Journal: Cell Death & Disease

Article Title: CXCR4 intracellular protein promotes drug resistance and tumorigenic potential by inversely regulating the expression of Death Receptor 5

doi: 10.1038/s41419-021-03730-8

Figure Lengend Snippet: A MCF7 cells were transfected with either empty vector pcDNA3.1 or vector containing the gene for the overexpression of either chemokine receptor CXCR4 or CXCR7 and made stable. These stable cell lines were stained with either APC-conjugated anti-human CXCR4 (CD184) or anti-human CXCR7 antibodies along with their respective isotype control antibodies and analyzed by flow cytometry. The surface expression levels of CXCR4 and CXCR7 are represented in histogram overlays (left panel). Western blot analysis of CXCR4 and CXCR7 in the lysate of control and CXCR4/CXCR7 overexpression MCF-7 cells; GAPDH and β-Actin were used as the protein loading control (right panel). B CXCR4 or CXCR7 overexpressing and control MCF7 cells were treated with Doxorubicin (250 nmol/L), Paclitaxel (25 nmol/L), Cisplatin (2.5 μmol/L), or 5-Fluorouracil (25 μmol/L) for 72 h and cytotoxicity was measured by SRB assay as described in materials and methods. Percent cell viability was tabulated. Columns, an average of triplicate readings of samples; error bars ± SEM. * p < 0.05, compared with control cells. C DiL (red) stained CXCR4 or CXCR7 overexpressing MCF7 cells and DiO (green) stained control MCF7 cells were mixed in equal numbers, seeded in 6-well plate and treated with vehicle or Paclitaxel (10 nmol/L) for 3 days and then analyzed via fluorescence microscopy; red arrows indicate DiL stained dead CXCR4 or CXCR7 overexpressing cells, while green arrows indicate DiO stained dead control cells. Photomicrographs are representative of three independent experiments. D DiL stained CXCR4 overexpressing or CXCR7 overexpressing MCF7 cells were equally mixed with DiO stained control MCF7 cells, and a small aliquot of mixture was acquired as day 0 reading by FACS. The rest of the cells were treated either with vehicle or Paclitaxel (10 nmol/L) for 5 days and subsequently analyzed by FACS. Data are representative of at least three independent experiments. E CXCR4 overexpressing and control MCF7 cells were treated with different concentrations of paclitaxel (12.5, 25, 50 nM) for 48 h, and cytotoxicity was evaluated by SRB assay. Percent cell viability was tabulated. Columns, an average of triplicate readings of samples; error bars ± SEM.* p < 0.05, compared to control cells treated with respective doses of paclitaxel. F Chili tagged HT-29 cells were made stable for the knockdown of CXCR4 via shRNA mediated lentiviral transduction; scramble shRNA transduced stable HT-29 cells were used as control. CXCR4 knockdown and control HT-29 cells were stained either with APC-conjugated anti-human CXCR4 (CD184) antibody or with appropriate isotype control antibody and analyzed by FACS. Histogram overlays represent the cell surface expression of CXCR4 (left panel). Western blot analysis of CXCR4 in the lysate of control and CXCR4 knockdown HT29 cells; β-Actin was used as the protein loading control (right panel). G Control and CXCR4 knockdown Chili tagged HT29 cells were mixed equally and analyzed by FACS either at day 0 or after three days of vehicle/paclitaxel (20 nmol/L) treatments. H CXCR4 knockdown and control HT-29 cells were treated with different concentrations of Paclitaxel (3.12, 6.25, 12.5, 25, 50 nM) for 48 h and cytotoxicity was measured by SRB assay. Percent cell viability was tabulated. Columns, an average of triplicate readings of samples; error bars ± SEM.* p < 0.05 compared to control cells. I Control and CXCR4 knockdown HT29 cells were treated with paclitaxel for 24 h and stained with FITC conjugated Annexin-V. Histogram overlays show the Annexin-V positive cells. J Western blot analysis of cleaved PARP and Caspase-8 in the lysate of 24 h post vehicle or Paclitaxel treated (10, 20, and 40 nM) control and CXCR4 knockdown HT-29 cells; β-Actin was used as the protein loading control. Western Blot densitometric quantification numbers are shown above the loading control blot of all immunoblot studies.

