Journal: Biomedicines

Article Title: Role of UPR Sensor Activation in Cell Death–Survival Decision of Colon Cancer Cells Stressed by DPE Treatment

doi: 10.3390/biomedicines9091262

Figure Lengend Snippet: Dose–time–response inhibition of proliferation and S-G2 cell cycle arrest induction by DPE in colon cancer cells: ( A ) cell viability of RKO and SW480 cell lines treated with different doses of DPE (25, 50, 100 and 200 μM) or left untreated as control (CT), as measured by a trypan blue exclusion assay after 24 or 48 h of treatment; ( B ) cell proliferation of RKO and SW480 cell lines treated with different doses of DPE (25, 50, 100, 200 μM and 400 μM) or control, as measured by MTT assay after 24 h; ( C ) cell viability of human colonic epithelial cell (HCoEpC) lines treated with DPE (100 μM) or left untreated as control, as measured by a trypan blue exclusion assay after 24 or 48 h of culture; ( D ) cell cycle analysis of RKO and SW480 treated with DPE (50 or 100 μM) or untreated for 24 or 48 h, as evaluated by FACS analysis after staining with PI. One representative out of three experiments is shown. The bars represent the means of the percentages of cells in each phase of the cell cycle (subG1, G1, S, and G2) plus S.D. of three experiments. Note: *** p < 0.001, ** p < 0.01, * p < 0.05. Not significant (n.s.).

Article Snippet: Human colonic epithelial cells (HCoEpC) were cultured in epithelial cell growth medium (iXCells Biotechnologies USA, MD-0041).

Techniques: Inhibition, Trypan Blue Exclusion Assay, MTT Assay, Cell Cycle Assay, Staining

Journal: Nature cell biology

Article Title: ATF4 couples MYC-dependent translational activity to bioenergetic demands during tumor progression

doi: 10.1038/s41556-019-0347-9

Figure Lengend Snippet: a. Immunoblot of B cells isolated from WT (n=3), pre-lymphoma Eμ-myc (n=3) or lymphoma bearing Eμ-myc mice (n=6), lysates from different mice per group were used. b . Immunoblot from two normal human B cells (NHBC) and Burkitt’s lymphoma cell lines. c. Immunoblot from normal human colonocytes (NHC) or colon cancer cell lines. d. Immunoblot from MCF10 (non-tumorigenic breast epithelial cell line) or breast cancer cell lines. e. Pearson correlation between EIF4EBP1 and ATF4 target gene expression in Diffused Large B cell Lymphoma (DLBCL, n=562), Colorectal Adenocarcinoma (COAD, n=329), Breast Cancer (BRCA, n=1218) and Sarcoma (SARC, n=265). The center lines depict linear regression lines and shaded regions are 95% confidence intervals for regression lines. Datasets analyzed are listed in the section. Previously known ATF4 target gene list used in this analysis is shown in table. f. Kaplan-Meier plots of progression free survival (left) and overall survival (right) of DLBCL patients with high or low EIF4EBP1 expression. The survival analysis using Kaplan-Meier and two-sided log-rank test between high and low EIF4EBP1 expression groups was performed on all the patients with records of progression-free survival (left, n= 173) and overall survival (right, n=171), respectively. g. Proposed model of ATF4 and MYC co-operation in tumor progression. All immunoblots are from n=1 and unprocessed scans of blots are shown in .

Article Snippet: Normal human colonocytes were purchased from Cell Applications Inc (732Cn-10a).

Techniques: Western Blot, Isolation, Targeted Gene Expression, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction

