Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Validation of CL16-RhoA binding by LC-MS/MS experiments (A) Workflow of LC-MS/MS experiments to detect RhoA-CL16 engagement in HCT116 cells. CL16-treated cells were harvested, digested and analyzed by LC-MS/MS. (B) Representative MS/MS showing CL16 modification on RhoA Cys16 in HCT116 cells. (C) Workflow of LC-MS/MS experiments using CL16-alkyne (a CL16-molecular probe) to study target profile of CL16. (D) Volcano plot revealing protein targets of CL16 identified by CL16-molecular probe. Statistical analyses were performed by two-tailed Student’s t-test by MS Excel. (E) Venn diagram summarizing the protein targets of CL16 identified in (A) and (C), highlighting RhoA as the primary target.

Article Snippet: Human: HCT116 (Wildtype/48Y/Male) , ATCC , #CCL-247.

Techniques: Biomarker Discovery, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Modification, Two Tailed Test

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Analysis of the selected senescence markers and anillin levels in HCT116 p53WT and MCF-7 cells induced to senesce by 1 day-treatment with doxorubicin and collected 5 days after senescence induction. ( A, B ) Densitometric analysis of protein levels in HCT116 p53WT ( A ) and MCF-7 ( B ), n=9; statistical analysis was performed using paired one-tailed t-Student test. Relative protein expression means fold change (in the expression of proteins relative to the expression of GAPDH) vs appropriate control. Boxes: Q1, median, Q3; error bars: Minimum, Maximum. ( C ) Representative images from Western blotting of HCT116 p53WT and MCF-7 cell lysates. ( D, E ) Analysis of fluorescence intensity of anillin (whole nucleus area) and representative images ( F, G ) of control and doxorubicin-treated HCT116 p53WT ( D, F ) and MCF-7 cells ( E, G ), n=3; statistical analysis was performed using Wilcoxon matched-pairs signed-rank test. Data on graphs represent individual values for analyzed cells, median, error bars: Minimum, Maximum. Red – anillin, blue – DAPI stained DNA. Scale 20 µm. Statistical significance relative to control: ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques: One-tailed Test, Expressing, Control, Western Blot, Fluorescence, Staining

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Analysis of selected senescence markers and anillin levels in HCT 116 p53KO cells induced to senescence by doxorubicin treatment for 1 day and collected 5 days after senescence induction. ( A ) Densitometric analysis of protein levels in control and doxorubicin-treated HCT116 p53KO based on Western blotting results, n=8; statistical analysis was performed using paired one-tailed t-Student test. Statistical significance is shown relative to control. ( B ) Representative images from Western blotting. ( C ) Comparison of anillin and p53 levels in p53-proficient (HCT116 p53WT and MCF7) and p53-deficient (HCT116 p53KO) cells treated with doxorubicin and analyzed using Western blotting, densitometric analysis (normalized to the level of anillin or p53 in control cells), n=3; statistical analysis was performed using Kruskal-Wallis test. Statistical significance is shown for differences between indicated cell lines. Relative protein expression means fold change (in the expression of proteins relative to the expression of GAPDH) vs appropriate control. ( D ) Representative images of immunostained control and doxorubicin-treated HCT116 p53KO cells. Red – anillin, green – lamin A/C, blue – DAPI stained DNA. Scale 20 µm. Boxes: Q1, median, Q3; error bars: Minimum, Maximum. Statistical significance: **** p ≤ 0.0001

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques: Control, Western Blot, One-tailed Test, Comparison, Expressing, Staining

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Correlation between p53 and anillin levels during senescence and the escape from senescence in breast cancer MCF-7 and colon cancer HCT116 p53WT cells (see ). ( A-B ). Representative Western blots showing the levels of anillin and p53 in HCT116 p53WT cells ( A ) and MCF-7 cells ( B ). ( C-F ) The level of anillin and p53 on subsequent days of cell culture after senescence induction by doxorubicin in HCT116 p53WT ( C and E ) and MCF-7 cells ( D and F ) n = 4; statistical analysis was performed using one-way ANOVA followed by post hoc analysis (Tukey’s honest significant difference test; HSD test). Statistical significance of differences between indicated days of treatment: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Boxes: Q1, median, Q3; error bars: Minimum, Maximum. ( G ) The heat map shows the levels of anillin and p53 during senescence and escape from senescence in MCF-7 and HCT116 p53WT cells. Heatmap: Original data points are standardized into z-scores.

