Fig 2: Exacerbation of DSS-induced colitis in Reg3b−/− mice. Two lines of 8- to 12-week-old of Reg3b−/− mice and respective control wild-type mice were administered with 1.5% DSS in drinking water for 5 days, which then was changed to regular water. (A) RNA was prepared from colon samples of wild-type mice on day 8 after DSS treatment, and the expression of Reg3b and Reg3g was determined by qPCR. Results are mean ± SEM (n = 4 mice). Statistical significance was determined using the two-tailed unpaired Student's t-test. *P < 0.05, **P < 0.01. (B, C) The average body weight is shown as the percentage relative to the initial value. Results are mean ± SEM (n = 9–10 mice). Pooled results of two independent experiments are shown. Statistical significance was determined using repeated measures ANOVA. *P < 0.05, ****P < 0.0001. (D) Colonic sections of the indicated mice on day 13 after DSS treatment were stained with H&E (n = 4 mice per genotype). Black arrows and arrowheads indicate submucosal edema and the inflamed colonic mucosa lacking epithelial cells, respectively. Scale bars, 100 μm. (E, F) Numbers of infiltrated neutrophils increased in the colon of Reg3b−/− mice on day 8 after DSS treatment. Single cell suspension was prepared from the colon of wild-type and Reg3b−/− mice. Cells were stained with the indicated antibodies, and percentages of CD11b+Ly-6G+ cells (neutrophils) among CD45.2-positive cells were calculated. The results are shown in the right upper corner. Representative profiles of flow cytometry (E). Results are mean ± SEM (n = 7 mice) (F). Pooled results of two independent experiments are shown. Statistical significance was determined using the two-tailed unpaired Student's t-test. *P < 0.05. (G) Expression of the indicated genes in the colon of wild-type and Reg3b−/− mice on day 8 after DSS treatment was determined by qPCR. Results are mean of ± SEM (n = 8 mice). Pooled results of two independent experiments are shown. Statistical significance was determined using the two-tailed unpaired Student's t-test. *P < 0.05, ns, not significant.

Fig 3: JAK2/STAT3 is not involved in REG3B-induced ADM.a Western blot analysis of related protein expression in 266-6 and AR42J cell lines 30 min and 72 h after different stimulations, with quantification data. b, c Expression level of JAK2/STAT3 signaling components in the 266-6 cell line under different conditions for 30 min (b) and 72 h (c). d, e Expression level of JAK2/STAT3 signaling components in the AR42J cell line under different conditions for 30 min (d) and 72 h (e) (n = 3, one-way ANOVA test). REG3B treatment alone did not increase p-JAK nor p-STAT3 expression at two timepoints in two cell lines but did increase their expression once the receptor EXTL3 was blocked.

Fig 4: Generation of Reg3g-/- mice. (A) Deletion of exon 3 of Reg3g genes in two lines of Reg3g-/- mice. Red characters indicate deleted nucleotides and mutated amino acids. (B) Tissue extracts were prepared from the small intestine of 8- to 12-week-old Reg3g+/+ and Reg3g-/- mice (line G151) and examined by Western blotting with the indicated antibodies. Each number indicates an individual mouse. Results represent two independent experiments. (C) In-frame deletion of exon 3 of Reg3b in two lines of Reg3g-/- mice. Red characters indicate the deleted nucleotides. (D) Paraffin-embedded tissue sections of the small intestine and colon of 8- to 12-week-old Reg3g+/+ and Reg3g-/- mice (line G151) were stained with H&E. Scale bars, 100 µm.

Fig 5: REG3B is elevated in caerulein-induced ADM in mice.a Scheme of caerulein injection to induce acute pancreatitis. Acute pancreatitis was induced by intraperitoneal (i.p.) injection with caerulein dissolved in PBS at a dose of 50 µg/kg at hourly intervals eight times daily, for two consecutive days. PBS injection alone served as control. b The size of pancreas of PBS-treated mice appear smaller than those of caerulein-treated mice (n = 6, student’s t-test). c Percentage of pancreas/body weight of caerulein-treated mice versus PBS-treated mice (n = 6, student’s t-test). d Abundant ADM formation in caerulein-treated mice (H&E, 40×, Scale bar: 500 µm). e Difference in the extent of ADM between PBS-treated mice and caerulein-treated mice, as measured by percentage of ADM area in total pancreatic area on H&E stained tissue sections (n = 6, student’s t-test). f Western blot analysis with quantifications showed higher REG3B protein level and lower AMYLASE protein level in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice (n = 6, student’s t-test). GAPDH served as a loading control. g RT-qPCR analysis showed higher Ck19 and lower Mist1 mRNA levels in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice (n = 5, student’s t-test). h H&E staining of caerulein-induced ADM in mice, with corresponding REG3B IHC staining. ADM areas were highlighted by dashed lines, with arrows indicating naïve ADM and asterisks indicating relatively mature ADM. Scale bar: 200 µm. Data are represented as means ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; Non-significant (n.s.) if P > 0.05.