Article Snippet: 3rd generation lentiviral vector pUltra-Chili-Luc (addgene no. 48688) with the bi-cistronic expression of tdTomato and Luciferase was used to make HT29 cells fluorescent.

Techniques: Transfection, Plasmid Preparation, Over Expression, Stable Transfection, Staining, Control, Flow Cytometry, Expressing, Western Blot, Sulforhodamine B Assay, Fluorescence, Microscopy, Knockdown, shRNA, Transduction

Journal: Cell Death & Disease

Article Title: CXCR4 intracellular protein promotes drug resistance and tumorigenic potential by inversely regulating the expression of Death Receptor 5

doi: 10.1038/s41419-021-03730-8

Figure Lengend Snippet: A HT-29 cells were treated with either different concentrations of paclitaxel (9, 18, 37, 75, 150 nM) or AMD3100 (5 μM) alone or in combinations for 48 h and cytotoxicity was measured by SRB assay. Percent cell viability was tabulated. Columns, an average of triplicate readings of samples; error bars ± SEM. B Control and CXCR4 overexpressed MCF-7 cells were treated with CXCR4 ligand CXCL12 (100 ng/ml) or CXCR4 antagonist AMD3100 (5 μmol/L) for 12 h, and subjected to Western blot analysis for DR5 and β-actin. C HT-29 cells were either pre-treated with vehicle or AMD3100 (5 μM) for 12 h, followed by treatment with CXCR4 ligand CXCL12 (100 ng/ml) for different time points (0.5, 1, and 2 mins) and subjected to Western blot analysis for p-ERK and β-actin. D CXCR4 + and CXCR4 − HT-29 cells were flow-sorted and plated. After 5 days of culture, cells were stained with either APC-conjugated CXCR4 (CD184) and PE-conjugated DR5 or their respective matched isotype control antibodies and analyzed by FACS. In the upper panel, dot plots represent CXCR4 staining in unsorted, CXCR4 + sorted and, CXCR4 − sorted cells. In the lower panel histograms represent DR5 staining in the above-mentioned respective cells. E DLD-1, HCT-116, A-549, and MDA-MB-468 cells were stained with either APC-conjugated anti-human CXCR4 (CD184) or isotype control antibodies and analyzed by FACS. The cell surface expression of CXCR4 is represented in histogram overlays. F DLD-1, HCT-116, A-549, and MDA-MB-468 cells were seeded on coverslips for 24 h and subjected to immunofluorescence staining for CXCR4 and analyzed by confocal microscopy; Scale bar 10 μm. G DLD-1, HCT-116, A-549, and MDA-MB-468 cells were made stable for CXCR4 knockdown via shRNA mediated lentiviral transduction and scramble shRNA transduced cells were used as control. Immunoblot analysis of CXCR4 and DR5 protein in control or CXCR4 knockdown cells are shown; β-Actin was used as an internal protein loading control. H – L MCF-7 and HCT-116 cells were transfected with scrambled, wild type CXCR4, CXCR4 L86P , or CXCR4 δ242-248 containing vectors and cultured. After 48 h, cells were either stained with APC-conjugated anti-human CXCR4 (CD184)/isotype control antibodies and analyzed by FACS, or subjected to western blot or total RNA isolation. H The cell surface expression of CXCR4 is represented in histogram overlays. I , K Immunoblot analysis of CXCR4 and DR5 protein in control, wild type CXCR4, CXCR4 L86P , or CXCR4 δ242-248 transfected MCF-7 and HCT-116 cells; β-Actin was used as an internal protein loading control. Western Blot densitometric quantification numbers are shown above the loading control blot of all immunoblot studies. J , L Fold change in DR5 mRNA expression was measured by RT-qPCR as described in Materials and Methods. Data are representative of three independent experiments, resulting from duplicate readings of two different samples; Columns, average value of DR5 mRNA expression; bars ± SEM. *, p < 0.05, compared with respective controls.

Article Snippet: 3rd generation lentiviral vector pUltra-Chili-Luc (addgene no. 48688) with the bi-cistronic expression of tdTomato and Luciferase was used to make HT29 cells fluorescent.

Techniques: Sulforhodamine B Assay, Control, Western Blot, Staining, Expressing, Immunofluorescence, Confocal Microscopy, Knockdown, shRNA, Transduction, Transfection, Cell Culture, Isolation, Quantitative RT-PCR