Journal: bioRxiv

Article Title: Muc6 -expressing gastric isthmus progenitors contribute to regeneration and metaplasia supported by myeloid-mesenchymal interactions

doi: 10.1101/2025.04.15.648856

Figure Lengend Snippet: (A) The epithelial cell clusters from WT mice were re-clustered and shown as a UMAP plot. Muc6 expression was shown as violin plot across 14 epithelial cell clusters and co-expression analysis among Muc6, Mki67, Stmn1 , and Bhlha15 (Mist1) was performed. Orange and yellow arrows indicate neck progenitor cells and co-expressing cells respectively. (B) Gene constructs of Muc6 -dsRED-FlpER; FSF- EGFP mice. (C) Muc6 -dsRED-FlpER; FSF- EGFP treated with low-dose (50 mg/kg) tamoxifen. GIF (green)/EGFP (green)/GSII (blue) staining are shown. (D) Gene constructs of Mist1 -CreERT; Muc6 -dsRED-FlpER; LSL-FSF-mTfp1 HA mice. (E-F) scRNA-seq data with WT and Mist1 -CreERT; LSL- Kras G12D mice treated with or without H. pylori. (E) Feature plot showing the indicated cell markers. (F) UMAP plot of the gastric epithelial cell clusters split by the mouse groups. Green arrows show the SPEM clusters. (G-H) Spatial transcriptional Visium HD analysis with Mist1 -CreERT and Mist1 -CreERT; LSL- Kras G12D mice 7 days after tamoxifen treatment. (G) Expression of cell markers shown in UMAP. (H) Expression of cell markers shown in Violin plots. (I) mTFP1(red)/AQP5(green) staining in Mist1 -CreERT; Muc6 -dsRED-FlpER; LSL-FSF-mTfp1 HA mice infected with H. pylori for 4 months. Scale bars; 100 μm. Mean ± S.E.M. *P < .05.

Article Snippet: The Loxp-STOP-Loxp (LSL)-Kras G12D , LSL -TdTomato, Mist1-creERT , Il1r1-/- , and Frt-STOP-Frt (FSF) -EGFP mice were purchased from Jackson laboratories.

Techniques: Expressing, Construct, Staining, Infection

Journal: bioRxiv

Article Title: Muc6 -expressing gastric isthmus progenitors contribute to regeneration and metaplasia supported by myeloid-mesenchymal interactions

doi: 10.1101/2025.04.15.648856

Figure Lengend Snippet: (A) GSII (green)/ Muc6 -dsRED (red), Ki67 (green)/ Muc6 -dsRED (red), and Stmn1 (green)/ Muc6 -dsRED (red) staining in Muc6 -dsRED-FlpER mice. Arrows indicate GSII-negative, Ki67-positive, or Stmn1-positive Muc6 + progenitor cells. (B) Ki67 (green)/ Muc6 -dsRED (red) and CD44v (green)/ Muc6 -dsRED (red) staining in WT, ATP, GIF, and GIFATP mice mated with Muc6 -dsRED-FlpER mice three days after DT treatment. (C) Lineage tracing images of Muc6 -dsRED-FlpER; FSF-EGFP mice treated with 300 mg/kg tamoxifen along the time course. (D) Lineage tracing images of Mist1 -CreERT; Muc6 -dsRED-FlpER; FSF-LSL-mTFP1 HA mice treated with 150 mg/kg tamoxifen along the time course. (E) Muc6 -dsRED-FlpER; FSF-EGFP mice mated with WT, ATP, GIF, GIFATP mice were treated with 50 mg/kg tamoxifen (day 0). DT was administered at day 3, and GFP(green)/Ki67 (red) expression was stained and quantified at day 10 (n=3 mice/group). (F) Mist1 -CreERT; Muc6 -dsRED-FlpER; FSF-LSL-mTFP1 HA mice mated with WT, ATP, GIF, GIFATP mice were treated with 150 mg/kg tamoxifen (day 0). DT was administered at day 3, and HKATPase (green)/GIF (blue)/mTFP1 (red) expression was stained and quantified at day 18 (n=3 mice/group). (G-I) Single cell RNA-seq with Mist1 -CreERT; LSL- Kras G12D mice treated with or without H. pylori. (G) Schema of the experiment. (H) Trajectory analysis and pseudotime calculation visualized in the UMAP plot. (I) Box plots showing Cytotrace2 scores across epithelial cell clusters. (J-K) Mist1 -CreERT; LSL- Kras G12D ; Muc6 -dsRED-FlpER; FSF-EGFP and Mist1 -CreERT; LSL- Kras G12D ; Muc6 -FlpER; LSL-FSF-mTFP1 HA were treated with 150 mg/kg tamoxifen and analyzed at day 30. (J) GIF(red)/GFP(green)/GIF(blue) and mTFP1(red)/GSII(green)/GIF(blue) staining are shown respectively. (K) CD44v(red)/GFP(green) and HA(red)/AQP5(green) staining are shown respectively. (L) Spatial transcriptomic Visium HD analysis of Mist1 -CreERT; LSL- Kras G12D mice 7 days after 150 mg/kg tamoxifen treatment. Spatial gene distribution and UMAP plot split by Mist1 -CreERT and Mist1 -CreERT; LSL- Kras G12D mice, and DEGs in the neck progenitors are shown. (M) HE, H/K-ATPase (red)/TFF2 (green), RFP (red)/CD44v (green), and Ki67 staining in Mist1 -CreERT; LSL- Kras G12D ; Gif-DTR mice treated with or without DT three times per week for two weeks (n=3 mice/group).

