Journal: Scientific Reports

Article Title: Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness

doi: 10.1038/s41598-020-68187-9

Figure Lengend Snippet: ( a ) Representative bright field images of parental cells and holoclones derived from DU145, PC-3, LNCaP and 22Rv1 prostate cancer cell lines at approximately 5–7 weeks following initial plating in high-salt agar. ( b – e ) Stemness gene expression profile in holoclones and parental cells derived from high-salt agar. ( b – d ) Relative quantification of change in expression of stem-associated genes (ALDH1, CD44, CD133, POU5F1, NANOG, CD24, c-Met, integrin α2, integrin β1 and integrin α6) in PC-3, 22Rv1 and LNCaP-derived holoclones, which were normalised to parental controls. ( e ) The associated heat map summarising changes in gene expression (holcolones versus parent) (Grey: undetected, green: downregulated, red: upregulated). Data represented as Mean ± SEM (*p < 0.05, unpaired Student’s two-tailed t test, n = 3).

Article Snippet: Appropriate antibodies, APC mouse anti-human CD133 (Miltenyi Biotec, Germany); PE-Cy7 mouse anti-human CD44, PE mouse anti-human CD29, FITC-mouse anti-human CD49b (BD Biosciences, San Jose, CA, USA) were added to each cell sample and incubated in the dark for 20 min. A further 100 μL PBA was added; cells were pelleted to remove unbound antibody and re-suspended in 200 μL PBA buffer.

Techniques: Derivative Assay, Gene Expression, Quantitative Proteomics, Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness

doi: 10.1038/s41598-020-68187-9

Figure Lengend Snippet: qRT-PCR primer information.

Article Snippet: Appropriate antibodies, APC mouse anti-human CD133 (Miltenyi Biotec, Germany); PE-Cy7 mouse anti-human CD44, PE mouse anti-human CD29, FITC-mouse anti-human CD49b (BD Biosciences, San Jose, CA, USA) were added to each cell sample and incubated in the dark for 20 min. A further 100 μL PBA was added; cells were pelleted to remove unbound antibody and re-suspended in 200 μL PBA buffer.

Techniques: TaqMan Assay

Journal: Cancers

Article Title: Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells

doi: 10.3390/cancers16142595

Figure Lengend Snippet: List of anti-human antibodies used for flow cytometry.

Article Snippet: CD44 , FITC , DB105 , 130-113-896 , Miltenyi Biotec.

Techniques: Cytometry

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The bar graphs show relative expression levels of CD44 in the indicated samples. (B–D) Maximum lifespan is plotted against CD44 expression levels in (B) cultured fibroblasts, (C) liver, and (D) skin. Results of Pearson and Spearman correlation tests are shown on the plots.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Expressing, Cell Culture

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in mouse and NMR OPCs. (B) A representative immunoblot of CD44 in NMR OPCs transfected with siLuciferase, siCD44–1, or siCD44–2. Cells were collected 3 days after transfection. (C) The heatmap shows the relative levels of genes that are differently expressed between control and CD44-knockdown NMR OPCs (q value <0.05). (D) The boxplot shows the effects of CD44 knockdown on the expression levels of the indicated gene sets in NMR OPCs. The effects are shown for whole genes, the genes that were differentially expressed in NMR OPCs compared with OPCs of all other species, and the genes that were differentially expressed in both NMR and human OPCs compared with mouse and rat OPCs. (E) Functional enrichment analyses of genes that were differentially expressed between control and CD44 knockdown NMR OPCs. (F) The results of transcription factor target enrichment analysis of genes that were downregulated by CD44 knockdown in NMR OPCs. Top five overrepresented transcription factors obtained by the search of ARCHS4 TFs. Co-expression database and TRRUST TF-target regulatory interaction database are shown. (G) The heatmap shows the relative expression levels of differentially expressed genes that are associated with the terms highlighted in (E). (H) A representative immunoblot of CD44 in mouse OPCs transfected with siLuciferase, siCD44–1, or siCD44–2. Cells were collected 3 days after transfection. (I) Cell survival rate of mouse and NMR OPCs transfected with siLuc, siCD44–1, and siCD44–2 after 6 days of 0.5 μg/mL tunicamycin treatment. Transfection was performed 2 days before starting tunicamycin treatment (n = 8). (J) A representative immunoblot of CD44 in mouse OPCs. Cells were transfected with empty vector or vector encoding mouse CD44, NMR CD44, or mouse LDLR 2 days before sample collection. (K) Cell survival rate of mouse OPCs after 6 days of 0.5 μg/mL tunicamycin treatment. Cells were transfected with empty vector or vector encoding mouse CD44, NMR CD44, or mouse LDLR 1 day before starting tunicamycin treatment (n = 8). Error bars are presented as mean ± SD values. *p < 0.05; Wilcoxon test for (D), one-way ANOVA with post hoc Sidak’s test for (I), and one-way ANOVA with post hoc Dunnett’s test for (K).

