Pstat3

The immunohistochemistry (IHC) analysis was performed by following the previous methodology.¹³ Primary antibodies including proliferating cell nuclear antigen (PCNA) (Santa Cruz, CA) and pSTAT3 (GeneTex Inc., CA) were stained in hepatic tissues of zebrafish. Data were analyzed by immunoreactive scores (IRSs) which were calculated by intensity (score 1‐3) multiply proportion of positive cells (score 1‐4).³⁶

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF) and proteins (20–40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, Rochester, NY). Antibodies of ATM, p-ATM, JAK1, p-JAK1, JAK2, p-JAK2, STAT3, and p-STAT3, were from Gene Tex (Irvine, CA), Antibodies of E-cadherin, N-cadherin, Vimentin, Snail, Zeb1, Twist and VEGF were obtained from Abgent (San Diego, CA) and antibodies of PD-L1, GAPDH, were from Cell Signaling (Danvers, MA).

For histological analysis, longitudinal sections from the large intestine were immediately fixed according to previously described protocols [8 (link)]. Colon sections with a thickness of 5 μm were stained with hematoxylin and eosin (H&E) to visualize morphology or with alcian blue to visualize goblet cells using an optical microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). For immunohistochemical and immunofluorescence staining, sections were incubated overnight at 4 °C with primary antibodies against p-STAT3, Ly6G (both from GeneTex, Irvine, CA, USA), iNOS, Arginase-1 (both from Cell Signaling Technology, Danvers, MA, USA) and then developed following a conventional technique. Samples were analyzed with a ZeissVert.A1 conventional epifluorescence microscope and a LEICA TCS SP8X confocal microscope.