Journal: The FASEB Journal

Article Title: Integrated analysis of the tumor microenvironment using a reconfigurable microfluidic cell culture platform

doi: 10.1096/fj.202200684RR

Figure Lengend Snippet: Cytotoxicity assays on cell lines in Stacks. (A) Workflow for cytotoxicity assays on cell lines. (B) Dose titration curve of 22rv1 prostate cancer cells treated with docetaxel for 48 h followed by analysis of viable cell number using quantification of Calcein AM staining with a microplate reader. Data expressed as percent of viable cells as compared to control condition ( n = 3). (C) Quantification of caspase‐3 activity in DU145 prostate cancer cells treated with 20 nM docetaxel for 48 h. Data expressed as fold change of caspase‐3 activity in docetaxel treated cells as compared to control condition (n = 3). (D) The H358 lung cancer cell line expressing the KRAS G12C oncogene was cultured in Stacks and treated with vehicle or a KRAS G12C inhibitor ( n = 2). Cells were stained with Hoechst (blue), anti‐EpCAM‐Alexa647 antibody (purple) and Image‐IT Dead™ reagent (green), and imaged in‐device using confocal fluorescence microscopy. (E) Dead cells and total number of cells was quantified with automated image analysis and the percentage of dead cells was significantly induced by inhibition of KRAS G12C in H358 lung cancer cells. Each data point represents percentage of Image‐IT Dead™‐positive cells in one microwell; * p < .05.

Article Snippet: The H358 lung cancer cell line expressing the KRAS G12C alteration was seeded on a collagen‐fibronectin matrix in Stacks and treated with 100 nM of adagrasib, a KRAS G12C small molecule inhibitor (Selleck Chemicals, Houston, TX, USA) or with DMSO for 48 h in serum‐free RPMI media.

Techniques: Titration, Staining, Control, Activity Assay, Expressing, Cell Culture, Fluorescence, Microscopy, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

doi: 10.1111/jcmm.13582

Figure Lengend Snippet: Downstream gene and metastatic regulator expression changes in H2030‐BrM3 cells after YAP knockdown. A, YAP knockdown by siRNA and shRNA decreased YAP protein expression in H2030‐BrM3 cells. B, C, YAP mRNA and mRNA expression of Hippo downstream genes CTGF and CYR61 significantly decreased in YAP shRNA‐transfected H2030‐BrM3 cells. D, Immunofluorescence stain assay showed that YAP staining decreased in YAP shRNA#1‐transfected H2030‐BrM3 cells. E, YAP knockdown by siRNA and shRNA decreased serpin I1 protein expression in H2030‐BrM3 cells. F, YAP knockdown by shRNA and siRNA significantly decreased serpin I1 mRNA expression in H2030‐BrM3 cells (error bars indicate standard deviations; * P < .05 and *** P ≤ .001)

Article Snippet: EGFR small interfering RNA (siRNA) and K‐ras siRNA were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, Knockdown, shRNA, Transfection, Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

doi: 10.1111/jcmm.13582

Figure Lengend Snippet: Analysis of cell migration and invasion abilities of H2030‐BrM3 cells after inhibiting YAP by YAP shRNA. A, Decrease in cell migration ability in H2030‐BrM3 cells after YAP knockdown by YAP shRNA#1. B, Quantitative analysis of migration assay result, indicating YAP knockdown by YAP shRNA#1 decreased cell migration ability in H2030‐BrM3 cells. C, Decrease in cell invasion ability in H2030‐BrM3 cells after YAP knockdown by YAP shRNA#1. D, Quantitative analysis of transwell invasion assay result, indicating YAP knockdown by YAP shRNA#1 decreased cell invasion ability in H2030‐BrM3 cells. E, Decrease in cell invasion ability in H2030‐BrM3 cells after YAP knockdown by YAP shRNA#2. F, Quantitative analysis of transwell invasion assay result, indicating YAP knockdown by YAP shRNA#2 decreased cell invasion ability in H2030‐BrM3 cells (error bars indicate standard deviations; ** P ≤ .01)

Article Snippet: EGFR small interfering RNA (siRNA) and K‐ras siRNA were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Migration, shRNA, Knockdown, Transwell Invasion Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

doi: 10.1111/jcmm.13582

Figure Lengend Snippet: Inhibition of YAP by shRNA suppressed metastatic ability of H2030‐BrM3 cells in a murine model (control shRNA n = 10, YAP shRNA#1 n = 5, YAP shRNA#2 n = 5). A, Bioluminescence images of mice inoculated with control shRNA‐transfected H2030‐BrM3 cells, YAP shRNA#1‐transfected and YAP shRNA#2‐transfected H2030‐BrM3 cells. B, Tumour metastasis burden in control mice and mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells based on photon flux (photons per second) (*** P ≤ .001). C, Kaplan‐Meier survival curves for control mice and mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells. D, Summary of the hypothetical model of our study. In H2030‐BrM3 cells, activation of YAP occurs at transcription mRNA level and increases YAP protein expression and mRNA expression of downstream genes CTGF and CYR61. CTGF/CYR61 forms an autocrine loop to activate the MAPK signalling pathway in H2030‐BrM3 cells. The enhancement of the MAPK signalling pathway increases the invasion and migration abilities and metastatic potential of H2030‐BrM3 cells

Article Snippet: EGFR small interfering RNA (siRNA) and K‐ras siRNA were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Inhibition, shRNA, Control, Transfection, Activation Assay, Expressing, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

doi: 10.1111/jcmm.13582

Figure Lengend Snippet: Histological analysis of mice brain tissue. A, H&E stain of brain tissues from control mice and mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells. B, YAP IHC stain of brain tissues from control mice and mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells. The scale bar = 100 μm

Article Snippet: EGFR small interfering RNA (siRNA) and K‐ras siRNA were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Staining, Control, shRNA, Transfection