Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Oxaliplatin-resistant SW620, SW480, HCT116, and HT29 colon cancer cell lines demonstrate similar or enhanced sensitivity to TRAIL compared to their parental counterparts after 24 hr of treatment. N = 3 (biological replicates); n = 9 (technical replicates). ( B ) IC50 values were calculated using a variable slope four-parameter nonlinear regression. ( C ) Representative Annexin-V/PI flow plots comparing SW620 parental and OxR cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. The four quadrants represent viable cells (bottom left), early apoptosis (bottom right), necrosis (top left), and late apoptosis (top right). ( D ) Representative flow plots of JC-1 assay after treatment with 1000 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. ( E ) Mitochondrial depolarization as a function of TRAIL concentration for SW620 parental and OxR cell lines. N = 3 (n = 9). For all graphs, data are presented as mean ± SD. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). Figure 1—source data 1. Raw viable cell counts from Annexin-V/PI assays and percent depolarized mitochondria in SW620 cells (panels A and E).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) SW620, SW480, HCT116, and HT29 cells were treated with various concentrations of oxaliplatin for 72 hr and cell viability was measured using an MTT assay. IC50 values were calculated using a variable slope four-parameter nonlinear regression. Data are presented as mean ± SEM. N = 2 (n = 12). ( B ) Counts of successfully invasive cells after a 4-day Transwell assay with an initial seeding of 200,000 cells. N = 2 (n = 6). * p<0.05; **p<0.01 (unpaired two-tailed t-test). Figure 1—figure supplement 1—source data 1. Raw data from MTT cell viability assays after oxaliplatin treatment (panel A). Figure 1—figure supplement 1—source data 2. Invasive cell counts from Transwell assays (panel B).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: MTT Assay, Transwell Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Sensitization of oxaliplatin-resistant SW620, HCT116, HT29, and SW480 cell lines compared to their parental counterparts as a function of TRAIL concentration. ( B ) Maximum TRAIL sensitization for each cell line between the tested concentrations of 0.1–1000 ng/ml. Data are presented as mean ± SEM. Figure 1—figure supplement 2—source data 1. TRAIL sensitization calculations.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Concentration Assay

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Representative flow plots of JC-1 assay after treatment with 200 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. ( B ) Mitochondrial depolarization as a function of TRAIL concentration for HCT116 parental and OxR cell lines. Data are presented as mean ± SD. N = 3 (n = 9). ****p<0.0001 (unpaired two-tailed t-test). Figure 1—figure supplement 3—source data 1. Percent depolarized mitochondria in HCT116 cells measured from JC-1 assays.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, B ) Volcano plots of RT-PCR Apoptosis Profiler arrays demonstrate downregulation of CASP10 in OxR phenotypes. N = 3. ( C ) CRISPR/Cas9 knockout of caspase-10 in SW620 parental cells was confirmed via western blot. sgRNA/Cas9 ribonucleoprotein complexes reduced caspase-10 expression by 93% compared to cells treated with Cas9 alone. ( D ) CASP10 knock-out (KO) cells demonstrate slight decreases in viability when treated with TRAIL compared to Cas9 control. Data are presented as mean ± SD. N = 3 (n = 9). ( E ) Representative Annexin-V/PI flow plots comparing SW620 parental (Cas9 only) and CASP10 KO cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. ( F ) Depletion of caspase-10 did not have a significant effect on TRAIL sensitization (unpaired two-tailed t-test). Data are presented as mean + SEM. N = 3 (n = 9). Figure 2—source data 1. Apoptosis microarray data in HCT116 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel A). Figure 2—source data 2. Apoptosis microarray data in SW620 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel B). Figure 2—source data 3. Western blot images (raw and annotated) confirming CASP10 KO (panel C). Figure 2—source data 4. Quantification of CASP10 KO from western blots (panel C). Figure 2—source data 5. Cell viability and TRAIL sensitization calculations in CASP10 KO cells (panels D and F).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Reverse Transcription Polymerase Chain Reaction, CRISPR, Knock-Out, Western Blot, Expressing, Control, Two Tailed Test, Microarray, Generated