Article Snippet: The Loxp-STOP-Loxp (LSL)-Kras G12D , LSL -TdTomato, Mist1-creERT , Il1r1-/- , and Frt-STOP-Frt (FSF) -EGFP mice were purchased from Jackson laboratories.

Techniques: Staining, Expressing, RNA Sequencing

Journal: Nature Communications

Article Title: Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer

doi: 10.1038/s41467-025-58923-y

Figure Lengend Snippet: a The schematic diagram of screening the candidate DUBs of regulating KRAS stability by using KRAS G12V -luciferase reporter system and DUBs siRNA library (The schematic diagram was created in BioRender. https://BioRender.com/w35e177 ). The KRAS G12V -luciferase reporter system was constructed by cloning the KRAS G12V coding sequences in front of the firefly-luciferase sequence, thus forming a fusion gene of KRAS G12V -Luciferase. CDS coding sequences, DUB deubiquitinases. b JOSD2 siRNA pools significantly minimized the luciferase activity of the KRAS G12V -luciferase reporter system. c JOSD2 depletion inhibited the luciferase activity of the KRAS G12V -luciferase reporter system in a dose-dependent manner. (means ± SD, n = 3; three independent experiments were performed). d , e JOSD2, but not JOSD1, positively regulated KRAS mutants’ protein levels. f JOSD2 depletion dramatically decreased KRAS mutants’ stability. g JOSD2 depletion downregulated KRAS levels through the ubiquitin-proteasome systems but not the autophagy-lysosome system. h , i JOSD2, but not JOSD1, positively regulated MAPK signaling pathways. WT, wild-type; Mut, mutation. The significance analysis of ( c ) was conducted by One-way ANOVA (Bonferroni method was utilized to correct for multiple comparisons). The samples derived from the same experiment and processed on different gels in parallel are shown below: d SW480(JOSD2): one gel for GAPDH/KRAS, another for JOSD2; e SW480(shJOSD2) & SW620(shJOSD2): one gel for GAPDH/JOSD2, another for KRAS. f Different gels for GAPDH and Myc(KRAS); g one gel for GAPDH/JOSD2, another for KRAS; h SW480(shJOSD1): one gel for p-MEK/GAPDH/MEK, another for p-ERK/JOSD1/ERK; SW620(shJOSD1): one gel for p-ERK/MEK, another for p-MEK/ERK, another for GAPDH/JOSD1; SW480(shJOSD2): one gel for p-MEK/JOSD2/GAPDH, another three for ERK, p-ERK and MEK, respectively; SW620(shJOSD2): one gel for p-MEK/GAPDH, another for p-ERK/JOSD2, another two for ERK and MEK, respectively; i SW480(JOSD1): one gel for p-MEK/KRAS/GAPDH/MEK, another for p-ERK/JOSD1, another for ERK; SW620(JOSD1): one gel for p-ERK/KRAS/p-MEK/GAPDH, another for MEK/JOSD1, another for ERK; SW480(JOSD2): one gel for ERK/KRAS, another for p-MEK/GAPDH, another for p-ERK/JOSD2, another for MEK; SW620(JOSD2): one for ERK/KRAS, another for p-MEK/GAPDH, another for p-ERK/JOSD2, another for MEK. Source data are provided as a Source Data file.