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing, Functional Assay, Plasmid Preparation

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (B) Cell survival rate of shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment (n = 10). (C) Functional enrichment analyses of genes that were up- or downregulated (q value <0.05) by both shCD44–1 and shCD44–2 expression in IMR90-hTert cells. (D) Real-time qPCR data of representative UPR genes in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells (n = 3). Data were normalized to GAPDH . (E) A representative immunoblot of HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (F) Relative density of HSPA5/b-Actin analyzed by ImageJ (n = 3). (G) The boxplot shows the effects of CD44 overexpression on the expression levels of the indicated genes in IMR90 cells. The effects are shown for the genes that are upregulated by ATF6 activating molecule AA147 and the genes co-expressed with ATF6, ATF4, or XBP1 (retrieved from Enrichr). (H) Percentages of overlap between CD44 co-expressing genes (retrieved from COXPRESdb v7) and the indicated genes. (I) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO) or 10 μM HA15 was added to the medium during tunicamycin treatment (n = 10). All immunoblots were repeated once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; one-way ANOVA with post hoc Dunnett’s test for (B and F), two-way ANOVA with post hoc Dunnett’s test for (D), Wilcoxon test for (G), Fisher’s exact test for (H), and two-tailed t test with Bonferroni-Dunn correction for (I).

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Western Blot, Functional Assay, Expressing, Over Expression, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Protein-protein interactions among the 41 CD44-associated proteins plus IRE1, PERK, and ATF6 were mapped using the STRING software. Only highest confidence interactions (interaction score ≥0.9) that are experimentally determined (purple line) or stored in curated databases (blue line) were included in the analysis. IRE1, PERK, and ATF6 are shown as red nodes and the 16 CD44-associated ER proteins are shown as blue nodes. CD44-associated proteins are defined here as proteins that are detected by cross-linked immunoprecipitation-mass spectrometry of IMR90 cells using anti-CD44 antibody with at least 10-fold greater abundance than in control experiment using normal rabbit IgG antibody. (B) Representative immunoblots of CALR, HSP47, and CD44 in whole cell lysate and in CD44-immunoprecipitates prepared from IMR90 cells. The experiment was repeated once with similar results. (C) Representative confocal images of IMR90 cells co-stained with antibodies against CD44 and organelle markers (mitochondrial marker mtTFA and ER markers ERp57, HSP47, and RPN2). (D) The dot plot shows Pearson correlation coefficients between immunofluorescence signals of CD44 and organelle markers. Each dot represents a single cell (n = 30). Error bars are presented as mean ± SD values. Scale bars, 10 μm.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Protein-Protein interactions, Software, Immunoprecipitation, Mass Spectrometry, Control, Western Blot, Staining, Marker, Immunofluorescence

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The heatmap shows relative abundances of ER-associated proteins detected by mass spectrometry of ER fractions isolated from shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. ER-associated proteins were defined here as the proteins whose ER localization have been confirmed by the Human Protein Atlas or associated with the GO term “endoplasmic reticulum lumen.” (B) Representative immunoblots of COL6A3, SEC62, and HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Whole cell lysates and culture supernatants were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. (C) Representative immunoblots of ATF6, IRE1, PERK, CALR, EGFR, and Vinculin in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were lysed in buffer containing either 1% Triton or 2% SDS. (D) A representative immunoblot of ATF6 in U2OS cells and in ATF6 KO U2OS cells infected with empty vector or vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N. (E) Cell survival rate of ATF6 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment (n = 8). Cells were infected with vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N and selected with 100 μg/mL hygromycin prior to the experiment. (F) Relative expression levels of HSP90B1 and HSP5A in CD44 KO U2OS cells (n = 3). Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before sample collection. Data were normalized to GAPDH . (G) Cell survival rate of CD44 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment. Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before starting tunicamycin treatment (n = 8). All immunoblots were repeated at least once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test (E) and two-tailed t test with (F) or without (G) Bonferroni-Dunn correction.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Mass Spectrometry, Isolation, Western Blot, Infection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Recombinant, Transfection, Protease Inhibitor, CyQUANT Assay, Proliferation Assay, Expressing, Mass Spectrometry, Software