Journal: Nature Communications

Article Title: 1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis

doi: 10.1038/s41467-026-68542-w

Figure Lengend Snippet: HIEC-6 ( a ) were treated with fluorescein labeled polysaccharides and then subjected to immunostaining for Rab5. Scare bar, 10 μm, 2 μm for the enlarged images. The colocalization analysis results are showed in ( b ). HIEC-6 cells were transfected with siRNAs against RAB5A , then treated with polysaccharides. Images of the internalized GFPBW1 and WGE were shown in ( c ), and the quantified results were exhibited in ( e ), and Western blotting results showed the siRNA efficiency ( d ). Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Control and RAB5A KO mice ( n = 5 mice for each group) were oral administrated with fluorescein labeled GFPBW1 ( f ) and WGE ( g ), images of the duodenum and jejunum of the mice were shown, the fluorescence intensity was analyzed by Fiji and shown in ( h , i ), respectively. Scare bar, 100 μm, 20 μm for the enlarged images. Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t test, *** p < 0.001, **** p < 0.0001. j – l Cells were transfected with RAB5A and the vector plasmid for 48 h, treated with fluorescein labeled GFPBW1 and WGE, and imaged using confocal microscopy, the quantified results are shown in ( l ). k The expression of Rab5 detected by Western blots was shown. Data are presented as mean ± SD from three biologically independent experiments. Unpaired two-tailed t test, * p < 0.05, ** p < 0.01. m , n SPR assay was performed to determine the interaction of polysaccharides and Rab5. Source data are provided as a file.

Article Snippet: A Biacore T200 instrument was used to measure the binding kinetics of human CLTC981-1100aa (abmart, Shanghai, China), human DNM11-271aa (Bioss, Beijing, China), human DNM1 (TP306284, OriGene, USA), human Rab5a (NBC1-18504, Novusbio, USA), human EGFR1-645aa, human Dectin-1, human BMPRIA1-152aa (Sino Biological Inc, Beijing, China) to the polysaccharides.

Techniques: Labeling, Immunostaining, Transfection, Western Blot, Two Tailed Test, Control, Fluorescence, Plasmid Preparation, Confocal Microscopy, Expressing, SPR Assay

Journal: Nature Communications

Article Title: 1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis

doi: 10.1038/s41467-026-68542-w

Figure Lengend Snippet: a Colocalization examination was conducted in T24 cells after the incubation of GFPBW1 and WGE for 1 h (green, polysaccharides, red, CLTC), the colocalization analysis results are shown in b . c The interaction of GFPBW1 to living cells in the absence and presence of CLTC antibodies (4 μg/mL). d–f T24 cells were transfected with si- CLTC and scrambled siRNA (Sham). The results of the uptake experiments are presented in d (red, polysaccharides). The efficiency of siRNA was detected by Western blotting ( e ), and the fluorescence intensity was quantified in ( f ). g , h The binding assays of fixed wild-type (Ctrl group) and CLTC-depleted T24 cells (si- CLTC group) to anti-CLTC were performed after the transfection with si- CLTC for 48 h. After regeneration of the chips, an injection of GFPBW1 (100 μg/mL) to both channels performed and the binding curves are displayed in ( h ). The immunostaining results of Rab5 after the treatment of polysaccharides and the colocalization analysis results were presented in i and j , respectively. k Uptake experiments of GFPBW1 and WGE were conducted after the transfection of si- RAB5A , the fluorescence intensity was quantified in l and the efficiency of siRNA was measured by immunoblot ( m ). n–q Further, cells treated with GFPBW1 and WGE were then fixed and stained with dynamin, the immunofluorescence images and the colocalization analysis were shown in ( n ) and o , respectively. p Images of the internalization of polysaccharides in T24 cells after the disturbing of si- DNM1 , the quantified fluorescence result was exhibited in q . Western blotting results showed the siRNA efficiency ( r ). Representative images of the polysaccharide uptake were shown in T24 cells after the treatment of dynasore ( s ), and the fluorescence intensity was quantified in ( t ). Scale bar, 10 μm, 2 μm for the enlarged images. Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a file.

