Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 2. Effects of integrin β4 knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co-culture system for 24 h. Then, the levels of integrin β4 in the VECs were examined using the immunofluorescence assay. The bar graph shows the relative intensity of integrin β4 in the single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of integrin β4 in VECs. The levels of integrin β4 were determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L integrin β4-specific siRNA or with scramble siRNA for 48 h. After the addition of SPC, the effects of the integrin β4 knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed by the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as the ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 3. Effects of Fyn knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co- culture system for 24 h. Then, the levels of Fyn in the VECs were examined using the immunofluorescence assay. The bar shows the relative intensity of Fyn in single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of Fyn in VECs. The value of Fyn was determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), the cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L Fyn-specific siRNA or with scramble siRNA for 48 h. After the SPC was added, the effects of the Fyn knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed using the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as a ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 4. Effects of integrin β4 knockdown on the changes in Fyn levels in VECs. (A) VECs were treated with 10–40 nmol/L integrin β4 siRNA or scramble siRNA for 48 h. The levels of integrin β4 in the VECs were determined by the immunofluorescence assay, and the bar graph shows the relative fluorescent intensity of integrin β4 per cell as determined by laser scanning confocal microscopy (bP<0.05 vs scramble ctrl, n=3). (B) After 10–40 nmol/L integrin β4 siRNA or scramble siRNA were treated for 48 h, the Fyn levels in the VECs were assessed using western blotting (cP<0.01 vs scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Immunofluorescence, Confocal Microscopy, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 5. Effects of integrin β4 knockdown on NO production in VECs. (A) VECs were cultured under normal conditions or with siRNA for 48 h and stimulated with SPC for 0, 3, 15, or 30 min, and the supernatant was used for the NO level assay using the NO Detection Kit. In the control group (ctrl), the cells were cultured in M199 medium with 0.3% (v/v) ethanol instead of SPC. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA in the presence of SPC. The bar graph shows the changes in NO levels in VECs that had been treated with SPC (bP<0.05 vs ctrl at 3 min, eP<0.05 vs ctrl at 15 min, n=5) or with siRNA in the presence of SPC (hP<0.05 vs scramble ctrl at 3 min, kP<0.05 vs scramble ctrl at 15 min, n=5). (B) The viabilities of VECs were measured by MTT, and no significant changes were observed (n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 6. Effects of integrin β4 knockdown in VECs on changes in ROCK levels in co-cultured VSMCs. VSMCs that had been co-cultured with VECs that were treated with or without integrin β4-specific siRNA (β4) or scramble control siRNA in the presence of SPC were immunostained for ROCK, α-SMA, or both. The bar graph shows the relative fluorescence intensity of ROCK per VSMC as determined by laser scanning confocal microscopy (bP<0.05 vs SPC-non-stimulated VSMCs; eP<0.05 vs SPC- stimulated scramble ctrl in co-cultured system, n=4).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Fluorescence, Confocal Microscopy

Journal: Nature Communications

Article Title: Priming mobilization of hair follicle stem cells triggers permanent loss of regeneration after alkylating chemotherapy

doi: 10.1038/s41467-019-11665-0

Figure Lengend Snippet: Bu-induced proliferation followed by Cy-induced apoptosis in organ-cultured HFs. a Schedule for Bu/– and Bu/Cy treatments and subsequent analysis at 2, 4, and 6 days after alkylating chemotherapy. b Hair shaft elongation and c , hair cycle score of organ-cultured human HFs after alkylating chemotherapy ( n = 38 biological replicates/timepoint, 3 independent experiments). Note that the score of the Bu/Cy group was lower than that of the control group on day 6. d Representative images of organ-cultured HFs of experimental groups. Control HFs maintained anagen morphology, and Bu-treated HFs prematurely entered catagen stage (black arrow); however, Bu/Cy-treated HFs showed completely shrunken bulb morphology (black arrowhead). Representative images and quantification of e , Ki67 + cells among K15 + HFSCs ( n = 5 biological replicates/group), f p53 + cells among integrin β4 + basal cells ( n = 6 biological replicates/group), and g , p53 + c-caspase-3 + cells ( n = 7 biological replicates/group) in the bulge after alkylating chemotherapy. HFSCs showed remarkable cellular proliferation after Bu treatment (white arrowhead) and were completely quenched with large-scale apoptosis after Bu/Cy treatment (white arrow; immunofluorescence; scale bar = 100 μm). Bu busulfan, Cy cyclophosphamide, Bu/Cy busulfan followed by cyclophosphamide, HF hair follicle, HPF high-power field, HFSC hair follicle stem cell, c-caspase-3 cleaved caspase-3. Data are mean ± SEM. Source data are provided as a Source Data file. * p < 0.05 (vs. Con); *** p < 0.001 (vs. Con, two-way analysis of variance with Dunnett’s multiple comparisons test) in b and c ; *** p < 0.001 (vs. Con, unpaired t test) in e , f , and g

