antibody (diluted 1:5000), and HRP-linked anti-mouse IgG antibody (diluted 1:4000). The signal was generated with SuperSignal® West Pico Luminol/Enhancer Solution (Pierce). We used Tanon 6600 Luminescent Imaging Workstation (Tanon Science & Technology, Shanghai, China) to scan the film. And band signal intensity was analyzed using Image J.