Article Snippet: GST fusion proteins were bound to GSH Sepharose (Sangon Biotech, C600031) at 4 °C for 2 h. The GSH Sepharose was washed with RIPA lysis buffer three times and incubated with recombinant His-KRAS-WT/G12C/G12D/G12V or indicated cell lysates at 4 °C for another 2 h. After washing with RIPA lysis buffer six times, the bound proteins were subjected to immunoblotting with the indicated antibodies.

Techniques: Luciferase, Construct, Cloning, Sequencing, Activity Assay, Mutagenesis, Derivative Assay

Journal: Nature Communications

Article Title: Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer

doi: 10.1038/s41467-025-58923-y

Figure Lengend Snippet: a The ability of KRAS G12C/G12D/G12V to bind to JOSD2 was stronger than that of KRAS WT . WT, wild-type; IP, immunoprecipitation. b The interaction between JOSD2 and KRAS was independent of the enzyme activity of JOSD2. CA&HY, JOSD2-C24A&H125Y. c – e JOSD2, not JOSD1, interacted with KRAS at semi-endogenous levels and endogenous levels. f In vitro GST pull-down assays to examine the interaction between JOSD2 and KRAS WT/G12C/G12D/G12V . 293T cells were transfected with the indicated expression plasmids for 24 h. Cell lysates were incubated with the Streptavidin magnetic beads conjugated with bacterial-expressed recombinant GST or GST-JOSD2 protein. Proteins retained on Streptavidin magnetic beads were subjected to WB analysis. g , h JOSD2, not JOSD1, removed the polyubiquitination chains of KRAS WT/G12C/G12D/G12V in an enzyme activity-dependent manner. i JOSD2 depletion promoted the polyubiquitination of KRAS WT/G12C/G12D/G12V . j JOSD2 removed the polyubiquitination chains of KRAS WT/G12C/G12D/G12V in vitro (The schematic diagram was created in BioRender. https://BioRender.com/w35e177 ). 293T cells were co-transfected with indicated expression plasmids, and cell lysates were incubated with anti-Flag affinity gel overnight. Then, proteins retained on anti-Flag affinity gel were incubated with bacterial-expressed recombinant human JOSD2 (rhJOSD2) protein at 37 °C for 3 h, followed by WB analysis. k JOSD2, not JOSD1, removed the polyubiquitination chains of KRAS G12V promoted by LZTR1. The samples derived from the same experiment and processed on different gels in parallel were shown below: d Input: one gel for JOSD1/JOSD2/GAPDH, another for Flag(G12V), IP: one gel for JOSD1/Flag(G12V), another for JOSD2; e SW620: Input: one gel for JOSD1/KRAS/GAPDH, another for JOSD2, IP: one gel for JOSD1/KRAS, another for JOSD2; HCT-116: Input: one gel for JOSD2/GAPDH, another for JOSD1/KRAS, IP: different gels for JOSD1, KRAS and JOSD2; f one gel for KRAS/GST-JOSD2, another for GST; g Input of KRAS-WT/G12C: one gel for HA(ub)/GAPDH/ Flag(KRAS), another for JOSD1&2, Input of KRAS-G12D/G12V: one gel for HA(ub)/GAPDH/JOSD1&2, another for Flag(KRAS); i Input of KRAS-WT/G12C/G12D/G12V: one gel for HA(ub)/GAPDH/JOSD2, another for Flag(KRAS); k Input: one gel for HA(ub)/GAPDH, another for Flag(KRAS), another for Myc(LZTR1)/JOSD1&JOSD2. Source data are provided as a Source Data file.

Article Snippet: GST fusion proteins were bound to GSH Sepharose (Sangon Biotech, C600031) at 4 °C for 2 h. The GSH Sepharose was washed with RIPA lysis buffer three times and incubated with recombinant His-KRAS-WT/G12C/G12D/G12V or indicated cell lysates at 4 °C for another 2 h. After washing with RIPA lysis buffer six times, the bound proteins were subjected to immunoblotting with the indicated antibodies.