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, B ) Confocal micrographs of HCT116 and SW620 cells, respectively. Red channel represents DR4, green is lipid rafts, and blue is DAPI (nuclei). Scale bar = 30 μm. ( C ) Quantification of DR4 area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. Data are presented as mean + SEM from N = 3 independent experiments. ***p<0.001; ****p<0.0001 (unpaired two-tailed t-test). ( D ) OxR cells had increased surface expression of DR4 in non-permeabilized cells analyzed via flow cytometry. # Significant according to a chi-squared test (see ). ( E ) Western blots for DR4 in whole cell lysates of parental and OxR cells. ( F ) Quantification of western blots from three independent experiments (N = 3). Data are presented as mean + SEM. *p<0.05 (unpaired two-tailed t-test). ( G ) Percentage of apoptotic SW620 cells after treatment with 0.01–10 µg/ml mapatumumab (sum of early and late-stage apoptotic cells from Annexin/PI staining). Data are presented as mean ± SD. N = 3 (n = 6). ****p<0.0001 (multiple unpaired two-tailed t-tests). ( H ) Cell viability of SW620 cells after mapatumumab treatment, determined by Annexin-V/PI staining. Data are presented as mean ± SD. N = 3 (n = 6). ( I ) Maximum mapatumumab sensitization within OxR cell lines compared to their parental counterparts. Data are presented as mean + SEM. Figure 3—source data 1. Quantification of DR4 area per cell in HCT116 and SW620 cells (panel C). Figure 3—source data 2. Western blot images (raw and annotated) for DR4 (panel E). Figure 3—source data 3. Quantification of DR4 from western blots (panel F). Figure 3—source data 4. Cell viabilty and percent apoptosis in SW620 cells after mapatumumab treatment (panels G-I).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Expressing, Flow Cytometry, Western Blot, Staining

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A–D ) Confocal micrographs and DR5 quantification of HCT116, SW620, SW480, and HT29 cells, respectively. Red channel is death receptor 5, green is lipid rafts, and blue is DAPI (nuclei). Scale bar = 30 μm. ** p<0.01; ****p<0.0001 (unpaired two-tailed t-test). Data are presented as mean + SEM. For each cell line, N = 75 cells were analyzed. ( E ) Oxaliplatin-resistant (OxR) cells only demonstrate increased surface expression of DR5 in non-permeabilized SW620 cells. # Significant according to a chi-squared test (see ). Figure 3—figure supplement 4—source data 1. Quantification of DR5 area per cell for all cell lines.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Expressing

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Cell viability of HCT116 cells after 0.01–10 µg/ml mapatumumab treatment, determined by Annexin-V/PI staining. ( B ) Percentage of apoptotic SW620 cells after mapatumumab treatment (sum of early and late-stage apoptotic cells from Annexin/PI staining). For all graphs, data are presented as mean + SD. N = 3 (n = 6). *p<0.05; ****p<0.0001 (multiple unpaired two-tailed t-tests). Figure 3—figure supplement 7—source data 1. Cell viabilty and percent apoptosis in HCT116 cells after mapatumumab treatment.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Staining, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Composite images and binary projections of DR4/LR colocalization areas in HCT116 and SW620 cell lines. Lipid raft and DR4 binary images were generated for a specified threshold, then multiplied by one another to generate images with positive pixels in double-positive areas. Red is DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. ( B ) Quantification of DR4 and lipid raft colocalization area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). ( C ) Correlation between the fold change in DR4/LR colocalization (OxR phenotype/parental) and maximum TRAIL sensitization observed by the OxR phenotype for each of the four cell lines (simple linear regression analysis). ( D ) Lipid raft fractions were isolated and analyzed for DR4 via western blot in parental and OxR cells. ( E ) Quantification of lipid raft DR4 blots in ( D ). *p<0.05 (unpaired two-tailed t-test). For all graphs, data are presented as mean + SEM. ( F ) Förster resonance energy transfer (FRET) efficiencies of FITC-labeled DR4 (donor) and Alexa 555-labeled lipid rafts (acceptor) in parental and OxR cells analyzed via flow cytometry. **p<0.01; ***p<0.001 (unpaired two-tailed t-test). Figure 4—source data 1. Quantification of LR-colocalized DR4 area per cell in HCT116 and SW620 cells (panel B). Figure 4—source data 2. Correlation analysis of LR-colocalized DR4 area per cell with TRAIL sensitization (panel C). Figure 4—source data 3. Western blot images (raw and annotated) for DR4 from LR isolates (panel D). Figure 4—source data 4. Quantification of LR DR4 from western blots (panel E). Figure 4—source data 5. FRET efficency calculations (panel F).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Generated, Two Tailed Test, Isolation, Western Blot, Förster Resonance Energy Transfer, Labeling, Flow Cytometry

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Quantification of DR5/LR colocalization in HCT116, SW620, SW480, and HT29 cells. *p<0.05; **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). For each cell line, N = 75 cells were analyzed. ( B ) Correlation of total DR5 area per cell and ( C ) DR5/LR colocalization with maximum TRAIL sensitization observed in OxR cells (linear regression analysis). For all graphs, data are presented as mean + SEM. ( D ) Western blots show DR5 was undetectable in lipid raft isolated fractions. Figure 4—figure supplement 2—source data 1. Quantification of LR-colocalized DR5 area per cell (panel A). Figure 4—figure supplement 2—source data 2. Correlation analysis of DR5 and LR-colocalized DR5 area per cell with TRAIL sensitization (panels B and C). Figure 4—figure supplement 2—source data 3. Western blot images (raw and annotated) for DR5 from LR isolates (panel D).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Western Blot, Isolation

