Journal: JCI Insight

Article Title: Intestinal epithelial BLT1 promotes mucosal repair

doi: 10.1172/jci.insight.162392

Figure Lengend Snippet: ( A ) RNAscope staining for LTB4R and CMKLR1 mRNA expression in frozen sections from colonic tissue of humans. ( B ) RNAscope staining for Ltb4r1 and Cmklr1 mRNA expression in frozen sections from colonic tissue of mice. Scale bars: 50 μm. ( C ) qPCR analysis of the expression of CMKLR1 and LTB4R mRNA in the SKCO-15, T84, and human 2D colonoids. The data are presented as the mean ± SEM. Cq, quantification cycle (measured as cycles). ( D ) Effect of BLT1 antagonist on the prorepair activity of RvE1 in the scratch wound assay using human primary IECs. After scratch wound was produced, IECs were incubated with RvE1 (100 nM) for 24 hours. BLT1 (CP105,696; 1 μM) or CMKLR1 (α-NETA; 10 μM) antagonist was applied 30 minutes before RvE1 treatment. Quantification of wound repair at 24 hours after wounding is shown. The data are presented as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. * P < 0.05, ** P < 0.01, compared with RvE1.

Article Snippet: BLT1 antagonist (CP105,696; MilliporeSigma) or CMKLR1 antagonist (α-NETA; Cayman Chemical) was applied 30 minutes before LTB4 or RvE1 treatment.

Techniques: RNAscope, Staining, Expressing, Activity Assay, Scratch Wound Assay Assay, Produced, Incubation

Journal: JCI Insight

Article Title: Intestinal epithelial BLT1 promotes mucosal repair

doi: 10.1172/jci.insight.162392

Figure Lengend Snippet: ( A ) The changes in the expression of Ltb4r1 mRNA in 3 mm punch biopsies of intact colonic tissues and colonic mucosal wounds on different days after injury. The data are presented as the mean ± SEM of 4–5 mice. Statistical analysis was performed using 1-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. * P < 0.05, ** P < 0.01, compared with intact tissue (IT). ( B – D ) RNAscope staining for Blt1 mRNA in frozen sections from intact tissues and wounded colonic tissues 2 days after injury. Arrows indicate upregulation of Ltb4r1 expression in the crypts next to the wound. W, wound. Scale bar is 50 μm. ( E ) The number of Ltb4r1 mRNA–positive dots in the crypt of intact colonic tissues and colonic mucosal wounds (adjacent to wound) 2 days after injury is shown. The data are presented as the mean ± SEM of 6 mice. Statistical analysis was performed using an unpaired (2-tailed) t test with Welch’s correction. * P < 0.05, compared with IT. AW, adjacent to wound. ( F and G ) RNAscope staining for LTB4R mRNA expression in frozen sections from healthy controls and patients with ulcerative colitis.

Article Snippet: BLT1 antagonist (CP105,696; MilliporeSigma) or CMKLR1 antagonist (α-NETA; Cayman Chemical) was applied 30 minutes before LTB4 or RvE1 treatment.

Techniques: Expressing, RNAscope, Staining

Journal: JCI Insight

Article Title: Intestinal epithelial BLT1 promotes mucosal repair

doi: 10.1172/jci.insight.162392

Figure Lengend Snippet: ( A – D ) Migration analysis by DiPer. ( A ) Plot at the origin graph of 20 cells. ( B ) Mean square displacement of 20 cells. The data are presented as the mean ± SEM. Statistical analysis was performed using 2-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. ** P < 0.01, **** P < 0.0001, compared with WT (vehicle). †† P < 0.01, ††† P < 0.001, †††† P < 0.0001, compared with Ltb4r1 –/– (vehicle). ( C ) Velocity autocorrelation was measured on at least 20 cells. Statistical analysis was performed using 2-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with WT (vehicle). †† P < 0.01, compared with Ltb4r1 –/– (vehicle). ( D ) Average cell speed was calculated on 20 cells. The data are presented as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. *** P < 0.001, **** P < 0.0001. ( E ) Immunoblotting was performed on lysates from scratch-wounded IEC monolayers treated with LTB 4 (100 nM) or vehicle. Levels of phosphorylated SRC (p-SRC) (Y416) and p-FAK (Y397, Y925) were compared with total Src, FAK, and GAPDH to assess activation. Numbers on the left represent kDa. ( F and G ) EdU incorporation analysis in murine 3D cultured colonoids stimulated with LTB 4 (10 nM) for 24 hours. ( F and G ) Effect of BLT1 antagonist. Pictures show representative images of EdU-incorporated (shown in green) colonoids. Blue, nuclei. Scale bar is 10 μm. The data are presented as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by post hoc Welch’s t test with Bonferroni’s correction. * P < 0.05, ** P < 0.01.

Article Snippet: BLT1 antagonist (CP105,696; MilliporeSigma) or CMKLR1 antagonist (α-NETA; Cayman Chemical) was applied 30 minutes before LTB4 or RvE1 treatment.

Techniques: Migration, Western Blot, Activation Assay, Cell Culture