Techniques: Immunoprecipitation, Activity Assay, In Vitro, Transfection, Expressing, Incubation, Magnetic Beads, Recombinant, Derivative Assay

Journal: Nature Communications

Article Title: Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer

doi: 10.1038/s41467-025-58923-y

Figure Lengend Snippet: a The intratumor JOSD2 levels of KRAS-mutant CRC patients were significantly higher than those of KRAS wild-type CRC patients. IHC, immunochemistry; WT, wild-type; Mut, mutation. (means ± SD, n = 6 patients/group; Scale bar, 100 μm). b , c KRAS mutants, not KRAS wild-type, positively regulated JOSD2 levels. d KRAS mutants posed little effect on JOSD2 mRNA levels (means ± SD, n = 3; three independent experiments were performed). e Overexpression of KRAS mutants, not KRAS wild-type, increased JOSD2 stability. CHX, cycloheximide. f KRAS mutants decreased the polyubiquitination levels of JOSD2. g There was no significant difference in the ubiquitination levels of JOSD2-WT and C24A when immunoprecipitated with KRAS-G12V. WB, western blot. h K48, K142, and K181 are three main ubiquitination sites on JOSD2 (The schematic diagram was created in BioRender. https://BioRender.com/w35e177 ). i The stability of JOSD2-3KR was significantly higher than JOSD2-WT. j KRAS mutants significantly increased the protein levels and stability of JOSD2-WT, but not that of JOSD2-3KR. DOX, doxycycline. k , l JOSD2 interacted with CHIP in vivo/in vitro. m , n CHIP positively regulated the polyubiquitination levels of JOSD2. o CHIP negatively regulated JOSD2 levels. p Overexpression of CHIP significantly decreased the protein levels of JOSD2-WT, but not of JOSD2-3KR. q Depletion of CHIP prevented the further upregulation of JOSD2 levels induced by KRAS G12V . (means ± SD, n = 3; three independent experiments were performed). The significance analysis of ( a ) was conducted by Two-tailed unpaired Student’s t -tests, and ( d , q ) were conducted by One-way ANOVA (Bonferroni method was utilized to correct for multiple comparisons). The samples derived from the same experiment and processed on different gels in parallel were shown below: b one gel for Flag(KRAS)/GAPDH, another for JOSD2; f Input: one gel for HA(ub)/GAPDH/Flag(JOSD2), another for Myc(KRAS); k Input: different gels for GAPDH and Flag(CHIP); IP: different gels for JOSD2 and Flag(CHIP); l different gels for GST and Myc(CHIP); q one gel for CHIP/Flag(G12V), another for GAPDH/JOSD2. Source data are provided as a Source Data file.

Article Snippet: GST fusion proteins were bound to GSH Sepharose (Sangon Biotech, C600031) at 4 °C for 2 h. The GSH Sepharose was washed with RIPA lysis buffer three times and incubated with recombinant His-KRAS-WT/G12C/G12D/G12V or indicated cell lysates at 4 °C for another 2 h. After washing with RIPA lysis buffer six times, the bound proteins were subjected to immunoblotting with the indicated antibodies.

Techniques: Mutagenesis, Over Expression, Immunoprecipitation, Western Blot, In Vivo, In Vitro, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer

