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: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: Colon adenocarcinoma DLD1 cells stably expressing EndoA3-mCherry were generated using retroviral transduction. (A) Increased EndoA3-mCherry expression (~3 fold over endogenous EndoA3, Supplementary Figure 2A) promotes endocytosis in DLD1 cells (blue) compared to DLD1 cells with empty vectors (gray) as measured by a FM1-43 dye intake assay (Betz et al., 1996; Meyers et al., 2003). Unpaired Student’s t-test was used for statistical analysis (*** p<0.001, n=3 independent replicates). (B) Automated cell growth assay shows that EndoA3 DLD cells (blue) proliferated faster compared to DLD1 cells carrying empty vectors (gray). (C) EndoA3-mCherry expression (blue) accelerates wound closure in a 2D scratch assay. Data from DLD1 cells with empty vectors are indicated in gray. (D-E) Knockdown of endogenous EndoA3 by siRNA reduces growth (D) and migration (E) of DLD1 cells. The error bars in panels B-E indicate SEM from 5 independent biological replicates. Two-way ANOVA tests were conducted to calculate statistical significance. *** indicates p<0.001 for the comparison between control and EndoA3 DLD1 cells.

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Stable Transfection, Expressing, Generated, Retroviral, Transduction, Growth Assay, Wound Healing Assay, Knockdown, Migration, Comparison, Control

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: (A) Representative immunofluorescence images of EndoA3 DLD1 (left) and parental DLD1 cells (middle) stained with Alexa488-phalloidin (green). White arrows indicate examples of filopodia. Scale bar 2 µm. Fold changes of actin-rich protrusions in DLD1 cells that express either EndoA3 or empty vectors (compared to those in parental DLD1 cells) were plotted in the right panel. “n” = number of images quantified. (B) Representative images of enhanced lamellipodia in DLD1 cells that stably expressed EndoA3-mCherry and Lifeact-eGFP are shown in the left panels. The images were taken from a movie used for lamellipodia quantification. Percentage of cells that show lamellipodia in 10 minutes were plotted in the right panel. “n” = number of movies quantified. Unpaired Student’s t-test, *** p<0.001. (C) Representative images show increased invasiveness of EndoA3 DLD1 cells. 3D spheroids were embedded into mixed matrigel and collagen gels (1:1 volumetric ratio), and images were collected at day 7 post embedding. Control DLD1 cells with empty vectors (left), and EndoA3 DLD1 cells (middle). Dotted circles represent the initial spheroid area at day 1. Fold increase of volume (day 7/day 0) from five different spheroids were used for the statistical analysis (Student’s ttest, n= number of spheroids used for quantification, * p<0.05).

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Immunofluorescence, Staining, Stable Transfection, Control

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: The comparison of (A) endocytosis, and (B) growth among cells expressing EndoA3ΔN-mCherry (orange), EndoA3WT-mCherry (blue), and control vectors (gray). Error bars indicate SEM. (C) The number of filopodia protrusions per cell is shown in the left panel, and the fraction of cells with lamellipodia is shown in the right panel. “n” = number of images quantified. (D) Wound closure of EndoA3ΔN DLD1 cells was accelerated in the 2D migration assay. Error bars indicate SEM. (E) Xenotransplantation of human cancer cells into zebrafish hindbrain for assessing cell dissemination in vivo. (Upper left panel) A representative image of a zebrafish that was injected with DLD1 cells expressing EndoA3WT-mCherry into the fish embryo hindbrain. The image was taken 96 hours after injection. The white arrowhead indicates DLD1 cells that have disseminated from the injection site (indicated by the white arrow) in the zebrafish embryo hindbrain. (Lower left panel) An image showing the zoomed area with disseminated DLD1 cells. (Right panel) Percentage of fishes with disseminated DLD1 cells expressing empty vectors, EndoA3 WT-mCherry, and EndoA3ΔN-mCherry. “n”, the number of animals analyzed over 3 independent experiments. One-way ANOVA tests were used for multiple comparisons followed by unpaired Student’s t-test used for error analysis (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Comparison, Expressing, Control, Migration, In Vivo, Injection

