Journal: BMC Gastroenterology

Article Title: Baseline levels of dynamic CD4 + T cell adhesion to MAdCAM-1 correlate with clinical response to vedolizumab treatment in ulcerative colitis: a cohort study

doi: 10.1186/s12876-020-01253-8

Figure Lengend Snippet: Dynamic expression of α4β1 integrin on CD4 + T cells in the peripheral blood of patients with UC treated with vedolizumab. a Timeline of vedolizumab treatment (T1–5) with representative flow cytometry and clinical data. Flow cytometry of integrin expression on CD4 + T-cells in the peripheral blood was performed before T1 and T3. Clinical response was determined at T5. Representative flow cytometric and clinical data from a responder to vedolizumab (upper panels) and a non-responder to vedolizumab (lower panels) are shown. b Left panel: Difference in the expression of α4β1 integrin on CD4 + T cells in the peripheral blood between week 0 (T0) and week 6 (T3) of vedolizumab treatment as determined by flow cytometry ( n = 26; 18 responders, 8 non-responders). Statistical comparison with two-tailed Mann-Whitney test. Right panel: Receiver-operator-characteristic (ROC) analysis for prediction of clinical response to vedolizumab treatment by changes in α4β1 expression between week 0 and week 6. Significance level (left) and p -value (right) are indicated. MCS – Mayo clinical subscore

Article Snippet: For the analysis of α4β1 expression on CD4 + T cells from the peripheral blood, PBMCs were isolated, stained with antibodies against CD4 (VioBlue, VIT4; Miltenyi Biotec), α4 integrin (FITC, MZ18-24A9; Miltenyi Biotec), β1 integrin (AF647, TS2/16; Biolegend), and fixed with the FoxP3/Transcription Factor Staining Buffer Set (eBioscience).

Techniques: Expressing, Flow Cytometry, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Oncogenesis

Article Title: Sec62 promotes early recurrence of hepatocellular carcinoma through activating integrinα/CAV1 signalling

doi: 10.1038/s41389-019-0183-6

Figure Lengend Snippet: a Left: Functional pathway analysis of the differentially expressed genes was conducted using commercially available IPA software. The change in integrin signalling in both Huh7-Sec62-shRNA cells and Huh7-Sec62-overexpressing cells is consistent with the expression of Sec62. The colour indicates the degree of down- (blue) or up- (orange) regulation following Sec62 knockdown or overexpression in Huh7 cells. Upper right: Cellular movement regulated by Sec62 was identified in both Huh7-Sec62-RNAi cells and Huh7-Sec62-overexpressing cells by GO functional analysis. Lower right: Interaction of Sec62 and integrin signalling after Sec 62 knockdown in Huh7 cells, which was determined based on the integrin signalling interactome in the Ingenuity IPA database overlaid with microarray data from Huh7-Sec62-RNAi cells. b Left: Integrin α5 and integrin αV were overexpressed in Sec62-knockdown (Sec62 − ) Huh 7 cells, and the healing ability of these cells was evaluated by a monolayer wounding assay. Western blot analysis of integrin α 5 and integrin αV expression in Sec62-knockdown cells. Middle: Transwell assay analysis of the migration ability of Sec62 − / integrin α5 (top) or Sec62 − / integrin αV (low) cells. The number of cells that invaded through the filter into the lower compartment was determined using a colorimetric crystal violet assay. Data are presented as the means ± SD of at least three independent experiments and were compared to the amounts of invaded cells from negative control transfected cells. * P < 0.01 versus the control group (Student’s t test). Right: Western blot of co-immunoprecipitated integrin αV (top) and integrin α5 (low) in Huh7 cells after Co-IP with an anti-Sec62 antibody. c The levels of integrin αV, integrin α 5, CAV1, calpains and MLCK expression in the integrin α/CAV1 pathway from HCC patient samples with recurrence ( n = 4) or without recurrence ( n = 4) were evaluated by Western blot analysis.

Article Snippet: Rabbit anti-integrin α2b (#13807), integrin α4 (#8440), integrin α5 (#98204), integrin αV (#60896), CAV1 (#3267), phospho-SHC (Tyr317, #2431), phospho-SHC (Tyr239/240, #2434), SHC (#2432), SOS1 (#5890), Bcar3 (#24032), Calpain 1 (#2556), Calpain 2 (#2539), antibodies were procured from Cell Signal Technology.