doi: 10.1038/s41467-025-58923-y

Figure Lengend Snippet: a , b JOSD2 depletion inhibits the cell proliferation of KRAS-mutant cells strongly than KRAS wild-type cells in vitro. HT-29 KRAS-WT/Mut cells were infected with indicated lentiviruses for 96 h, then these cells were seeded with 2000 cells per well in 96-well/6-well plates, followed by SRB staining. Ctrl, control. a The representative cells proliferation images of the indicated group; Scale bar, 6 mm. b The quantitative analyses of cells proliferation, P = 0.0965, WT-Ctrl vs . WT-shJOSD2; P = 4E-8, G12C-Ctrl vs . G12C-shJOSD2; P = 6E-8, G12D-Ctrl vs . G12D-shJOSD2; P = 1E-6, G12V-Ctrl vs . G12D-shJOSD2. (means ± SD, n = 3; three independent experiments were performed). c , d JOSD2 depletion inhibits the colony formation of KRAS-mutant cells strongly than KRAS wild-type cells in vitro (means ± SD, n = 3; three independent experiments were performed). c The representative colony formation images of the indicated group; Scale bar, 5 mm. d The quantitative analyses of colony formation. e – h JOSD2 depletion posed a more potent anti-tumor effect on KRAS-mutant xenografts compared to KRAS wild-type in vivo (means ± S.E.M., n = 7 mice/group). e The tumor images of the indicated groups. f The tumor volume of the indicated groups. Tumors were measured every two days. g The tumor weight of the indicated groups. h Knocking down JOSD2 downregulated intratumor KRAS levels of KRAS-mutant xenografts are stronger than those of KRAS wild-type xenografts. The proteins isolated from the tumor were subjected to WB analysis. The significance analysis of ( b , d , f , g ) was conducted by One-way ANOVA (Bonferroni method was utilized to correct for multiple comparisons). The samples derived from the same experiment and processed on different gels in parallel are shown below: h one gel for JOSD2/GAPDH and another for KRAS. Source data are provided as a Source Data file.

Article Snippet: GST fusion proteins were bound to GSH Sepharose (Sangon Biotech, C600031) at 4 °C for 2 h. The GSH Sepharose was washed with RIPA lysis buffer three times and incubated with recombinant His-KRAS-WT/G12C/G12D/G12V or indicated cell lysates at 4 °C for another 2 h. After washing with RIPA lysis buffer six times, the bound proteins were subjected to immunoblotting with the indicated antibodies.

Techniques: Mutagenesis, In Vitro, Infection, Staining, Control, In Vivo, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: Splice-switching ASOs targeting an Alu-derived exon in the AURKA 5’UTR collapse an SRSF1-AURKA-MYC oncogenic circuit in pancreatic cancer

doi: 10.1101/2025.04.14.648775

Figure Lengend Snippet: ASO-A collapses the SRSF1-AURKA-MYC circuit, induces apoptosis, and overcomes KRAS G12D inhibitor resistance in PDAC (A) Radioactive RT-PCR of AURKA 5’UTR from SUIT2 cells after ASO transfection. NTC, non-treated control (Lipofectamine 3000 only); SC, scrambled control; A, ASO-A. (B-E) PSI quantification of +E2 isoforms (B) and RT-qPCR of total AURKA (C), SRSF1 (D), and MYC (E) normalized to RPS13 from (A) (n = 3 biological replicates). (F) Western blotting of AURKA, SRSF1, MYC, and VINC after ASO treatment as in (A). (G) Cell-viability assay of SUIT2 cells after ASO treatment as in (A), normalized to day 0 (n = 3 biological replicates). (H) Apoptosis assay of SUIT2 cells after ASO treatment as in (A). Staurosporine (STS) at 10 μM for 24 h as a positive control (n = 3 biological replicates). (I) Dose-response in SUIT2 vs. HPDE cells. Points are the mean ± SD (n = 3 biological replicates). (J) Western blotting of AURKA, SRSF1, MYC, phosphorylated and total ERK1/2 (p-ERK and t-ERK, respectively), and VINC from SUIT2 parental cells and SUIT2 RMC-9805-resistant cells. Cells were either treated with DMSO, RMC-9805 [1 μM], or a combination of RMC-9805 [1 μM] and SC or ASO-A [65 nM] for 48 h. (K) Relative protein expression compared to each indicated control, for AURKA (left), SRSF1 (middle), and MYC (right) from (J) normalized to VINC (n = 3 biological replicates). (L) Quantification of the ratio of p-ERK/t-ERK from (J) normalized to VINC; p-ERK was then normalized to t-ERK, and the ratio was then normalized to each indicated control (n = 3 biological replicates). (M) Cell-viability assay from SUIT2-parental and SUIT2-RMC-9805-resistant cells treated as in (I) normalized to day 0 (n = 3 biological replicates). (N) Schematic of SRSF1-AURKA-MYC circuit. Data shown as mean ± SD. ns = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 ****p ≤ 0.0001 one-way ANOVA (B, C, D, E, G, H, K, L, M). See also Figures S10 and S11.

