Fig 2: Cell lineage differentiation 3 days after acute Notch inhibition.(A) PAS-stained jejunum of DBZ-treated control and VilCreERT2 Myo5Bfl/fl mice. Arrows indicate luminal mucus covering the villi. (B–E) Immunostaining for tuft cell marker DCLK1 (B), goblet cell marker TFF3 (C), Paneth cell marker lysozyme (D), and proliferating cell marker PCNA (E). Nuclei were counterstained with Hoechst in cyan or blue. Scale bars: 100 µm.

Fig 3: Let‐7i in BMSC suppresses proliferation and metastasis of lung cancer cells in nude mice. A, Statistical analysis of tumour volume of nude mice at various time‐points (n = 6). B & C, Representative pictures of tumours and statistical analysis of tumour weight of nude mice on the 30th d. D, Statistical analysis of nodule metastasis in lung tissues detected by HE staining (× 400). E & F, The expression of let‐7i, KDM3A, DCLK1, and FXYD3 in tumour tissues determined by RT‐qPCR and Western blot analysis. G, The expression of vimentin, N‐cadherin and E‐cadherin in tumour tissues determined by Western blot analysis. *P < .05 compared to model mice, #P < .05 compared to nude mice treated by si‐FXYD3. Data are all measurement data and expressed as the mean ± standard deviation

Fig 4: Evidence of altered autophagy in the intestinal/colon tumorsParaffin-embedded sections prepared from the intestinal or colonic (upper panels) tumors isolated from uninfected (Un), CR infected (CR), AOM-treated or CR+AOM-treated Apc1638N/+ mice were subjected to co-localization studies with antibodies for p62 and Dclk1, respectively. DAPI was used as nuclear stain. Co-localization of p62 and Dclk1 appears as yellow/orange puncta in the merged images. Scale bars = 150-250µm; n = 3 independent experiments. Middle panel. In vitro evidence of autophagy flux.HCT116 and SW480 cells treated for 24hr with either Chloroquin (CQ, 30µM) to block autophagy and promote efficient autophagic flux or LPS to mimic CR infection. Cells were processed and co-localization of p62 with Dclk1 and p62 with LC3B was evaluated. DAPI was used as nuclear stain (n = 3 independent experiments). Co-localization of p62 and Dclk1 appears as yellow/orange puncta in the merged images. Lower panel. Left:Presence or absence of autophagic vesicles was further confirmed via electron microscopy wherein, the effect of both autophagy inhibitor CQ or LPS was examined (n = 3 independent experiments). Middle: Western blotting revealed a dose-dependent increase in Dclk1, LC3-II and p62, respectively. ß-Actin and H3 were used as loading controls. Right: Dclk1 promoter assay. HCT116 cells transfected with Dclk1-L or Dclk1-S reporters were either untreated or treated with varying dosages of CQ or LPS followed by measurement of reporter activity (*p-values<0.05 vs control; n=3 independent experiments).

Fig 5: BMSC-EV-derived let-7i can inhibit the DCLK1/FXYD3 axis by regulating KDM3A, thus inhibiting the pathogenesis of lung cancer. A, The expression of let-7i after co-culture of si-NC-transfected lung cancer cells EVs-treated lung cancer cells measured by RT-qPCR, *P < .05 compared with cells transfected with si-NC, &P < .05 compared with cells transfected with si-FXYD3 + EV-inhibitor-NC. B, The KDM3A, DCLK1 and FXYD3 protein expression determined by Western blot analysis, *P < .05 compared with cells transfected with si-NC, #P < .05 compared with cells transfected with si-FXYD3, &P < .05 compared with cells transfected with si-FXYD3 + EV-inhibitor-NC. C, The proliferation of lung cancer cells after EV treatment determined by CCK-8, *P < .05 compared to cells transfected with si-NC, #P < .05 compared to cells transfected with si-FXYD3, &P < .05 compared to cells transfected with si-FXYD3 + EV-inhibitor-NC. D, The migration and invasion of lung cancer cells after EV treatment determined by Transwell (× 200), *P < .05 compared to cells transfected with si-NC, #P < .05 compared to cells transfected with si-FXYD3, &P < .05 compared with cells transfected with si-FXYD3 + EV-inhibitor-NC. E, Flow cytometry analysis of lung cancer cell apoptosis after EV treatment, *P < .05 compared with cells transfected with si-NC, #P < .05 compared with cells transfected with si-FXYD, &P < .05 compared with cells treated by si-FXYD3 + EV-inhibitor-NC. The experiment was repeated 3 times