Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Rab11 vesicles perform kiss-and-run dependent on FERARI. Movie stills showing kiss-and-run of endogenously GFP-tagged Rab11 vesicles (arrow) in ctr KO ( n = 41) and vipas39-KO ( n = 25) HeLa cells (see also Supplementary Movie ). Scale bar: 2 µm. b Rab11 vesicles dock on SNX1 networks with residence times in distinct intervals. Groups of vesicles with similar residence times appear as peaks (see also Supplementary Fig. for single-vesicle graph, mock : n = 41, vipas39-KO : n = 25). This pattern is abolished in vipas39 -KO cells (see Supplementary Fig. for ehd1-KO and rab11fip5-KO additional data). One-way multiple comparison ANOVA test P -values: mock-vipas39-KO P = 1.98E-09, mock-ehd1-KO P = 1.6E-10, mock-rab11fip5-KO P = 1.58E-09. c List of FERARI genes in C. elegans and H. sapiens . The human nomenclature is used throughout the manuscript. The model organism is indicated by a human or worm icon next to the data. d Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in wild-type worms ( n = 53). Scale bar: 2 µm. e Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in ehd1(RNAi) worms ( n = 33) (see also Supplementary Movie ). Scale bar: 2 µm. f Rab5 vesicles dock on SNX1 networks with residence times in distinct intervals (see also Supplementary Fig. for single-vesicle graph, n = 53). This pattern is abolished in ehd1 ( n = 33) and fip5 ( n = 35) knock-downs (see Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 4.63E-05, mock-fip5 P = 3.83E-05. g Residence times intervals in Rab5 vesicles docking to mCherry-EHD1 compartments ( wild-type from f as comparison, n = 63) (see Supplementary Fig. ). Unpaired two-tailed t -test: P = 0.8467. h Rab5 exhibits kiss-and-run behavior in HeLa cells stably expressing mApple-Rab5 and transiently expressing GFP-SNX1. Movie stills show representative vesicles for ctr KO ( n = 41) and vipas39- KO ( n = 23) cells (arrow) (see also Supplementary Movie ). Scale bars: 2 µm. i Binning of vesicles from (h) reveals distinct intervals of residence times for Rab5 vesicles ( mock : n = 41, vipas39-KO : n = 23). This pattern is abolished in vipas39-KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000118.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Two Tailed Test, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a GFP-Rab10 compartments docking onto mCherry-SNX1 networks in worm intestine (see also 3D projection, Supplementary Movie ). Regions of co-localization are indicated by arrows. Quantification of co-localization by Mander’s coefficients ( n = 10). Data are presented as mean values +/– SD. M1 denotes the overlap between SNX1 and Rab10, and M2 between Rab10 and SNX1. One-way multiple comparison ANOVA test P -values: M1 SNX1-Rab10 vs. SNX1-DHS-3 P = 1.94E-08, M1 SNX1-Rab10 vs. SNX1-MANS P = 9.62E-09, M2 SNX1-Rab10 vs. SNX1-DHS-3 P = 4.37E-13, M2 SNX1-Rab10 vs. SNX1-MANS P = 4.43E-13. Scale bar: 2 µm. b Genetic interaction between FERARI subunits and Rab10. rab10(ok1494) causes accumulation of SNX1 compartments. RNAi of vps45 and vipas39 but not vps33B (CHEVI) show additional enlargement of SNX1 (arrowheads). n = 20 worms from 3 experiments (for quantification see Supplementary Fig. ). Scale bar: 10 µm. c Western blot of CRISPR–Cas9-mediated KO of Rab10 in HeLa cells ( n = 3 independent experiments). d Size of the endogenous SNX1 structure is enlarged in rab10 KO cells. SNX1 was detected by immunofluorescence. Violin plot showing the enlarged volume of the SNX1 structure in ctr and rab10 KO cells (volume of >14,700 particles was measured from each group from three independent experiments, median indicated by the red line). Unpaired two-tailed t -test: P = 1.46E-17. Scale bars 10 µm. e Movie stills for Rab10 vesicles (arrow) showing kiss-and-run in ehd1(RNAi) ( n = 40) and wild-type worms ( n = 51) (Supplementary Movie ). “N”: nucleus with high mCh-SNX1 signal. Scale bar: 2 µm. f Rab10 vesicles show residence times with quantal increases ( n = 51). This behavior is abolished in ehd1 ( n = 40) and fip5 ( n = 36) knock-downs (Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 7.23E-08, mock-fip5 P = 4.29E-10. g Kiss-and-run of Rab10 on sorting compartments is conserved in metazoans. Residence times of Rab10 vesicles exhibit characteristic intervals abolished in vipas39- KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000128. h Movie stills of mCherry-tagged Rab10 vesicles (arrow) showing kiss-and-run on SNX1 (GFP-tagged) compartment in ctr KO and vipas39- KO HeLa cells ( n = 3 independent experiments, mock : n = 53, vipas39-KO : n = 22 vesicles) (see Supplementary Movie ). Scale bar: 2 µm.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Western Blot, CRISPR, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Co-localization between GFP-Rab11 and endogenous SNX1 is reduced in snx5 + 6 double knock out cells. Antibodies against GFP and SNX1 were used to detect Rab11 and SNX1 by immunofluorescence, respectively. Scale bars 10 µm; 5x magnified. Pearson’s coefficient from n = 65 and n = 71 cells from ctr KO and snx5 + 6 KO cells, respectively. Data are presented as single data points with median and min to max whiskers. Unpaired two-tailed t -test P = 2.5316E-07. b Size of the GFP-Rab11-positive structures is increased in snx5 + 6 KO cells in comparison with the ctr KO cells ( n = 3 independent experiments) Scale bars 10 µm; magnification 5x. c Volume of the GFP-Rab11-positive particles in both ctr and KO cells were determined (volume of >15,000 particles were measured from each group from three independent experiments, median is shown by the red line). Unpaired two-tailed t -test P = 3.05E-73. d CI-MPR trafficking is impaired in snx 5 + 6 KO cells. CI-MPR (red) is mostly localized in TGN (green) area in ctr KO cells and is dispersed in snx 5 + 6 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Pearson’s coefficient measurements are shown on the right. Unpaired two-tailed t -test P = 1.97E-51. Scale bars 10 µm. e CI-MPR localization remain unchanged in vipas39 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Scale bars 10 µm; magnification 5x. Co-localization was quantified by Pearsons’s coefficients. Unpaired two-tailed t -test P = 0.0536. f Immunoprecipitation data showing that endogenous SNX6 (left panel), but not SNX5 (right panel), binds to FERARI members Rabenosyn-5 and VIPAS39 in HEK-293 cells ( n = 3 independent experiments). g C. elegans SNX6 interacts with RABS-5 (but not with other FERARI subunits) in Y2H assays. Quantification is shown in Table ( n = 3 growth experiments with n = 6 independent yeast colonies each).

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Knock-Out, Immunofluorescence, Two Tailed Test, Immunoprecipitation

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. Cadherin-11 protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Staining, Invasion Assay, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Negative Control, Invasion Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: shRNA, Quantitative RT-PCR, In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Labeling, Invasion Assay, Quantitation Assay, In Vitro, Micro-MRI, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: 1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing, Luciferase, Over Expression, In Vivo, In Vitro