Article Snippet: For KRAS G12D inhibition RMC-9805 was purchased from MedChemExpress and dissolved in DMSO.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Quantitative RT-PCR, Western Blot, Viability Assay, Apoptosis Assay, Positive Control, Expressing

Journal: bioRxiv

Article Title: Tff2 marks gastric corpus progenitors that give rise to pyloric metaplasia/SPEM following injury

doi: 10.1101/2025.04.09.647847

Figure Lengend Snippet: ( A ) Schematic of the experimental design for the effect of H.p infection on the fate of Tff2 + progenitors. In brief, Tff2-CreERT; R26-tdTomato mice were treated with tamoxifen (75 mg/kg) to induce nuclear transfer of Cre recombinase, then the mice were orally gavaged with H. p (SS1, 2*109 CFU, every 2 days*3 doses) or vehicle 1 day later. Corpus tissues were then sampled for histological analysis 60 days after oral gavage (2M). SAC, scarify. ( B ) (Top and middle) H&E staining and (bottom) immunofluorescence staining for GS-II (green), GIF (red), and DAPI (blue) in the corpus glands of Tff2-CreETR; tdTomato mice 2 months after H.p infection (H.p 2M) or nontreated controls (Ctrl). Yellow dashed box indicates mucinous metaplasia, red dashed box indicates normal chief cell. Scale bars, 100 μm. ( C ) Immunofluorescence staining for Ki-67 (green), tdTomato (red), and DAPI (blue) as in (B). Scale bars, 100 μm. ( D ) Quantification of the number of positive cells per gland (tdTomato + cells and Ki-67 + cells) as in (C). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. *P < 0.05, **P < 0.01. ( E ) Schematic of the experimental design for the effect of Kras mutation on the fate of Tff2 + progenitors. In brief, Tff2-CreERT; Kras LSL-G12D/+ ; R26-tdTomato (Kras) mice or Tff2-CreERT; Kras +/+ ;R26-tdTomato (Ctrl) mice were treated with tamoxifen (75 mg/kg) to induce Kras mutation and/or nuclear transfer of Cre recombinase. Corpus tissues were then sampled for histological analysis 60 days after induction (2M). ( F ) Immunofluorescence staining for Ki-67 (green), tdTomato (red), and DAPI (blue) in the corpus glands of Kras and Ctrl mice 2 months after induction. Scale bars, 100 μm. ( G ) Quantification of the number of positive cells per gland (tdTomato + cells and Ki-67 + cells) as in (F). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. *P < 0.05. ( H and I ) Histopathology score according to the criteria for mucinous metaplasia (H) and dysplasia (I) in corpus tissues of Tff2-CreERT; Kras +/+ ;R26-tdTomato mice 6 months after H.p infection (H.p) or vehicle (Ctrl), or Tff2-CreERT; Kras LSL-G12D/+ ; R26-tdTomato mice 6 months after Kras mutation (Kras) or Kras mutation plus H.p infection (Kras+H.p). Schematic of the experimental design as shown in (Fig. S7G). Data are shown as mean ± SD. n = 3 to 12 mice per group. Statistical significance was assessed with one-way ANOVA. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ( J , K , L ) (Top) Alcian blue staining for acidic mucins (blue) in the corpus glands of Tff2-CreERT; Kras +/+ ;R26-tdTomato mice at 5 days after tamoxifen induction (Ctrl) (J), or Tff2-CreERT; Kras LSL-G12D/+ ; R26-tdTomato mice 6 months after Kras (K) and Kras+H.p (L). Cell nuclei were counterstained with hematoxylin (purplish blue). (Bottom) Periodic acid Schiff (PAS) staining neutral mucins (magenta) in the corresponding groups described in (top) panel. Cell nuclei were counterstained with hematoxylin (purplish blue). Scale bars, 100 μm.

Article Snippet: 6-to 8-week-old Tff2-CreERT; Kras LSL-G12D ; ROSA26-tdTomato mice or Tff2-CreERT; ROSA26-tdTomato mice were infected by oral gavage with H. pylori (SS1, 2×10 CFU, every 2 days×3 doses, from) in 0.2ml brucella broth (Becton Dickinson, Cat# 211088).

