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. 2024 Apr 9;12:RP89776. doi: 10.7554/eLife.89776

Figure 2. Goblet cells of intestine display dominant expression of Rab7.

(A, B) Confocal imaging of healthy and dextran sulfate sodium (DSS)-treated mice (7 d) colon sections showing Rab7 (red) expression within goblet cells (UEA1-FITC) marked with asterisk. Inset shows zoomed areas of the image (scale bar = 20 µm). Graph shows Rab7 fluorescence intensity within UEA1 positive goblet cells measured within region of interest (ROI). 20 goblet cells were selected randomly from three different fields of each mouse (n = 3). (C, D) Representative co-immunofluorescence images of human ulcerative colitis (UC) and control colon biopsy sections stained for Rab7 (red) and Muc2 (green) for goblet cells (marked with asterisk) (scale bar = 50 µm). Inset shows zoomed areas of the image (scale bar = 20 µm). Graph shows Rab7 fluorescence intensity within Muc2-positive goblet cells measured within ROI. 20 goblet cells were selected randomly from five different fields of each sample (n = 3). (E) Co-immunofluorescence of Rab7 (green) and Muc2 (red) in undifferentiated and differentiated HT-29 cells (scale bar = 50 µm). Graphs show Rab7 and Muc2 fluorescence intensity measured in 10 different fields of three independent experiments each. (F) Rab7 protein visualized in HT29-MTX-E12 cells by immune-EM. Arrows indicate presence of Rab7 on vacuoles of cells (scale bar = 1 µm). Inset showed zoomed area of image (scale bar = 200 nm). Error bars represent mean + SEM. Statistical analysis by Student’s t-test.

Figure 2.

Figure 2—figure supplement 1. Expression of Rab7 in goblet cells of different types and regions of gastrointestinal tract.

Figure 2—figure supplement 1.

(A) Immunohistochemistry images of staining of Rab7 in different regions of intestine: small intestine, proximal colon and distal colon; showing Rab7 expression in vacuolated cells (marked with asterisk) (n = 3). Scale bar = 50 µm. (B) Confocal images of mice distal colon stained with Rab7 (red) and goblet cell specific marker UEA1 (green) showing the presence of Rab7 in UEA1 positive cells (marked with asterisk) (n = 3). Scale bar = 100 µm. (C, D) Single-channel images of DAPI, UEA1/Muc2, and Rab7 staining shown in Figure 2A and C. (E) RT-PCR analysis of relative fold expression of Lgr5, Muc2, and Rab7 genes in differentiated HT29 cells relative to undifferentiated cells. HPRT was used for normalization. (F) Immunoblot showing Rab7 protein expression in differentiated HT29 cells relative to undifferentiated cells. Graph represents densitometric analysis of Rab7 expression calculated by normalizing to loading control (β actin). (G) TEM analysis of HT29-MTX-E12 cells showing presence of vacuoles (V) in the cytoplasm (Cy) along with nucleus (Nu) nucleolus (Ns) and mitochondrion (M). Each dot represents one individual experiment. Error bars represent mean + SEM. Statistical analysis by Student’s t-test. ns = nonsignificant.
Figure 2—figure supplement 1—source data 1. Original files for the western blot analysis in Figure 2—figure supplement 1F (anti-Rab7 and anti-actin).