EMBO/soda-nel huggingface dataset:
SARS-CoV-2 Spike Spurs Intestinal Inflammation via VEGF Production in Enterocytes (2022)
Figure 1

Figure 1. Serum VEGF level correlates with GI symptoms and disease progression of COVID-19.

A H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm.

B The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated.

C Cytokine levels in the plasma of COVID-19 patients with (n=12) or without (n=6) GI symptoms by cytokine assay.

D Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=10) disease progression by cytokine assay.

E Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n=8) and without (n=10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19).

F Levels of plasma VEGF in COVID-19 patients with (n=7) or without (n=6) interstitial vasodilation by cytokine assay.

G Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=5) interstitial edema by cytokine assay.

Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.
SARS-CoV-2 Spike Spurs Intestinal Inflammation via VEGF Production in Enterocytes (2022)
Figure 2

Figure 2. VEGF is elevated in small intestine upon abdominal stimulation of SARS-CoV-2 spike.

A Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n=5) or healthy controls (n=5) by RNA-seq.

B Illustration depicts generation of the animal model with SARS-CoV-2 spike RBD-induced intestinal inflammation. Mice were fasted for 24 hours before acetic acid clysis. 16 hours after acid challenge, mice were given Control-Fc or Spike RBD-Fc intraperitoneally. Six hours later, TRITC-dextran or Evans Blue dye was injected intravenously for evaluation of permeability.

C, D Representative H&E images (C) and the quantitative analysis of inflammation (D) of the intestinal tissues from animals treated by the protocol shown in (B). The inflammatory infiltrates were indicated by a yellow arrow. Scale bar, 100 µm. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).

E qRT-PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).

F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).

G ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).

H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice).

Data information: All data are shown as mean ± SD. For (A), (F) and (H), P values are determined by Student's t-test; for (E), P values are determined by Mann-Whitney test.
SARS-CoV-2 Spike Spurs Intestinal Inflammation via VEGF Production in Enterocytes (2022)
Figure 3

Figure 3. SARS-CoV-2 spike RBD promotes VEGF production via the Ras-Raf-MEK-ERK pathway.

A qRT-PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n=3. Data are from 3 independent biological experiments.

B ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n=4. Data are from 4 independent biological experiments.

C Immunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm.

D Levels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments.

E ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments.

F, G Immunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen).

H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (human specimen).

I The levels of Ras, c-Raf, pMEK, ERK, pERK and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments.

Data information: All data are shown as mean ± SD. For (A), (B), (D), P values are determined by Paired Student's t-test; (F) and (G), P values are determined by Student's t-test; for (E), P values are determined by Mann-Whitney test; for (H) and (I), P values are determined by one-way ANOVA.
SARS-CoV-2 Spike Spurs Intestinal Inflammation via VEGF Production in Enterocytes (2022)
Figure 4

Figure 4. SARS-CoV-2 spike protein disrupts the vascular barrier via inhibiting endothelial VE-cadherin.

A The normalized fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc or Spike RBD-Fc after 24 hours. For each group, n=3. Data are from 3 independent biological experiments.

B The expression of junctional proteins ZO-1, VE-cadherin (VE-cad), phosphorylated VE-cad (pVE-cad; 658) and pVE-cad (731) in HUVECs co-cultured with the supernatant of Caco-2 cells that were treated with either Control-Fc or Spike RBD-Fc detected by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments.

C, D Immunohistochemical staining shows levels of VE-cad (C) and pVE-Cad (731) (D) in the duodenum tissue from COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen).

E Immunofluorescence analysis of VE-cad expression and subcellular localization in Caco-2 cells that were treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE-cad staining in green and nuclear staining in blue. Scale bar, 20 µm.

F Fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates.

G Immunohistochemical staining shows levels of VE-cad and pVE-Cad (731) in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice).

Data information: All data are shown as mean ± SD. For (A) and (B), P values are determined by Paired Student's t-test; for (C) and (D), P values are determined by Student's t-test; for (F) and (G), P values are determined by one-way ANOVA.
SARS-CoV-2 Spike Spurs Intestinal Inflammation via VEGF Production in Enterocytes (2022)
Figure 5

Figure 5. Blockage of ERK/VEGF axis can alleviate SARS-CoV-2 spike RBD-induced hyperpermeability and intestinal inflammation.

A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice).

B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice).

C, D Representative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice).

E Model for the SARS-CoV-2 spike RBD-mediated vascular hyperpermeability and intestinal inflammation. ① Binding of spike RBD and receptors on enterocytes; ② Activation of the Ras-Raf-MEK-ERK pathway; ③VEGF over-production; ④VEGF-triggered phosphorylation of Y731 and internalization of VE-cadherin; ⑤Increase of endothelial permeability accompanied by interstitial edema and inflammation in the intestinal tissue.

Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 1

Figure 1. siRNA-based screening for RAB GTPases indispensable for matrix degradation invasive program

(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.

(b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment).

(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones). Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm.

(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 2

Figure 2. RAB2A is not only required for invasion through ECM, but also promotes ECM digestion

(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.

(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.