Techniques: Infection, Staining, Immunofluorescence, Mutagenesis, Histopathology

Journal: bioRxiv

Article Title: Tff2 marks gastric corpus progenitors that give rise to pyloric metaplasia/SPEM following injury

doi: 10.1101/2025.04.09.647847

Figure Lengend Snippet: ( A and B ) Immunofluorescence staining for TROP2 (green), tdTomato (red), and DAPI (blue) in the corpus glands of Tff2-CreETR; tdTomato mice 6 months after tamoxifen induction (Ctrl) (A) or Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato mice 6 months after Kras mutation plus H.p infection (Kras+ H.p) (B). Scale bars, 100 μm. ( C ) Quantification of the percentage of tdTomato + TROP2 + lesions per TROP2 + lesion as in (A and B). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. *P < 0.05. ( D ) H&E staining in the corpus glands of Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato mice 6 months after Kras mutation. Yellow dashed box indicates metaplasia, red dashed box indicates dysplasia. Scale bars, 100 μm. ( E ) Immunofluorescence staining for CD44v9 (green), TROP2 (red), and DAPI (blue) in the corpus glands of Tff2-CreETR; tdTomato mice 6 months after H.p infection or Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato mice 6 months after Kras mutation. Yellow dashed box indicates metaplasia, red dashed box indicates dysplasia. Scale bars, 100 μm. ( F ) (Top) In situ hybridization of Tff2 (green) and immunofluorescence staining for CD44v9 (red), and DAPI (blue) in the corpus glands of Tff2-CreETR; tdTomato mice 6 months after H.p infection. Scale bars, 100 μm. (Bottom) In situ hybridization of Tff2 (green) and immunofluorescence staining for TROP2 (red), and DAPI (blue) in the corpus glands of Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato mice 6 months after Kras+ H.p. Scale bars, 100 μm. ( G ) Quantification of the number of Tff2-mRMA + cells per gland as in (F). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed with one-way ANOVA. *P < 0.05, ****P < 0.0001. ( H ) Quantification of the percentage of mucinous metaplasia (top) and dysplasia (bottom) to the total number of corpus glands of Tff2-CreERT; R26-tdTomato mice at different time points (0M, 2M, 3M, 6M, 12M) after H.p infection or Tff2-CreERT; Kras LSL-G12D/+ ; R26-tdTomato mice after Kras mutation. Schematic of the experimental design as shown in (Fig. S4H). Data are shown as mean ± SD. n = 3 to 12 mice per group. Statistical significance was assessed with multiple unpaired t tests. *P < 0.05, **P < 0.01. ( I ) Quantification of the percentage of tdTomato + Tff2-mRMA + cells to total Tff2-mRMA + cells in the corpus glands of Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato mice 2 and 3 months after Kras mutation. Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. **P < 0.01.

Article Snippet: 6-to 8-week-old Tff2-CreERT; Kras LSL-G12D ; ROSA26-tdTomato mice or Tff2-CreERT; ROSA26-tdTomato mice were infected by oral gavage with H. pylori (SS1, 2×10 CFU, every 2 days×3 doses, from) in 0.2ml brucella broth (Becton Dickinson, Cat# 211088).

Techniques: Immunofluorescence, Staining, Mutagenesis, Infection, In Situ Hybridization

Journal: bioRxiv

Article Title: Tff2 marks gastric corpus progenitors that give rise to pyloric metaplasia/SPEM following injury