Article Snippet: A Biacore T200 instrument was used to measure the binding kinetics of human CLTC981-1100aa (abmart, Shanghai, China), human DNM11-271aa (Bioss, Beijing, China), human DNM1 (TP306284, OriGene, USA), human Rab5a (NBC1-18504, Novusbio, USA), human EGFR1-645aa, human Dectin-1, human BMPRIA1-152aa (Sino Biological Inc, Beijing, China) to the polysaccharides.

Techniques: Incubation, Transfection, Western Blot, Fluorescence, Binding Assay, Injection, Immunostaining, Staining, Immunofluorescence, Two Tailed Test

Journal: Life science alliance

Article Title: CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.

doi: 10.26508/lsa.202302045

Figure Lengend Snippet: Figure 7. Modulation of CFTR trafficking. (A) Diagram of RAB5, RAB11 siRNA, and Brefeldin A effect on CFTR trafficking. (B) RAB5 and RAB11 siRNA treatments. Lytic and extracellular assays were performed after 48 h of siRNA treatments. (C) Brefeldin A inhibition of protein transport from ER to GA. Lytic and extracellular assays were performed after 6 h treatment. The effect of siRNA and Brefeldin A treatments on viability was measured by MTS assay (mean ± SD). The luminescent signal was measured after 30 min (mean ± SD). All experiments were done in three independent biological replicates (n = 9; technical replicates). P-values were calculated using one-way ANOVA-Tukey’s test, ns = nonsignificant, *P ≤0.05, **P < 0.005, ***P < 0.001.

Article Snippet: Rab5 (RAB5A) and RAB11 Human siRNA Oligo Duplex (Origene) were used in 10 and 50 nM concentration for RAB5/11 knockdown.

Techniques: Inhibition, MTS Assay

Journal: PLoS ONE

Article Title: Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

doi: 10.1371/journal.pone.0019414

Figure Lengend Snippet: (A) Functional inactivation of recycling endosomes inhibits tTG secretion. NIH3T3-tTG fibroblasts were incubated with HRP-transferrin or HRP, followed by DAB/H 2 O 2 treatment, to ablate the recycling endosomes or lysosomes, respectively . (B) Proteolysis of VAMP by Tetanus toxin inhibits tTG secretion. The light (catalytic) chain of Tetanus toxin (TeTx-LC) was expressed in NIH3T3-tTG fibroblasts. The TeTx-LC and VAMP3 levels were tested by immunoblotting. (C) Interference with SNAP23 function reduces tTG secretion. Wild type SNAP23 and its dominant negative δc9-SNAP23 mutant were expressed in NIH3T3-tTG fibroblasts as myc-tagged proteins and their levels were defined by immunoblotting. (D) Downregulation of Rab11A/Rab11B inhibits tTG secretion. shRNAs for Rab4A/B, Rab11A/B, Arf6, Rab5A and Rab22A and scrambled control were expressed in NIH3T3-tTG fibroblasts. tTG synthesis in (A–D) was induced for 4 h prior to cell surface biotinylation and isolation of surface proteins. Cell surface and total levels of tTG, β1 integrin and tubulin were defined by immunoblotting. The relative surface levels of tTG (A–D) and β1 integrin (A) were compared to those in DAB/H 2 O 2 -treated cells (A) or control transfectants (B–D). Shown in (A–D) are representative of three independent experiments. Bars show means ± SEM, *p<0.05, **p<0.005. (E) Interference with GTPase activity of Rab11 decreases tTG externalization. NIH3T3-tTG fibroblasts were transfected with wild type Rab11A, Rab11B, or their S25N dominant negative mutants fused to GFP. 48 h later the transfectants were induced to synthesize tTG for 4 h and live cells were stained at 4°C for surface tTG. Two-color flow cytometry of surface tTG (phycoerythrin) and transfected Rab11 proteins (GFP) levels is shown for gated live transfectants. Note a reduction in surface tTG levels in the transfectants that express high levels of Rab11A and Rab11B dominant negative mutants (right quadrants). Shown is representative of four independent experiments.

Article Snippet: Transient expression of pre-designed and verified shRNAs for mouse Rab4A/B, Rab11A/B, Arf6, Rab5A, Rab22A, Tsg101, and Vps24 in pGFP-V-PS vector in these cells was achieved by their transfection with Turbofectin 8.0 (all from Origene Technologies).

Techniques: Functional Assay, Incubation, Western Blot, Dominant Negative Mutation, Mutagenesis, Isolation, Activity Assay, Transfection, Staining, Flow Cytometry