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: (A) EndoA3 shows robust binding with recombinant TIAM1. A scheme diagram of the GST pull-down assay is shown in the left panel. GST-tagged BAR domains of EndoA1, EndoA2, and EndoA3 (12 µg) were incubated with the recombinant TIAM1 fragment (20 µM, residues 841-1418) for 4 hours. Proteins bound to the GST beads were analyzed using SDS-PAGE and coomassie blue staining (right). “TIAM1 input” indicates 13% of the recombinant TIAM1 protein used in GST pull-down experiments. * indicates proteolytic fragments of the recombinant TIAM1. (B) Endogenous interactions between EndoA3 and TIAM1 in DLD1 cells. Co-immunoprecipitation experiments were performed using antibodies against EndoA3 (top) and TIAM1 (bottom). IgG was used as controls for immunoprecipitation experiments. Samples were analyzed using western blotting and enhanced chemiluminescence. (C) A scheme diagram showing that free phosphates (indicated as P) are released upon Rac1 GTP hydrolysis (left). The recombinant EndoA3 BAR domain (middle), but not the EndoA2 BAR domain (right), stimulates the TIAM1-dependent Rac1 GTPase activity in vitro. Free phosphates were detected using the malachite green absorbance. Unpaired Student’s t-tests were used for statistical analysis (n=5). *** p<0.001, ** p<0.01, and * p<0.05. (D) Expression of EndoA3 increases the levels of active Rac1 GTPase activity in DLD1 cells. Active Rac1 GTPases that bound to immobilized GST-tagged p21 activated kinase binding domain (PAK PBD) was detected using monoclonal anti-Rac1 antibody (Cytoskeleton Inc.) and enhanced chemiluminescence. Band intensities were quantified using densitometry (normalized to the EndoA2/tubulin ratio) (left). Increases of active RAC1 (fold) were quantified using data from three independent experiments (right). (E) Knockdown of TIAM1 (left) and the Rac1/Cdc42 inhibitor ML141 (right) blocked protrusion formation. Unpaired Student’s t-tests were used for 3 independent experiments (*** p<0.001).

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Binding Assay, Recombinant, Pull Down Assay, Incubation, SDS Page, Staining, Immunoprecipitation, Western Blot, Activity Assay, In Vitro, Expressing, Knockdown