doi: 10.1101/2025.04.09.647847

Figure Lengend Snippet: (A) Schematic representing the experimental design for screening of Tff2 + progenitors for organoid culture after DMP777-induced acute injury or nontreated controls (Ctrl). In brief, Tff2-CreERT; R26-tdTomato mice were treated with tamoxifen (75 mg/kg) to induce Cre recombinase 1 day before DMP777 treatment or nontreated. Flow cytometric selecting of tdTomato + EpCAM + cells as Tff2 + progenitors 1 day after DMP777 treatment or Ctrl and tested them for 3D organoid growth. ( B ) Organoid growth quantification evaluated as grown area (log10 scale) as in (A). Data are shown as mean ± SD. n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. **P < 0.01. ( C ) Immunofluorescence staining for Ki67 (red) and DAPI (blue) 5 days after the third passage (P3-D5) of organoids comparing the proliferation capacity of Tff2 + progenitors from DMP777 and Ctrl mice as in (A). Scale bars, 100 μm. ( D ) Quantification of the percentage of Ki-67 + cells to total DAPI + cells in organoids as in (C). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. **P < 0.01. ( E ) Immunofluorescence staining for Cleaved Caspase 3 (red) and DAPI (blue) 5 days after the third passage (P3-D5) of organoids comparing the anti-apoptotic capacity of Tff2 + progenitors from DMP777 and Ctrl mice as in (A). Scale bars, 100 μm. ( F ) Quantification of the percentage of Cleaved Caspase 3 + cells to total DAPI + cells in organoids as in (E). Data are shown as mean ± SD; n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. *P < 0.05. ( G ) Schematic representing the experimental design for screening and organoid culture of Tff2 + progenitor or SPEM cells. In brief, Tff2-CreERT; Kras LSL-G12D/+ ; R26-tdTomato mice (Kras G12D ) or Tff2-CreERT; Kras +/+ ;R26-tdTomato mice (Kras WT ) were used. Flow cytometric selecting of CD44v9 - tdTomato + EpCAM + cells as Tff2 + Kras G12D or Tff2 + Kras WT progenitors 1 day after low-dose tamoxifen induction, selecting of CD44v9 + tdTomato + EpCAM + cells as Tff2 + Kras G12D or Tff2 + Kras WT SPEM cells 5 days after HDT treatment, and tested them for 3D organoid growth. ( H ) Organoid growth quantification evaluated as grown area (log10 scale) with the same setting as in (G). Data are shown as mean ± SD. n = 3 mice per group. Statistical significance was assessed with one-way ANOVA. ns, not significant, ***P < 0.001, ****P < 0.0001. ( I and J ) In situ hybridization of Tff2 (green), Olfm (red), immunofluorescence staining for tdTomato (white), and DAPI (blue) in the corpus glands of Tff2-CreETR; Kras LSL-G12D/+ ; tdTomato (Kras) mice 3 (I) or 6 (J) months after Kras mutation. White arrow indicates Olfm-mRMA + cells derived from tdTomato + progenitors. Yellow asterisk indicates tdTomato + Tff2-mRMA + progenitors. Scale bars, 100 μm. ( K ) (Top, left) Schematic representing the experimental design for screening of tdTomato + progenitors and tdTomato − epithelial cells for 2D cell culture. In brief, Tff2-CreERT; Kras LSL-G12D/+ ; tdTomato (Kras G12D ) and Tff2-CreERT; Kras +/+ ; tdTomato (Kras WT ) mice were treated with tamoxifen (75 mg/kg) to induce Cre recombinase for 1 day. Flow cytometric selecting of tdTomato + EpCAM + cells as Tff2 + progenitors and tdTomato - EpCAM + cells as other epithelial cells 1 day after tamoxifen-induced and tested them for 2D cell growth. (Bottom, left) 2D cell growth quantification evaluated as grown area (log10 scale) with the same setting as in (top, left). Data are shown as mean ± SD. n = 3 mice per group. Statistical significance was assessed by unpaired Student’s t test. **P < 0.01. (Right) Bright field and fluorescence images of epithelial 2D cell cultures at 5 days (D5) for tdTomato + progenitors and tdTomato − epithelial cells as in (top, left). Scale bars, 100 μm. ( L ) Immunofluorescence staining for Cytokeratin 19 (CK19) (green) and DAPI (blue) at 9 days (D9) for tdTomato + Kras WT progenitors and tdTomato − Kras WT epithelial cells as in (K). Scale bars, 100 μm. ( M ) Immunofluorescence staining for CK19 (green) and DAPI (blue) at day 9 (D9) for both tdTomato + Kras G12D progenitors (top) and tdTomato + Kras WT progenitors treated with N-Methyl-N-nitrosourea (MNU, 20 nmol/L) for 24hours starting at day 7 (bottom). Scale bars, 100 μm.

Article Snippet: 6-to 8-week-old Tff2-CreERT; Kras LSL-G12D ; ROSA26-tdTomato mice or Tff2-CreERT; ROSA26-tdTomato mice were infected by oral gavage with H. pylori (SS1, 2×10 CFU, every 2 days×3 doses, from) in 0.2ml brucella broth (Becton Dickinson, Cat# 211088).

Techniques: Immunofluorescence, Staining, In Situ Hybridization, Mutagenesis, Derivative Assay, Cell Culture, Fluorescence