Article Snippet: For immunofluorescence, frozen tissues were sectioned at a thickness of 4 μm and incubated overnight at 4 °C with the following primary antibodies diluted in antibody diluent reagent (Invitrogen): Fas (ab82419, 1:100; Abcam, MA, USA), Ki67 (SP6, 1:200; Spring Bioscience, Pleasanton, CA, USA or MIB-1, 1:200; Dako), K15 (LHK15, 1:200; Thermo Fisher), p53 (DO-7, 1:800; Novocastra, Newcastle, England or 1C12, 1:1000; Cell Signaling, Danvers, MA, USA), K14 (GTX104124, 1:1000; GeneTex, Irvine, CA, USA), cleaved caspase-3 (5A1E, 1:400; Cell Signaling), γ-H2AX (#2577, 1:800; Cell Signaling), Shh (EP1190Y, 1:500; Abcam), Lhx2 (C-20, 1:200; Santa Cruz Biotechnology), integrin β4 (H-101, 1:200; Santa Cruz Biotechnology), phospho Akt (#9275, 1:800; Cell Signaling), phospho p38 (#9211, 1:800; Cell Signaling), TYR (T311, 1:100; Thermo Fisher, Waltham, MA, USA), TRP1 (SC25543, 1:100; Santa Cruz Biotechnology), and MITF (C5, 1:100; Thermo Fisher).

Techniques: Cell Culture, Control, Immunofluorescence

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 1. Increased expression of ITGB4 in KRAS-mutant colorectal cancer (CRC). A, The expression profile of ITGB4 mRNA across cancer types in TCGA PanCancer Atlas studies. B, ITGB4 mRNA expression in colorectal cancer by KRAS status in TCGA PanCancer Atlas studies. C, Western blot analysis of ITGB4 protein in human colorectal cancer cell lines. Caco-2 cells (5 105 cells/100 mm dish) and HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Data are representative of three independent experiments. D, Quantitative RT-PCR analysis of Itgb4 mRNA expression in mouse colonic organoids. Bar graph shows the mean SD of four independent experiments. E, Western blot analysis of ITGB4 protein in mouse colonic organoids. Data are representative of three independent experiments. D and E, Mouse colonic organoids (at density of 3 103 cells/50 mL of Matrigel/24 well) were cultured for 5 days. F, IF staining and quantification of ITGB4 and E-cadherin– positive cells in human colorectal cancer tissues. Each dot represents 1 patient with colorectal cancer. Bar graphs show the mean SD for each group (KRAS-wt, n ¼ 24; KRAS-mt, n ¼ 14). Scale bars, 100 mm. D and F, Student t test. , P < 0.05; ns, not significant.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Expressing, Mutagenesis, Western Blot, Cell Culture, Quantitative RT-PCR, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 2. Mutant KRAS increases ITGB4 and ITGA6 expression in HCT-116 cells through the MEK/ERK signaling pathway. Western blot analysis (A) and quantitative RT-PCR analysis (B–E) of ITGB4 and ITGA6 expression in HCT-116 cells treated with MEK kinase inhibitor (PD0325901, 10 mmol/L) or PI3K inhibitor (LY294002, 10 mmol/L). HCT-116 cells (1 106 cells/100 mm dish) were cultured for 2 days and treated with the inhibitors for 24 hours. A, Data are representative of three independent experiments. B–E, Bar graphs show the mean SD of three independent experiments. Student t test. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Mutagenesis, Expressing, Western Blot, Quantitative RT-PCR, Cell Culture

