Ab272692

VTN (459 a.a, HEK293, His), RSL3 (HY-100218A), Ferrostatin-1 (HY-100579), DIP (HY-B0312), and Rofi (HY-15455S2) were purchased from MedChemExpress (Shanghai, China). Antibodies targeting GPX4 (67763-1-Ig, 1 : 200 for immunofluorescence (IF), 1 : 2,000 for western blotting (WB)), SLC7A11 (26864-1-AP, 1 : 200 for IF, 1 : 2,000 for WB), CDX2 (60243-1-Ig, 1 : 200 for IF, 1 : 2,000 for WB), CREB1 (12208-1-AP, 1 : 2,000 for WB), PRKACA (27398-1-AP, 1 : 2,000 for WB), α-tubulin (66031-1-Ig, 1 : 2,000 for WB) were from Proteintech Company (Wuhan, China). Phospho-CREB1 (Ser133) (YP0075, 1 : 2,000 for WB), Phospho-PKA (Thr198) (YP0226, 1 : 2,000 for WB), Phospho-PDE4 (YP0668, 1 : 100 for IF, 1 : 2,000 for WB), EpCAM (YM0219, 1 : 200 for IF), EpCAM (YM6053, 1 : 200 for IF) were from Immunoway Research (Plano, USA). Antibodies against MUC2 (ab272692, 1 : 2,000 for WB) and PDE4 (ab99409, 1 : 2,000 for WB) were from Abcam (Cambridge, UK). Lamin A/B (sc-376248, 1 : 3,000 for WB) was from Santa Cruz Biotechnology (Dallas, Texas, USA). All unconjugated secondary antibodies were from Beijing Ray Antibody Biotech (Beijing, China). All ultrapure reagents were from Promega (Madison, WI, USA).GPX4 plasmid was purchased from Youbio (Hunan, China), siRNA was synthesized by Genepharma (Shanghai, China).

Intestinal barrier function was assessed by measuring the protein expression of Muc2, ZO-1, and occludin in colon samples. The degree of acute inflammatory cell infiltration was evaluated by measuring MPO expression. The protein expression of STAT3 and FOXO3 was also measured. Paraffin-embedded mouse colon samples were labeled with rabbit anti-MUC2 antibody (1/2000; Abcam, ab272692), ZO-1 (1/100; Abcam, ab216880), occludin (1/100; Abcam, ab168986), MPO (1/1000 dilution; Abcam, ab208670), STAT3 (1/1200 dilution; ABclonal, A19566), and FOXO3 (1/1000; CST, 2497 S), followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated polyclonal secondary antibody (ab214880) for 30 min at room temperature. Immunostaining was performed using the DAB Detection Kit (Gene Tech, Shanghai, China). Protein expression was scored by two independent researchers based on the percentage of positively-stained cells (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) and staining intensity (0, none; 1, mild; 2, moderate; and 3, strong) [31 ]. The total score was the product of staining extent and intensity scores. A score of 6 was the threshold for high protein expression levels.

The small intestinal tissue of mice was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into sections at a thickness of 5 μm (The histopathology associated index of intestine was evaluated though the criterion in Supplementary Table 1). For immunohistochemical staining, the intestinal sections were subjected to deparaffinization, hydration, antigen retrieval, quenching of endogenous peroxidase, and blocking procedures. All slices were then incubated with the primary antibodies against pSTAT3 (CST #9145) and Ki-67 (Abcam, England, Ab16667) at 4°C overnight followed by incubation with biotinylated secondary antibodies for 30 min and visualization using a 3,3′-Diaminobenzidine Kit (ZSGB-BIO, Beijing, China). For immunofluorescence staining for the detection of MUC2 (Abcam, England, Ab272692), Lgr5 (NBP1-28904SS), ZO-1 (Abcam, England, Ab221547), and Claudin-1 (Abcam, England, Ab242370), the colonic sections were processed by the methods described above. Periodic Acid-Schiff stain (PAS) staining (Biossci, China) was performed following the manufacturer’s protocols.

Colonic tissue was fixed in cold 4% paraformaldehyde, and then the samples were dehydrated in ethanol and toluene, embedded in paraffin, and sectioned at 5 μm thickness. Samples were transferred to microscope slides treated with 3-aminopropyl-triethoxysilane-treated for immunofluorescence staining. Briefly, sections were deparaffinized and rehydrated, and antigen retrieval was performed by exposure to microwaves for 15 min in 0.01% sodium citrate buffer (pH 6.0). Sections were blocked with 10% normal donkey serum and incubated overnight at 4 °C with primary antibodies (Abcam, UK) against Ki67 (1:200, ab15580), MUC2 (1:200, ab272692), ZO-1 (1:200, ab221547), or E-cadherin (1:200, ab231303). Next, sections were incubated with Alexa Fluor 488- or 555- conjugated secondary antibodies (1:100, Invitrogen, Life Technologies, USA) for 1 h at 37 °C and Hoechst 33,342 (B2261; Sigma Aldrich, USA) as a nuclear counterstain. Samples were observed under an A1 confocal laser microscope (Nikon, Japan).

Rabbit anti-Ki67 (1:300; ab15580, Abcam), rabbit anti-Mucin 2 (1:300; ab272692, Abcam), rabbit anti-lysozyme (1:300; ab108508, Abcam), rabbit anti-chromogranin A (1:300; ab15160, Abcam), mouse anti–E-cadherin (1:1000; 610182, BD Biosciences), mouse anti–α-catenin (1:500; 610194, Biosciences), mouse anti–β-catenin (1:500; 610154, Biosciences), rabbit anti-BMPR1A (1:1000; sc-20736, Santa Cruz Biotechnology), rabbit anti-Smad4 (1:1000; sc7154, Santa Cruz Biotechnology), rabbit anti-Olfm4 [1:300; 39141, Cell Signaling Technology (CST)], mouse anti-vimentin (1:500; 550513, BD Biosciences), rabbit anti–IL-1α (1:50; ab300499, Abcam), rabbit anti–IL-1α (1:50; 16765-1-AP, Proteintech), mouse anti–IL-1β (1:200; sc-52012, Santa Cruz Biotechnology), rabbit anti-MOT10 (1:1000; YT7177, ImmunoWay), rabbit anti-CHP2 (1:1000; YT0915, ImmunoWay), rabbit anti–p-Smad1/5 (1:100; 9516, CST), mouse anti-Ki67 (1:200; 9449, CST), and rabbit anti-Gli1 (1:80; 3538, CST).

Immunohistochemistry of colonic sections was performed as described previously [23 (link)]. After microwave antigen retrieval, the colonic slides were incubated overnight with primary antibodies of Ki-67 (Abcam, ab21700, Cambridge, UK) and proliferating cell nuclear antigen (PCNA) (Abcam, ab29, Cambridge, UK) at 4 °C. For immunofluorescence staining, colonic slides were incubated overnight with primary antibodies of β-catenin (Abcam, ab32572, Cambridge, UK) and Muc-2 (Abcam, ab272692, Cambridge, UK) at 4 °C.