Journal: bioRxiv

Article Title: Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

doi: 10.1101/2023.05.17.541154

Figure Lengend Snippet: (A, B) Immunohistochemical (IHC) staining for Cbl (A) and Cbl-b (B) on formalin-fixed paraffin-embedded (FFPE) tissue sections from 6-weeks old mice reveal their expression in a gradient from crypt to villus in WT animals (left panels); Lack of epithelial staining in sections from Cbl -/- or Cblb -/- knockout mice (right panels) demonstrates the specificity of antibody staining. (C) Crypt-villus fractionation of intestinal mucosa followed by immunoblotting confirmed the crypt enrichment of CBL and CBL-B expression. Blotting for crypt (pH3 and CyclinD1) and villus (Villin-1) associated markers confirmed the purity of villus and crypt fractions. Hsc-70 served as a loading control. (D) Immunoblot analysis of crypt and villus fractions prepared from age- and gender-matched WT , Cbl -/- and Cbl-b -/- animals. Within the crypt compartment, deletion of Cbl-b resulted in an increase in the expression of Cbl while Cbl-b expression was unchanged upon deletion of Cbl . The reverse was seen in the villus compartment, with increase in Cbl-b expression upon Cbl deletion whereas Cbl expression was unchanged in Cbl-b -/- animals. Scale bars in A and B parts are 100 µm; shown are representative of 3 experiments. Altered intestinal epithelial cell proliferation and differentiation in Cbl -/- mice. (E) WT and Cbl -/- were mice injected with BrdU 4 hours prior to euthanasia and FFPE sections of intestines were stained with anti-BrdU antibody (Red) and DAPI (Blue); scale bar= 20 mm. (F) Quantification of the number of BrdU+ cells per crypt shows increased proportion of cells in S phase of the cell cycle in Cbl null mice. (G-I) PAS staining of tissue sections from age matched WT , Cbl -/- and Cblb -/- mice revealed a significant increase in the number of goblet cells (PAS+ cells in the villus) in both Cbl -/- and Cblb -/- mice (H) and a mild reduction in the Paneth cell (PAS+ cells in the crypt) number in Cbl -/- mice (I), scale bar= 100 µm. (J, K) Chromogranin A staining (Red), showed no significant difference in the number of enteroendocrine cells between WT and Cbl -/- mice; scale bar= 50 mm. Data are represented as +/- SEM of 4 experiments. Statistical analysis used student’s two-tailed t-test ; ns, p ≤ 0.05, *; p ≤ 0.01, **.

Article Snippet: The following antibodies were procured from commercial sources: Anti-Cbl (Clone 17/c-Cbl, BD Bioscience); anti-Cbl-b (Clone D3C12, Cell Signaling Technology); anti-HSC70 (Clone B6, Santa Cruz Biotechnology Inc. Santa Cruz, CA), phospho Akt (Ser473) (Cat# 4060, Cell Signaling Technology); Akt (pan, Cat# 4691, Cell Signaling Technology); phospho S6 (Ser235/236), Cat# 2211, Cell Signaling Technology); Total S6 (5G10, Cat#2217, Cell Signaling Technology), phospho-histone 3 (Ser10) (Cat# 9701, Cell Signaling Technology), Villin-1 (Cat# 2369, Cell Signaling Technology).

Techniques: Immunohistochemical staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Expressing, Staining, Knock-Out, Fractionation, Western Blot, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