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 3. ITGB4 is necessary for the stability of ITGA6 protein in HCT-116 cells. Western blot analysis (A) and quantification (B) of total ITGA6 protein in KRAS-wt and KRAS-mt Caco-2 cells with or without ITGB4 o/e. B, Bar graphs show the mean SD of three independent experiments. C, Quantitative RT-PCR analysis of ITGA6 mRNA expression in Caco-2 cells. Bar graph shows the mean SD of three independent experiments. A–C, Caco-2 cells (5 105 cells/ 100 mm dish) were cultured for 3 days. D, Western blot analysis of total ITGA6 protein and two splice isoforms (ITGA6A and ITGA6B) in KRAS-wt and KRAS-mt HCT-116 cells with or without ITGB4-KO. Quantification of total ITGA6 protein (E), splice isoform ITGA6A protein (F), and splice isoform ITGA6B protein (G) in HCT-116 cells using Western blot analysis in D. Bar graphs show the mean SD of three independent experiments. H, Quantitative RT-PCR analysis of ITGA6 mRNA expression in HCT-116 cells. Bar graph shows the mean SD of three independent experiments. D–H, HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. I, Western blot analysis of ITGA6 protein stability in KRAS-mt HCT-116 cells with or without ITGB4-KO. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 2 days and treated with protein synthesis inhibitor, CHX (50 mg/mL), and harvested at indicated timepoints. Graph shows the mean SD of four independent experiments. Each ITGA6 protein level was normalized to respective untreated control (0 hour). Multiple t test. , P < 0.05. J, Co-immunoprecipitation of ITGB4 and ITGB1 with anti-ITGA6 antibody from HCT-116 cells. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Representative of three independent experiments. Whole-cell lysates were used as input control. B, C, and E–H, One-way ANOVA. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; ns, not significant; o/e, overexpression.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Control, Immunoprecipitation, Over Expression

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 4. KO of ITGB4 does not affect the proliferation rate and the tumor growth of KRAS-mutant HCT-116 cells and mouse colonic organoids. A, Western blot analysis of ERK1/2 and AKT phosphorylation in ITGB4 KO HCT-116 cells. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Data are representative of three independent experiments. Proliferation rates of HCT-116 cells (B), mouse organoids (C), and Caco-2 (D) were determined at day3 by MTT, WST-1, and MTT, respectively. Bar graphs show the mean SD of three independent experiments. MTT or WST-1 values at day 3 were normalized to day 0 of each cell line. Student t test. , P < 0.05; ns, not significant. E and F, HCT-116 cells were injected into the both flanks of nude mice subcutaneously. Tumor growths were measured on indicated days. E, Growth of HCT-116 KRAS-mt tumors with or without ITGB4-KO. Graph shows the mean SD for each group (HCT-116 KRAS-mt, n ¼ 10; HCT-116 KRAS-mt/ITGB4-KO, n ¼ 10 tumors from each 5 mice). F, Representative H&E staining images of tumor tissues. Scale bars, 50 mm. G and H, Mouse organoids were injected into both flanks of C57BL/6 mice subcutaneously. Tumor growths were measured on indicated days. G, Growth of APK and APK/ITGB4-KO in C57BL/6 mice. Graph shows the mean SD for each group (APK, n ¼ 8 tumors from 4 mice; APK/ITGB4-KO, n ¼ 9 tumors from 5 mice). H, Representative H&E staining images of tumor tissues. Scale bars, 50 mm.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Mutagenesis, Western Blot, Phospho-proteomics, Cell Culture, Injection, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 5. KO of ITGB4 decreases migration and invasion of KRAS mutant HCT-116 cells in vitro. Transwell migration assay (A) and transwell invasion assay (B) of HCT116 cells. HCT-116 cells were seeded on the transwell membrane without A or with B matrigel coating. After 24 hours, cells on the opposite surface of the transwell membrane were fixed, stained, and quantified. Bar graphs show the mean SD of four independent experiments. Representative images are shown below. Scale bars, 100 mm. C, 3D invasion assay of HCT-116 cells in organotypic culture system. HCT-116 cells were seeded onto 3D gels composed of collagen, matrigel, and fibroblasts. After 10–12 days, gels were fixed and paraffin embedded, and cells invading into the gel were stained and quantified. Representative images of IF staining of E-cadherin, ITGB4, and Vimentin are shown. Scale bars, 100 mm. Quantification of maximum invasion depth (D), total invasion area (E), and total invaded cell numbers (F) of 3D invasion assay in C. Bar graphs show the mean SD of five independent experiments. A, B, and D–F, Student t test. , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Migration, Mutagenesis, In Vitro, Transwell Migration Assay, Transwell Invasion Assay, Membrane, Staining, Invasion Assay