doi: 10.1038/s12276-024-01354-4

Figure Lengend Snippet: a A schematic image illustrating how GzmA affects colonic epithelial cells through PARs signaling. b Overall scheme of the T cell isolation and senescence process (upper panel). Isolated pan-T cells after stimulation with anti-CD3/28 cocktail and rhIL-2 were assessed for cumulative population doubling level (PDL) (lower panel). Young cells were defined as 2 < cPDL < 5 and senescent cells as cPDL > 9. c The C 12 FDG staining analysis using flow cytometry (upper panels). The mRNA expression level of representative T cell senescence markers (p21 Waf1 , p16 INK4A , CD28, and CD57) are shown (lower panel). ND not detected. d The mRNA (upper panel) and protein (lower panel) levels of GzmA in young and senescent T cells are performed by real-time PCR and ELISA, respectively. e The IHC analysis of cleaved caspase-3 in normal colon tissues from young and elderly individuals (upper panel) and 4-month and 18-month-old mouse colon tissues (lower panel). The right panels display the quantification data, respectively. f , g Human primary colon epithelial cells were co-cultured with young or senescence T cells ( f ) or treated with CM of young or senescence T cells ( g ) for 3 days. The luminescence-based assay and ICC to determine cleaved caspase-3/7 activity and cleaved caspase-3 expression level were performed, respectively. h IHC analysis for IL8 was performed in normal colon from young and elderly tissues (upper panel). The IHC analysis results are categorized into “Low,” “Moderate,” and “High” based on the intensity of the immunostaining, with representative images provided for each category. The left lower panel shows the quantification data for IHC analysis. The right lower panel shows a dot plot illustrating the expression of CXCL8 (IL8) in colonic epithelial cells from young and elderly individuals in the GSE178341 data set. i The CXCL8 (IL8) mRNA expression was analyzed by real-time PCR in human colon epithelial cells, which were co-cultured with young or senescent T cells (upper panel) and treated with CM of young or senescent T cells (lower panel) for 3 days. A p -value in ( h ) is calculated using the Chi-square test. The rest of the p -values are calculated using the Mann–Whitney U test. The graph in ( b ) is shown as mean ± standard deviation, while the rest of the bar graphs are shown as mean + standard deviation. “Young” and “Sen” in ( c , d , f , g , i ) indicate the young and senescent T cells, respectively. “Young” and “Old” in ( e , h ) indicate the young and the elderly individuals, respectively.

Article Snippet: These mixtures were treated to human colonic epithelial cells (HCoEpiC, Cell Biologics, Inc.) for 1 day.

Techniques: Cell Isolation, Isolation, Staining, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Luminescence Assay, Activity Assay, Immunostaining, MANN-WHITNEY, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction

doi: 10.1038/s12276-024-01354-4

Figure Lengend Snippet: A schematic image illustrating how senescent T cells can affect colonic epithelial function via the PARs signaling pathway.

Article Snippet: These mixtures were treated to human colonic epithelial cells (HCoEpiC, Cell Biologics, Inc.) for 1 day.

Techniques:

Journal: Cancers

Article Title: Intestinal Epithelial Cells Adapt to Chronic Inflammation through Partial Genetic Reprogramming

doi: 10.3390/cancers15030973

Figure Lengend Snippet: Human colonic epithelial cells adapt to chronic inflammation-associated oxidative stress in vitro. ( A ) HCEC-hTERT cells (left panel) were subjected to chronic inflammation by culturing them in the presence of activated macrophage supernatant (+AMS) or tert-Butyl-hydroperoxide (TBHP). Cells committed to senescence after a week, demonstrated by the detection of SA-β-galactosidase activity (central panel), and maintained their senescent phenotype for three weeks before resuming proliferation (right panel). ( B ) Assessment in both experimental settings (left AMS, right TBHP) of phospho-serine 10 histone H3 (pS10-H3), p53, acetylated-p53 (Ac-p53), and p21 CIP1 (p21) by Western blot (protein levels were quantified relative to the β-actin and HCEC-hTERT cells) and of SA-β-galactosidase-positive cells in parental HCEC-hTERT cells (1) and senescent cells (2). A non-parametric Student’s t -test, mean, and SD of one experiment performed in triplicate are shown. *** p < 0.001, ** p < 0.01. Uncropped Western blots are provided in .

Article Snippet: Human primary colonic epithelial cells (HCEC) were purchased from Applied Biological Materials (abm, Richmond, Canada) and immortalized through transduction of hTERT (HCEC-hTERT).

Techniques: In Vitro, Activity Assay, Western Blot

Journal: Cancers

Article Title: Intestinal Epithelial Cells Adapt to Chronic Inflammation through Partial Genetic Reprogramming

doi: 10.3390/cancers15030973

Figure Lengend Snippet: Escape from chronic-inflammation-induced senescence does not rely on p53 inactivation. ( A ) Confirmation of the wild-type status of the TP53 gene in HCEC-hTERT cells and their derivatives. TP53 exons within parental HCEC-hTERT cells and their Esc-Inf derivatives (of two independent experiments, Esc-Inf A and B) were fully sequenced. No mutation in TP53 was found, as shown by the sequences encompassing the exons encoding the residues R175, R248, and R273 that constitute the main mutation hot-stops reported in colitis-associated colon cancers. ( B ) The functionality of the p53 pathway in both parental HCEC-hTERT and its Esc-Inf derivatives was compared by submitting cells to an increasing dose of Fluorouracil (5-FU) over 24 h. The phosphorylation of the Ser10 of p53 (pS15-p53) and the levels of p53 and p21 CIP1 (p21) were assessed by Western blot. Protein levels were quantified relative to the β-actin and HCEC-hTERT cells. ( C ) The ectopic expression of p53 variants does not facilitate the escape of HCEC-hTERT cells from chronic-inflammation-induced senescence. Upper panels: cells were successively infected with mutant p53 retroviral-expressing vectors (as indicated on top), cultured in the presence of activated macrophage supernatants (+AMS) for ten days, and stained with crystal violet. Esc-Inf cells were used as an internal positive control. Percentages of stained surface are indicated. Lower panel: analysis of p53 by Western blot. Protein levels were quantified relative to the β-actin and HCEC-hTERT cells. Uncropped Western blots are provided in .