doi: 10.1101/2023.05.17.541154

Figure Lengend Snippet: Equal number of crypts isolated from Cbl flox/flox ; Cblb flox/flox (FF Control) and Cbl flox/flox ; Cblb flox/flox ; R26Cre ERT2 (FF/creERT) mice were plated in 100% Matrigel in the presence of growth factors to form organoids. Once formed, organoids were re-plated at a 1:4 split ratio and treatment with 400 nM of 4-OH TAM initiated after 24 h to induce Cbl/Cblbb deletion. (A) Bright field imaging showed that while FF control mouse intestinal organoids showed steady increase in budding (crypt domains) over time (up to 72 hours of observation), the FF/creERT mouse organoids showed increased budding until 48 h after 4-OH-TAM induction but rapid loss of crypt domains by 72 h post-induction; scale bar = 40 µm. (B) Representative H&E-stained images of FF control and FF/CreERT organoids confirm the loss of morphological features in the latter; scale bar = 50 µm. (C) Ten distinct organoids per genotype were followed up to 72 hours and change in the number of buds (crypt domains) at each time point relative to time 0 was quantified. (D) FF Control and FF/creERT organoids grown in 4-OH-TAM for 72 h were re-passaged and imaged after 48 h. Note the lack of organoid structures with intact morphology in FF/creERT organoid cultures, supporting a loss of self-renewal contrary to growth and intact morphology of control organoids; scale bar = 400 µm. (E) Immunoblotting of organoid lysates at different time-points confirmed effective Cbl/Cblb deletion by 48 hours and 72 h time points in FF/creERT organoids. HSC70, loading control. FF/creERT organoids derived from two independent female mice (2F and 3F) were cultured in the presence (iDKO) or absence (control) of 400 nM 4-hydroxy-TAM for 72 hours. Single cells isolated from the organoids were processed through the GemCode Single Cell Platform (10X Genomics) to perform single cell RNAseq and analyzed using Seurat. (F) tSNE map of combined controls and combined Cbl/Cblb iDKO mouse organoid cells. Cells are grouped into seven clusters based on transcriptome profiles and are colored accordingly. The cell types were assigned based on gene expression profile of stem cells ( Lgr5, Ascl2, Axin2, Olfm4, Gkn3 ), transit-amplifying (TA) cells ( Mki67, Cdk4, Mcm5, Mcm6, Pcna ), enterocytes ( Alpi, Apoa1, Apoa4, Fabp1 ), Paneth cells ( Lyz1, Defa17, Defa22, Defa24, Ang4 ), enteroendocrine cells ( Chga, Chgb, Tac1, Tph1, Neurog3 ), Goblet cells ( Muc2, Tff3, Agr2 ) and Tuft cells ( Dclk1, Trpm5, Gfi1b ). (G) Heat map shows the expression levels of top cluster-specific genes in each cluster. Yellow represents the highest expression while purple represents low or no expression. (H) Histogram depicting the percentage of cells in each cluster. (I) Validation of Cbl and Cblb deletion in the 2F and 3F organoids by qPCR. (J) Gene set enrichment analysis (GSEA) of each cell type between control and iDKO organoids shows the upregulation of PI3K-Akt-mTOR signaling pathway upon Cbl/Cblb iDKO in stem cells, goblet cells, enteroendocrine cells and enterocytes. NES (normalized enrichment score), FDR (false discovery rate) and p values are indicated in the GSEA plots. (K) To validate the alterations in PI3K-mTOR signaling upon Cbl/Cblb iDKO, organoid lysates were collected at different time-points of culture in the presence of 4-OH-TAM and analyzed by immunoblotting. Clear upregulation of p-AKT/mTOR pathway components (p-Akt and p-S6) is seen. Hsc-70 served as a loading control. (L) Intestinal tissue sections from Control and Cbl/Cblb iDKO mice 10 d after initiating TAM treatment were stained with antibodies against p-S6 to assess the impact of Cbl/Cblb deletion. Sections stained with the secondary antibody alone were used as a negative control. p-S6 levels were sharply increased in iDKO as compared to control sections; scale bar= 20 µm. Quantified data are presented as mean +/- SEM of three separate experiments with statistics using student’s two-tailed t-test . ns, not significant; p ≤ 0.05, *; p ≤ 0.01, **; p≤ 0.001, ***.

Article Snippet: The following antibodies were procured from commercial sources: Anti-Cbl (Clone 17/c-Cbl, BD Bioscience); anti-Cbl-b (Clone D3C12, Cell Signaling Technology); anti-HSC70 (Clone B6, Santa Cruz Biotechnology Inc. Santa Cruz, CA), phospho Akt (Ser473) (Cat# 4060, Cell Signaling Technology); Akt (pan, Cat# 4691, Cell Signaling Technology); phospho S6 (Ser235/236), Cat# 2211, Cell Signaling Technology); Total S6 (5G10, Cat#2217, Cell Signaling Technology), phospho-histone 3 (Ser10) (Cat# 9701, Cell Signaling Technology), Villin-1 (Cat# 2369, Cell Signaling Technology).

Techniques: Isolation, Imaging, Staining, Western Blot, Derivative Assay, Cell Culture, Expressing, Negative Control, Two Tailed Test