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 6. KO of ITGB4 decreases orthotopic implantation rate of APK organoids. A–D, APK and APK/ITGB4-KO organoids were transplanted into the lumen of C57BL/6 mouse rectum. Tumor growth was assessed by endoscopy at 2 and 4 weeks after transplantation, and rectal tissues were collected after second endoscopy (APK, n ¼ 15; APK/ITGB4-KO, n ¼ 14). A, Schematic illustration of endoluminal tumor model. B, Representative endoscopic images and H&E staining of rectums with adenocarcinoma. Scale bar: 200 and 50 mm. C, Graph shows the percentage of tumor burden at 4 weeks after transplantation. Fisher exact test. P ¼ 0.0421. D, Graph shows the percentage of tumor size within lumen. Mean SEM for each group (APK, n ¼ 5; APK/ITGB4-KO, n ¼ 14).

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Transplantation Assay, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 7. KO of ITGB4 decreases number of pulmonary metastatic foci of APK organoids. A–E, APK-LM and APK-LM/ITGB4-KO organoids derived from liver metastasis were transplanted into NSG mice via tail vein. Lung and liver tissues were collected at 5 weeks after transplantation. A, Schematic illustration of lung metastatic tumor model. B, Representative images of H&E staining and IF staining of lung tissues. GFP is a marker of transplanted organoids. Scale bar: 2000, 200, and 50 mm. C, Quantification of metastatic foci and metastatic area in B are shown. D, Representative images of H&E staining of liver. Scale bar: 2,000, 500, and 50 mm. E, Quantification of metastatic foci and metastatic area in D are shown. C and E, Each dot in the individual graphs represents one mouse. Bar graphs show the mean SD for each group (APK-LM, n ¼ 11; APK-LM/ITGB4-KO, n ¼ 11). Student t test. , P < 0.01; ns, not significant.

Article Snippet: Organoids were transfected with ITGB4 [CRISPR/Cas9 KO plasmid (m)] (Santa Cruz Biotechnology, sc-431434) and ITGB4HDRPlasmid (m) (Santa Cruz Biotechnology, sc-431434-HDR) using OMNIfect Transfection Reagent (TransOMIC, OTR1001) with OPTI-MEM media according to the manufacturer’s instruction.

Techniques: Derivative Assay, Transplantation Assay, Staining, Marker

Journal: Cell Stem Cell

Article Title: Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming

doi: 10.1016/j.stem.2017.10.013

Figure Lengend Snippet:

Article Snippet: The following antibodies were used with indicated dilution: ICAM1-biotin conjugate (1:100, eBioscience, #13-0541), CD44-allophycocyanin (APC) conjugate (1:300, eBioscience, #17-0441), E-CADHERIN-eFluor 660 (1:300, eBioscience, #50-3249-82), MEFSK4-biotin conjugate (1:100, Miltenyi, 130-101-875), CD47-biotin conjugate (1:100, BioLegend, 127505), CD73-Alexa Fluor 647 (1:300, BD Biosciences, 561543) and CD104-APC (1:300, Miltenyi, 130-106-924) and Strepdavidin PE-Cy7 (1:1500, eBioscience, #25-4317).