Techniques: Functional Assay, Software, shRNA, Expressing, Over Expression, Microarray, Western Blot, Transwell Assay, Migration, Crystal Violet Assay, Negative Control, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncogenesis

Article Title: Sec62 promotes early recurrence of hepatocellular carcinoma through activating integrinα/CAV1 signalling

doi: 10.1038/s41389-019-0183-6

Figure Lengend Snippet: Luciferase-labelled Huh7 cells with or without stable Sec62 knockdown were subcutaneously injected into the right axillary, and then, the xenografts were orthotopically implanted into the livers of nude mice. Mice underwent HCC resection on day 14 after implantation. a Left: tumour recurrences were detected after tumour resection with the IVIS system. Then, luciferase-labelled Huh7 cells with or without stable Sec62 overexpression were subcutaneously injected into the right axillary, and then, the xenografts were orthotopically implanted into the livers of nude mice. Mice underwent HCC resection on day 10 after implantation. a Right: tumour recurrences were detected after tumour resection with the IVIS system. b Quantitative fluorescence data in mice. * P < 0.01 versus the control group (Student’s t test). c Western blot analysis of Sec62, integrin αV, integrin α 5, CAV1, calpains and MLCK expression in the resected tumour is shown.

Article Snippet: Rabbit anti-integrin α2b (#13807), integrin α4 (#8440), integrin α5 (#98204), integrin αV (#60896), CAV1 (#3267), phospho-SHC (Tyr317, #2431), phospho-SHC (Tyr239/240, #2434), SHC (#2432), SOS1 (#5890), Bcar3 (#24032), Calpain 1 (#2556), Calpain 2 (#2539), antibodies were procured from Cell Signal Technology.

Techniques: Luciferase, Injection, Over Expression, Fluorescence, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Identification of Molecular Determinants of Primary and Metastatic Tumor Re-Initiation in Breast Cancer

doi: 10.1038/ncb3148

Figure Lengend Snippet: ( a ) MDA-TE3 control cells or MDA-TE3 LAMA4 -knockdown cells were sorted at clonal density (one cell per well) into low-attachment 96-well plates. LAMA4 -depletion led to reduced proliferation. n = 212 (shControl), n = 219 (shLAMA4_1), n = 224 (shLAMA4_2) wells. ( b ) MDA-parental, MDA-TE3, or MDA-LM2 cells were sorted at clonal density into low-attachment plates. MDA-TE3 and MDA-LM2 cells proliferated more extensively than MDA-parental cells. n = 152 (parental), n = 163 (TE3), n = 141 (LM2) wells. ( c ) Cell-cycle analysis of MDA-TE3 control cells or MDA-TE3 LAMA4 -knockdown cells (left). Representative DNA content histograms (right). n = 6 independent samples. ( d ) Cell-cycle analysis of MDA-parental, MDA-TE3, or MDA-LM2 cells (left). Representative DNA content histograms (right). n = 4 independent samples. ( e ) Relative p27 protein levels in MDA-TE3 control cells or MDA-TE3 LAMA4 -knockdown cells quantified by Western. p27 values normalized to β-tubulin. n = 3 independent samples. Representative blot in . ( f ) The fraction of Ki67-positive cells was assessed in MDA-TE3 control cells or MDA-TE3 LAMA4 -knockdown cells using flow-cytometry (left). Representative plots (right). n = 6 independent samples. ( g ) Incubation with β1-integrin blocking antibody suppressed the proliferation of MDA-parental cells over-expressing LAMA4 upon sorting at clonal density into low-attachment plates. Counts on day 3. n = 4 independent experiments. ( h ) The capacity of recombinant LAMA4 -containing protein (laminin-411) to rescue the proliferation of MDA-TE3 LAMA4 -knockdown cells was assessed in the presence/absence of β1-integrin blocking antibody upon sorting at clonal density into low-attachment plates. Counts on day 3. n = 3 independent experiments. ( i ) Incubation with β1-integrin blocking antibody suppressed the proliferation of MDA-TE3 cells upon sorting a clonal density into low-attachment plates. Counts on day 3. n = 3 independent experiments. ( j – k ) 1×10 3 MDA-Parental control cells or LAMA4 -over-expressing (LAMA4oe) cells were injected directly into the lung parenchyma in the presence of IgG control or β1-integrin blocking antibody to assess ectopic tumor re-initiation capacity. Lung bioluminescence was measured on day 39 ( j ). n = 7 independent mice. On day 39 lungs were sectioned, vimentin-stained, and the number of macroscopic nodules per lung was counted ( k ). n = 7 lungs from 7 independent mice. Scale bars: 1mm. Insets are magnified 5x. * P <0.05, ** P <0.01, *** P <0.001 obtained using one-sided Mann-Whitney test ( a – b , j – k ), or one sided Student’s t-test ( c – i ). Experiments ( a – f ) are representative and were replicated at least 2 times. All data are represented as mean +/± S.E.M.

Article Snippet: For immunofluorescence, paraffin sections underwent antigen retrieval and were stained with primary antibodies against human Vimentin (1:100, Vector Laboratories) and Ki-67 (1:200, ab15580, abcam), or for detection of endogenous laminin-α4, the mouse anti-human laminin-α4 antibody (1:50, clone 839084, R&D) was used and the isotype-matched mouse IgG1 antibody (1:50, clone 11711, R&D) was used as a control at an equivalent concentration.

Techniques: Cell Cycle Assay, Western Blot, Flow Cytometry, Incubation, Blocking Assay, Expressing, Recombinant, Injection, Staining, MANN-WHITNEY