Article Snippet: Human primary colonic epithelial cells (HCEC) were purchased from Applied Biological Materials (abm, Richmond, Canada) and immortalized through transduction of hTERT (HCEC-hTERT).

Techniques: Mutagenesis, Phospho-proteomics, Western Blot, Expressing, Infection, Retroviral, Cell Culture, Staining, Positive Control

Journal: Cancers

Article Title: Intestinal Epithelial Cells Adapt to Chronic Inflammation through Partial Genetic Reprogramming

doi: 10.3390/cancers15030973

Figure Lengend Snippet: Escape from chronic-inflammation-induced or TBHP-induced senescence relies on the induction of ZEB genes. ( A ) Relative expression of SNAI1 , TWIST2 , ZEB1, and ZEB2 in cells that escaped from chronic-inflammation- (left panel) or TBHP-induced senescence (right panel), as assessed by qRT-PCR. Levels are expressed relative to the housekeeping HPRT1 gene and are normalized against HCEC-hTERT cells. One sample t and Wilcoxon test and the mean and SD of three independent experiments are shown. * p < 0.05 Central panel: analysis of ZEB1 in Esc-Inf cells by Western blot. EMT-committed Hs578T breast cancer cells were used as a positive protein detection control. Protein levels were quantified relative to the α-tubulin and Esc-Inf cells. ( B ) Ectopic expression of murine Zeb1 or Zeb2 facilitates escape from chronic inflammation-induced (+AMS, left panels) or chemical, peroxide-induced (+TBHP, right panels) senescence, as assessed in a crystal violet assay. Percentages of stained surface are indicated. Uncropped Western blots are provided in .

Article Snippet: Human primary colonic epithelial cells (HCEC) were purchased from Applied Biological Materials (abm, Richmond, Canada) and immortalized through transduction of hTERT (HCEC-hTERT).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Crystal Violet Assay, Staining

Journal: Cancers

Article Title: Intestinal Epithelial Cells Adapt to Chronic Inflammation through Partial Genetic Reprogramming

doi: 10.3390/cancers15030973

Figure Lengend Snippet: The ZEB-driven resistance relies on a partial genetic reprogramming. ( A ) Venn diagram highlighting the number of genes commonly up- or down-regulated (cut-off > 1.5) in HCEC-hTERT cells which had either escaped from chronic-inflammation-induced senescence (Esc-Inf A or B from two independent experiments), TBHP-induced senescence (Esc-TBHP A or B from two independent experiments), or ectopically expressed either Zeb1 or Zeb2 (HCEC- Zeb 1 and HCEC- Zeb 2) when compared to their HCEC-hTERT parental counterparts. Common up- and down-regulated genes constitute the basis for the OSAP program. ( B ) Listing of the genes commonly up-regulated (in red) or down-regulated (in green) in Esc-Inf (A and B), Esc-TBHP (A and B), HCEC- Zeb1 , and HCEC- Zeb2 compared to the parental HCEC-hTERT cells (cut-off ≥ 1.5). ( C ) Expression analysis of APOE and QPRT in cells that escaped either from chronic-inflammation-induced (upper panels) or TBHP-induced (lower panels) senescence. Left panels: expression analysis by qRT-PCR. Levels are expressed relative to the housekeeping HPRT1 gene and are normalized against HCEC-hTERT cells. One sample t and Wilcoxon test and the mean and SD of three independent experiments are shown. Right panels: analysis of APOE and QPRT by Western blot. Protein levels were quantified relative to the β-actin and HCEC-hTERT cells. ** p < 0.01, * p < 0.05. Uncropped Western blots are provided in .

Article Snippet: Human primary colonic epithelial cells (HCEC) were purchased from Applied Biological Materials (abm, Richmond, Canada) and immortalized through transduction of hTERT (HCEC-hTERT).

Techniques: Expressing, Quantitative RT-PCR, Western Blot