Techniques: Recombinant, Software

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 1. Increased expression of ITGB4 in KRAS-mutant colorectal cancer (CRC). A, The expression profile of ITGB4 mRNA across cancer types in TCGA PanCancer Atlas studies. B, ITGB4 mRNA expression in colorectal cancer by KRAS status in TCGA PanCancer Atlas studies. C, Western blot analysis of ITGB4 protein in human colorectal cancer cell lines. Caco-2 cells (5 105 cells/100 mm dish) and HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Data are representative of three independent experiments. D, Quantitative RT-PCR analysis of Itgb4 mRNA expression in mouse colonic organoids. Bar graph shows the mean SD of four independent experiments. E, Western blot analysis of ITGB4 protein in mouse colonic organoids. Data are representative of three independent experiments. D and E, Mouse colonic organoids (at density of 3 103 cells/50 mL of Matrigel/24 well) were cultured for 5 days. F, IF staining and quantification of ITGB4 and E-cadherin– positive cells in human colorectal cancer tissues. Each dot represents 1 patient with colorectal cancer. Bar graphs show the mean SD for each group (KRAS-wt, n ¼ 24; KRAS-mt, n ¼ 14). Scale bars, 100 mm. D and F, Student t test. , P < 0.05; ns, not significant.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Expressing, Mutagenesis, Western Blot, Cell Culture, Quantitative RT-PCR, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 2. Mutant KRAS increases ITGB4 and ITGA6 expression in HCT-116 cells through the MEK/ERK signaling pathway. Western blot analysis (A) and quantitative RT-PCR analysis (B–E) of ITGB4 and ITGA6 expression in HCT-116 cells treated with MEK kinase inhibitor (PD0325901, 10 mmol/L) or PI3K inhibitor (LY294002, 10 mmol/L). HCT-116 cells (1 106 cells/100 mm dish) were cultured for 2 days and treated with the inhibitors for 24 hours. A, Data are representative of three independent experiments. B–E, Bar graphs show the mean SD of three independent experiments. Student t test. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Mutagenesis, Expressing, Western Blot, Quantitative RT-PCR, Cell Culture

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 3. ITGB4 is necessary for the stability of ITGA6 protein in HCT-116 cells. Western blot analysis (A) and quantification (B) of total ITGA6 protein in KRAS-wt and KRAS-mt Caco-2 cells with or without ITGB4 o/e. B, Bar graphs show the mean SD of three independent experiments. C, Quantitative RT-PCR analysis of ITGA6 mRNA expression in Caco-2 cells. Bar graph shows the mean SD of three independent experiments. A–C, Caco-2 cells (5 105 cells/ 100 mm dish) were cultured for 3 days. D, Western blot analysis of total ITGA6 protein and two splice isoforms (ITGA6A and ITGA6B) in KRAS-wt and KRAS-mt HCT-116 cells with or without ITGB4-KO. Quantification of total ITGA6 protein (E), splice isoform ITGA6A protein (F), and splice isoform ITGA6B protein (G) in HCT-116 cells using Western blot analysis in D. Bar graphs show the mean SD of three independent experiments. H, Quantitative RT-PCR analysis of ITGA6 mRNA expression in HCT-116 cells. Bar graph shows the mean SD of three independent experiments. D–H, HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. I, Western blot analysis of ITGA6 protein stability in KRAS-mt HCT-116 cells with or without ITGB4-KO. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 2 days and treated with protein synthesis inhibitor, CHX (50 mg/mL), and harvested at indicated timepoints. Graph shows the mean SD of four independent experiments. Each ITGA6 protein level was normalized to respective untreated control (0 hour). Multiple t test. , P < 0.05. J, Co-immunoprecipitation of ITGB4 and ITGB1 with anti-ITGA6 antibody from HCT-116 cells. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Representative of three independent experiments. Whole-cell lysates were used as input control. B, C, and E–H, One-way ANOVA. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; ns, not significant; o/e, overexpression.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Control, Immunoprecipitation, Over Expression