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: (A) Addition of recombinant TIAM1 fragments reduces membrane bound EndoA3 BAR. Cosedimentation assays were used to detect membrane-bound (pellet, P) and unbound (supernatant, S) fractions of EndoA3. Liposomes (1%PIP2, 24%PS, 70%PC, 5%NBD-PE, 0.5mM total lipids) and his6-tagged EndoA3 BAR domain (2 µM) were incubated with TIAM1 (0, 5, 10, and 20 µM, respectively) for 30 minutes. Samples were centrifuged at 75,000 rpm for 30 min. Supernatants and pellets were analyzed by western blotting and enhanced chemiluminescence. (B) Addition of TIAM1 reduces the tubulation activity of EndoA3. Liposomes (1%PIP2, 24%PS, 75%PC, 0.5mM total lipids) were incubated with his6-tagged EndoA3 BAR (1µM), with and without the recombinant TIAM1 fragment (20 µM), for 30 min at room temperature. Samples were analyzed using negative stain transmission electron microscopy. Representative images were shown at the left. The fraction of liposomes with membrane tubules were quantified and shown at the right. Unpaired Student’s t-tests were used for statistical analysis of 3 independent experiments. * p<0.05. (C) Membrane inhibits EndoA3-dependent, but not EndoA3ΔN-dependent, Rac1 activation. His6-tagged EndoA3 BAR (WT or ΔN; 8 µM) and TIAM1 fragments (0.5 µM) were incubated with Rac1 (2µM) in the absence and presence of liposomes (1%PIP2, 25%PS, 74%PC, 0.5mM total lipids). Fold activation of Rac1 GTPase activity was calculated using results from control experiments in the absence of his6-tagged EndoA3 BAR. Malachite green assay was used to quantify free phosphate groups released from GTP by Rac1. Student’s t-tests (** indicates p<0.01). (D) Anchoring EndoA3ΔN to the plasma membrane using an N-myristoylation tag reduces the levels of active TIAM1 in DLD1 cells. Lysates from DLD1 cells expressing EndoA3ΔN-GFP and Myr-EndoA3ΔN-GFP were analyzed. Active TIAM1 was detected using Rac1 G15A agarose beads. TIAM1 signal was normalized to tubulin levels. The ratio of active TIAM1 levels between EndoA3ΔN-GFP and Myr- EndoA3ΔN-GFP cells were indicated. (E) The ability of EndoA3ΔN to stimulate cell migration is significantly reduced when it is anchored to the plasma membrane. Migration (left) and growth (right) of DLD1 cells expressing either myr-EndoA3ΔN-GFP (green) or EndoA3ΔN-GFP (orange) was quantified using the automated assays described in Figure 2. Error bars indicate SEM. Statistical analysis was done using two-way ANOVA (** p<0.001). (F) EndoA3 localization was examined using fluorescence microscopy. EndoA3 cells were treated with DMSO (left), PTEN inhibitor 1 (bpV(HOpic), 200nM; middle), or PTEN inhibitor 2 (VO-OHpic, 200nM; right). (G) Ratio between membrane-bound and cytoplasmic EndoA3 was measured using ImageJ as previously described in Materials and Methods. “n” = the number of cells analyzed. ** p<0.01, one-way ANOVA. (H) Inhibition of PTEN accelerates the growth of EndoA3 DLD1 cells (4 independent experiments, * p<0.05). (I) Inhibition of PTEN reduces filopodia abundance in EndoA3 DLD1 cells (“n” = number of images analyzed). Three independent repeats. *** p<0.001, one-way ANOVA).

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Recombinant, Membrane, Liposomes, Incubation, Western Blot, Activity Assay, Staining, Transmission Assay, Electron Microscopy, Activation Assay, Control, Malachite Green Assay, Clinical Proteomics, Expressing, Migration, Fluorescence, Microscopy, Inhibition

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: Experimental metastasis was examined in nude mice injected with DLD1 cells expressing vector control (gray), EndoA3-mCherry (blue), and EndoA3ΔN-mCherry (orange), respectively. (A) Number of metastatic nodules per mice in lungs upon tail vein injection of DLD1 cells. Metastatic nodules were visualized by immunofluorescence staining against Ck8/18. (B) Average area per nodule per mice in lung was measured. “n” indicates the number of animals. (C) The extent of cell proliferation was assessed using Ki-67 immunohistochemistry of pulmonary lobes. One-way ANOVA was used for multiple comparisons followed by unpaired Student's t-test was used for statistical analysis (*** p<0.001, ** p<0.01, * p<0.05).

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Injection, Expressing, Plasmid Preparation, Control, Immunofluorescence, Staining, Immunohistochemistry

Journal: Developmental cell

Article Title: Competition between TIAM1 and membranes balances Endophilin A3 activity in cancer metastasis

doi: 10.1016/j.devcel.2018.05.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human colon adenocarcinoma cells: DLD1 , ATCC , Cat#: CCL-221.

Techniques: Virus, Recombinant, Blocking Assay, Imaging, Retroviral, Mutagenesis, Plasmid Preparation, Software, Microscopy, Microarray

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: Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers

doi: 10.1016/j.xcrm.2021.100421

Figure Lengend Snippet:

Article Snippet: Additional utilized tissue sections include normal human tissue microarrays obtained from US Biolab XHN801 (tongue) and XHN803 (tongue) as well as US Biomax OR208a (tongue) (range 22-73 years; n = 12 male and n = 4 female), BN1002b (esophagus) (range 22-42 years; n = 4 male and n = 1 female), and LP208 (Larynx (range 16-48 years; n = 3 male and n = 1 female), oro/hypo-pharynx (range 15-43 years; n = 2 male and n = 2 female)).

Techniques: Microarray, Recombinant, Blocking Assay, Sequencing, Expressing, Multiplex Assay, Software

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

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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