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, G ) SW620 oxaliplatin-resistant (OxR) and HCT116 OxR cells, respectively, treated for 24 hr with a combination of TRAIL and 5 µM nystatin. ( B, H ) SW620 OxR and HCT116 OxR cells, respectively, showed a significant decrease in TRAIL sensitization when treated in combination with nystatin. N = 3 (n = 9). ( C, I ) Treatment with 5 µM nystatin significantly decreased DR4/LR colocalization area in SW620 OxR and HCT116 OxR cells, respectively. For each cell line, N = 40 cells were analyzed. ( D, J ) SW620 Par and HCT116 Par cells, respectively, treated for 24 hr with a combination of TRAIL and 70 µM resveratrol. N = 3 (n = 9). ( E, K ) SW620 Par and HCT116 Par cells, respectively, showed a significant increase in TRAIL sensitization when treated in combination with resveratrol. N = 3 (n = 9). ( F, L ) Treatment with 70 µM nystatin significantly increased DR4/LR colocalization area in SW620 Par and HCT116 Par cells, respectively. For each cell line, N = 40 cells were analyzed. ( M ) Representative composite images and binary projections of DR4/LR colocalization in SW620 OxR cells before and after nystatin treatment. ( N ) Representative composite images and binary projections of DR4/LR colocalization in parental SW620 cells before and after resveratrol treatment. Red represents DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test for all graphs). ( A, D, G, J ) Data are presented as mean ± SD. ( B, C, E, F, H, I, K, L ) Data are presented as mean + SEM. Figure 5—source data 1. Cell viability after TRAIL combination treatments with resveratrol and nystatin (panels A, D, G, J). Figure 5—source data 2. TRAIL sensitization calculations after resveratrol and nystatin (panels B, E, H, K). Figure 5—source data 3. Quantification of LR-colocalized DR4 after resveratrol and nystatin treatment (panels C, F, I, L).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: For each cell line, N = 40 cells were analyzed. Data are presented as mean + SEM. *p<0.05 (unpaired two-tailed t-test). Figure 5—figure supplement 1—source data 1. Quantification of the effects of resveratrol and nystatin on DR5 colocalization with LRs in HCT116 and SW620 cells.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: MTT Assay, Recombinant, Software, Membrane, Gene Knockout, Labeling, Control, Neutralization, Clinical Proteomics, Isolation, Magnetic Beads

Journal: Cell Reports Medicine

Article Title: Combinatorial targeting of glutamine metabolism and lysosomal-based lipid metabolism effectively suppresses glioblastoma

doi: 10.1016/j.xcrm.2024.101706

Figure Lengend Snippet:

Article Snippet: Authenticated (short tandem repeat profiling) human GBM cell lines, U251 (Male) and U373 (Male) from Sigma, U87 (HTB-14) (Female), T98 (CRL-1690) (Male), LN (CRL-2611) (Female) cells American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% HyClone fetal bovine serum (FBS), and 4 mM glutamine.

Techniques: Plasmid Preparation, Virus, Microarray, Recombinant, Modification, Isolation, Clinical Proteomics, Acid Assay, Amplex Red Cholesterol Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Lysis, Protease Inhibitor, Luciferase, Transfection, cDNA Synthesis, Control, Software

Journal: Oncotarget

Article Title: Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

doi:

Figure Lengend Snippet: A. P62 mRNA expression in human cancers compared to corresponding normal tissues. The graph was derived from microarray data of Oncomine. B-D. Expression of oncogenes leads to p62 accumulation. MCF10A human mammary epithelial cells were transduced with retroviruses (B,C) or lentiviruses (D) expressing the corresponding oncogenes, and (after brief selection) levels of p62 were assessed by immunoblotting with anti-p62 antibody. Duplicates (separate infections) are shown in B and D, and effect of increased levels of RAS virus in C. Anti-actin antibody was used as a loading control.

Article Snippet: MCF10A were from ATCC; MCF10A cells expressing PIK3CA were kindly provided by Dr. T. Waldmann.

Techniques: Expressing, Derivative Assay, Microarray, Transduction, Selection, Western Blot, Virus, Control

Journal: Cancer cell

Article Title: Apoptotic cell-derived extracellular vesicles promote malignancy of glioblastoma via intercellular transfer of splicing factors

doi: 10.1016/j.ccell.2018.05.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HBEC5i , ATCC , Cat# CRL-3245.

Techniques: Virus, Microarray, Clinical Proteomics, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Plasmid Preparation, Labeling, Polymer, Membrane, Immunoprecipitation, Flow Cytometry, Bradford Protein Assay, RNA Sequencing, shRNA, Sequencing, Cloning, Software