Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 1. Kras secreted tumor exosomes reveal regulation of SMARCE1/NCOR1 chromatin remodeling genes. (a) Nanoparticle tracking analysis was performed for isolated cir- culating Kras exosomes from metastatic lung cancer patients. The size distribution and relative concentration were calculated by Nanosight software (n = 3). (b) Exosome characterization from Kras (MT/WT) patients was performed using Transmition Electron Microscopy from patient biopsies. (c) Immunoblotting of exosomal-related proteins CD63, and HSPA8. (d). Uptake of PKH67-labeled exosomes by LC/DR cells. Confocal microscopy image of PKH67-labeled cells (green) and exosomes (red). Images were captured using Carl Zeiss fluorescence confocal microscope (Scale bar, 100 nm). (e) Exosomal concentration of Kras (MT/WT) in LC-DR cells. (f–g) The protein expression levels of ARID1A, CHD4, NCOR1 and SMARCE1 chromatin remodeling genes were assayed by Western blot. LC-DR cells were trans- fected with siRNA against Kras (100 nM) and then treated with GW-4869 (25 μM) for 24 h. Densitometric analysis of each pro- tein level was calculated from the average of three experiments. Each value was expressed as the ratio of the measured protein to GAPDH level (p < 0.001). The results represent the mean ± SD of three independent experiments. Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks (*P < 0.05, **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Isolation, Concentration Assay, Software, Electron Microscopy, Western Blot, Labeling, Confocal Microscopy, Microscopy, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 3. Kras-derived exosomes prompt tumor growth and guide lymph node metastasis in mice model. (a) Representative images of colony formation assay ofcells (magnification × 200). (b) Quantitative analysis of the colony formation rates. SiKRAS and GW-4869 co-treatment significantly inhibited the colony-forming ability of LC-DR cells following 24 h treatment (Scale bar, 20 μm). (c-d) Effect of siKRAS tranfection and exosomal inhibition on cell migration using transwell migration assay. Ex vivo cultured cells tranfected with siRNA against Kras (100 nM, 48 h) then treated with GW-4869 (25 μM) for 24 h and subjected to migration assay to determine cellular migration (magnification × 200) (Scale bar, μm). (e–f) Effects of GW-4869-treatment on LC-DR-luciferase cell-derived orthotopic xenograft tumors in athymic nude mice. Luciferase labeled LC-DR cells (2 × 106) were implanted in athymic nude mice. GW-4869 (2 mg/kg body weight) and vehicle treatment was started 3 days post-implantation. There were eight mice per group. Bioluminescence representative of anesthetized mice (wk 8) from control and GW- 4869-treated groups. The bioluminescence values (photons/sec/cm2/sr) of the lung region were quantified for each group of mice and mean values ± SE were plotted. Mice from both groups were sacrificed at week 8, their lungs and lymph nodes were excised and imaged immediately. Bar graph represents the biolumi- nescence values (photons/sec/cm2/sr) of the excised lungs from control and GW-4869-treated mice. Each value in the graph is the mean ± SE from eight mice. *p < 0.05 was considered as significant. (g–h) In vivo metastatic analysis of lymph node metastasis from control and GW-4869-treated groups. Images showed representative lymph node metastatic foci highlighted in yellow from different groups. Statistical analysis of the number of metastatic foci of each group. (i-g) Representative photographs of excised tumors from mice after treatment with si-KRAS and GW-4869. Tumor growth curve of mice treated in the different groups. (k) Survival rates of tumor-bearing mice after a 60-day tumor challenge in each group. Data were given as the mean ± SD (n = 6). (*P < 0.05, **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Derivative Assay, Colony Assay, Inhibition, Migration, Transwell Migration Assay, Ex Vivo, Cell Culture, Luciferase, Labeling, Control, In Vivo

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 5. Blockage of Kras exosomal pathway triggers carboplatin related RIP3/TNFa dependent necroptosis. (a–b) Protein expression levels of necroptosis markers RIP-1 and RIP-3 were evaluated by Western blot. LC-DR cells were co-treated with siRNA against Kras (100 nM) and then treated wih GW-4869 (25 μM) for 24 h. Densitometric analysis of each protein level was calculated from the average of three experiments. (c–d) Representative scatter plots of flow cytometry analysis for the fraction of necrotic LC-DR Kras (MT) cells following treatment with siKRAS/GW-4869 for 24 h. The cells distributed in the upper left quadrant were the PI- positive cells and considered the necrotic cell fraction. (d) Quantitative analysis of apoptotic and necrotic rates; (*P < 0.05, **P < 0.01). (e) Mitochondrial ROS generation of treated LC-DR Kras (MT) cells stained with MitoSox Red (5 μM) and analyzed by flow cytometry. (f–g) Immunofluorescence analysis of TNF-a ex- pression in LC-DR cells(magnification × 200). Ex vivo cultured cells were treated with siKras (100 nM, 48 h) and GW-4869 (25 μM) for 24 h. Data represent the mean ± SD of three separate experiments. (*P < 0.05; **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Expressing, Western Blot, Cytometry, Staining, Ex Vivo, Cell Culture

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