(c-e) EV and shRAB2A MDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).

(f-h) Doxycycline-inducible murine RAB2A(mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 3

Figure 3. RAB2A is an independent predictor of metastatic recurrence in human BC patient

Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.

(a) Images are representative of RAB2A expression scoring according to intensity staining in TMA. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma.

(b) Correlation of RAB2A expression with clinicopathological parameters. Only for 520 of 622 samples expression data for RAB2A was available. Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma.

(c) Kaplan–Meier Distant Recurrence Free Interval survival (DRFI [70]) and (d) Univariate and Multivariate Cox-regression analysis, shows that in BC patients’ high levels of RAB2A protein predict a poor prognosis, and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 4

Figure 4. RAB2A localizes on late endosomes positive for MT-1MMP, and is required for MT1-MMP exocytosis

(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm).

(c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm).

(d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscope. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 5

Figure 5. The VPS39-RAB2A axis is crucial for MT1-MMP-dependent ECM proteolytic and invasive activity

(a) Total cellular lysates from HEK293T cells transfected with myc-VPS39 or VPS41 were incubated with GST-empty vector (EV), RAB2A-S20N (SN), or RAB2A-Q65L (QL) proteins bound to glutathione sepharose beads. Immunoblotting was performed with anti-myc antibody. Input lysates are also shown. GST proteins loaded in gels were stained with CBB.

(b) FLAG-EV or RAB2A were transiently co-transfected with EGFP-VPS39 or VPS41 into HEK293T cells. Immunoprecipitated (IP) fraction from cell lysates with anti-FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right.

(c, d) MDA-MB-231 cells were transfected with EGFP-fused empty EV, VPS39, or VPS41. Cells were immunostained with RAB7 (c, red) or anti-MT1-MMP (d) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. Bars, 10 μm, (inset 5 μm).

(e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm.

(j-l) Inverse invasion assay of MDA-MB-231 cells transfected with siRNA oligonucleotides targeting interest genes. Invasive activity is the numbers of cells/field that migrate through Matrigel further than 50 µm (k). Efficacy of targeted gene silencing was verified by qPCR (l). Data are mean ± SD from 3 different fields. Bar, 250 µm.

(m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 6

Figure 6. Silencing RAB2A promotes cell compaction and increases cell surface E-cadherin and ZO-1

(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm.

(b-d) MCF10.DCIS.com cells (104 cells) transfected with scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A) were plated onto 6-well dish. Representative phase contrast images (b) and the distribution of cell numbers per cluster of the indicated cell number (c) are shown. Data are means ± SD of 10 fields (n > 60 cells/field) of independent 4 independent experiments. Bars, 100 μm. Efficacy of RAB2A gene silencing was verified by qPCR (d). Bar, 200 µm.

(e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue).

(f, g) MCF-10AneoT (e) and MCF10-CA1a (f) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.

(h) Total cell lysates from (e), (f), and (g) were immunoblotted by anti-E-cadherin, RAB2A, and anti-actin antibodies. ***, P < 0.001, NS, not significant.
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs (2016)
Figure 7

Figure 7. RAB2A overexpression impairs trafficking of E-cadherin from Golgi to cell surface

(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.

(c) CTR and RAB2A MCF10A cells (104 cells) were plated onto 6-well dish. Representative phase contrast images (left) and the distribution of cell numbers per cluster composed of the indicated number of cells are shown. Data are means ± SD of 10 fields (n > 20 cells/field) from 3 independent experiments. Bar, 100 μm.

(d) The circularity of CTR and RAB2A MCF10A cells and the percentage of cells with protrusive structure were quantified. Data are means ± SD of ten independent fields (n > 100 cells/field). n, number of cells scored for each cell population.

(e) Total cell lysate from (c) were immunoblotted with antibodies anti-E-cadherin, which recognize either an epitope in the cytoplasmic domain (IC), an epitope in the extracellular domain of mature E-cadherin (SHE78-7), respectively, or anti-RAB2A, and anti-actin. An arrow indicates the unprocessed form of E-cadherin.

(f-h) MCF10A cells were transfected with scrambled siRNA (siNC) or siRNA against Furin, and immunostained with anti-E-cadherin (green) and anti-TGN46 (red) antibodies. Merged images with DAPI (blue) are shown on the right (f). Bar, 20 μm. Total cell lysate were immunoblotted with antibodies indicated on the right (g). Efficacy of Furin silencing was verified by qPCR (h).

(i) Representative electron migrographs of Golgi stacks (arrows) of CTR and RAB2A MCF10.DCIS.com cells. Bars, 1 μm.

(j) CTR and RAB2A MCF10A cells were transfected with Str-KDEL_SBP-EGFP-E-cadherin (green). At the indicated time points after the addition of biotin, cells were fixed, and stained without permeabilization with anti-GFP antibody, followed by Alexa568-conjugated secondary antibody (red). The cell surface and total levels of SBP-EGFP-E-cadherin at 60 min after the addition of biotin were quantified using ImageJ software on non-saturated images, and surface/total ratio was expressed as a surface GFP index (relative to control cells). Bar, 20 µm. ***, P < 0.001.