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 4. KO of ITGB4 does not affect the proliferation rate and the tumor growth of KRAS-mutant HCT-116 cells and mouse colonic organoids. A, Western blot analysis of ERK1/2 and AKT phosphorylation in ITGB4 KO HCT-116 cells. HCT-116 cells (1 106 cells/100 mm dish) were cultured for 3 days. Data are representative of three independent experiments. Proliferation rates of HCT-116 cells (B), mouse organoids (C), and Caco-2 (D) were determined at day3 by MTT, WST-1, and MTT, respectively. Bar graphs show the mean SD of three independent experiments. MTT or WST-1 values at day 3 were normalized to day 0 of each cell line. Student t test. , P < 0.05; ns, not significant. E and F, HCT-116 cells were injected into the both flanks of nude mice subcutaneously. Tumor growths were measured on indicated days. E, Growth of HCT-116 KRAS-mt tumors with or without ITGB4-KO. Graph shows the mean SD for each group (HCT-116 KRAS-mt, n ¼ 10; HCT-116 KRAS-mt/ITGB4-KO, n ¼ 10 tumors from each 5 mice). F, Representative H&E staining images of tumor tissues. Scale bars, 50 mm. G and H, Mouse organoids were injected into both flanks of C57BL/6 mice subcutaneously. Tumor growths were measured on indicated days. G, Growth of APK and APK/ITGB4-KO in C57BL/6 mice. Graph shows the mean SD for each group (APK, n ¼ 8 tumors from 4 mice; APK/ITGB4-KO, n ¼ 9 tumors from 5 mice). H, Representative H&E staining images of tumor tissues. Scale bars, 50 mm.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Mutagenesis, Western Blot, Phospho-proteomics, Cell Culture, Injection, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 5. KO of ITGB4 decreases migration and invasion of KRAS mutant HCT-116 cells in vitro. Transwell migration assay (A) and transwell invasion assay (B) of HCT116 cells. HCT-116 cells were seeded on the transwell membrane without A or with B matrigel coating. After 24 hours, cells on the opposite surface of the transwell membrane were fixed, stained, and quantified. Bar graphs show the mean SD of four independent experiments. Representative images are shown below. Scale bars, 100 mm. C, 3D invasion assay of HCT-116 cells in organotypic culture system. HCT-116 cells were seeded onto 3D gels composed of collagen, matrigel, and fibroblasts. After 10–12 days, gels were fixed and paraffin embedded, and cells invading into the gel were stained and quantified. Representative images of IF staining of E-cadherin, ITGB4, and Vimentin are shown. Scale bars, 100 mm. Quantification of maximum invasion depth (D), total invasion area (E), and total invaded cell numbers (F) of 3D invasion assay in C. Bar graphs show the mean SD of five independent experiments. A, B, and D–F, Student t test. , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Migration, Mutagenesis, In Vitro, Transwell Migration Assay, Transwell Invasion Assay, Membrane, Staining, Invasion Assay

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 6. KO of ITGB4 decreases orthotopic implantation rate of APK organoids. A–D, APK and APK/ITGB4-KO organoids were transplanted into the lumen of C57BL/6 mouse rectum. Tumor growth was assessed by endoscopy at 2 and 4 weeks after transplantation, and rectal tissues were collected after second endoscopy (APK, n ¼ 15; APK/ITGB4-KO, n ¼ 14). A, Schematic illustration of endoluminal tumor model. B, Representative endoscopic images and H&E staining of rectums with adenocarcinoma. Scale bar: 200 and 50 mm. C, Graph shows the percentage of tumor burden at 4 weeks after transplantation. Fisher exact test. P ¼ 0.0421. D, Graph shows the percentage of tumor size within lumen. Mean SEM for each group (APK, n ¼ 5; APK/ITGB4-KO, n ¼ 14).

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Transplantation Assay, Staining

Journal: Molecular Cancer Research

Article Title: KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-21-0994

Figure Lengend Snippet: Figure 7. KO of ITGB4 decreases number of pulmonary metastatic foci of APK organoids. A–E, APK-LM and APK-LM/ITGB4-KO organoids derived from liver metastasis were transplanted into NSG mice via tail vein. Lung and liver tissues were collected at 5 weeks after transplantation. A, Schematic illustration of lung metastatic tumor model. B, Representative images of H&E staining and IF staining of lung tissues. GFP is a marker of transplanted organoids. Scale bar: 2000, 200, and 50 mm. C, Quantification of metastatic foci and metastatic area in B are shown. D, Representative images of H&E staining of liver. Scale bar: 2,000, 500, and 50 mm. E, Quantification of metastatic foci and metastatic area in D are shown. C and E, Each dot in the individual graphs represents one mouse. Bar graphs show the mean SD for each group (APK-LM, n ¼ 11; APK-LM/ITGB4-KO, n ¼ 11). Student t test. , P < 0.01; ns, not significant.

Article Snippet: To generate ITGB4-overexpressing cells, Caco-2 cells were transduced with ITGB4 Lentiviral Activation Particle (h; Santa Cruz Biotechnology, sc400573-LAC) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Transplantation Assay, Staining, Marker