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques: Western Blot, Cell Culture

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Inverse correlation between ANLN and p53 during senescence and escape from senescence in cancer cells. Downregulation of anillin is a consequence of the induction of p53 due to cellular senescence. After some time, senescent cells resume divisions, which is associated with a decrease in p53 levels and an increase in anillin. Performed with Biorender.

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques:

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Analysis of the selected senescence markers and anillin levels in HCT116 p53WT and MCF-7 cells induced to senesce by 1 day-treatment with doxorubicin and collected 5 days after senescence induction. ( A, B ) Densitometric analysis of protein levels in HCT116 p53WT ( A ) and MCF-7 ( B ), n=9; statistical analysis was performed using paired one-tailed t-Student test. Relative protein expression means fold change (in the expression of proteins relative to the expression of GAPDH) vs appropriate control. Boxes: Q1, median, Q3; error bars: Minimum, Maximum. ( C ) Representative images from Western blotting of HCT116 p53WT and MCF-7 cell lysates. ( D, E ) Analysis of fluorescence intensity of anillin (whole nucleus area) and representative images ( F, G ) of control and doxorubicin-treated HCT116 p53WT ( D, F ) and MCF-7 cells ( E, G ), n=3; statistical analysis was performed using Wilcoxon matched-pairs signed-rank test. Data on graphs represent individual values for analyzed cells, median, error bars: Minimum, Maximum. Red – anillin, blue – DAPI stained DNA. Scale 20 µm. Statistical significance relative to control: ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques: One-tailed Test, Expressing, Control, Western Blot, Fluorescence, Staining

Journal: Aging and Disease

Article Title: Anillin Recedes in p53-Dependent Senescence of Tumor Cells and Reappears in Cells Escaping from Senescence

doi: 10.14336/AD.2025.0402

Figure Lengend Snippet: Correlation between p53 and anillin levels during senescence and the escape from senescence in breast cancer MCF-7 and colon cancer HCT116 p53WT cells (see ). ( A-B ). Representative Western blots showing the levels of anillin and p53 in HCT116 p53WT cells ( A ) and MCF-7 cells ( B ). ( C-F ) The level of anillin and p53 on subsequent days of cell culture after senescence induction by doxorubicin in HCT116 p53WT ( C and E ) and MCF-7 cells ( D and F ) n = 4; statistical analysis was performed using one-way ANOVA followed by post hoc analysis (Tukey’s honest significant difference test; HSD test). Statistical significance of differences between indicated days of treatment: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Boxes: Q1, median, Q3; error bars: Minimum, Maximum. ( G ) The heat map shows the levels of anillin and p53 during senescence and escape from senescence in MCF-7 and HCT116 p53WT cells. Heatmap: Original data points are standardized into z-scores.

Article Snippet: The human HCT116 p53-proficient (referred to as HCT116 p53WT) colon cancer cell line and the breast cancer cell line MCF-7 were obtained from ATCC (HCT116 CCL-247; MCF-7 HTB-22).

Techniques: Western Blot, Cell Culture

Journal: Biochemistry and Biophysics Reports

Article Title: Integrated transcriptomic and proteomic validation identifies SLC35D3 as a tumor-selective surface antigen for colorectal and neuroendocrine carcinomas

doi: 10.1016/j.bbrep.2026.102587

Figure Lengend Snippet: Immunohistochemical analysis of SLC35D3 expression in human tumors, normal tissues, and positive-control samples. (A) SLC35D3 staining in human tumors: (a) primary colon tumor; (b) corresponding lymph-node metastasis; (c) normal adjacent tissue (NAT) of a; (d) primary rectal tumor; (e) corresponding lymph-node metastasis; (f) NAT of d; (g–h) small-cell lung carcinoma (SCLC); (i) pancreatic neuroendocrine neoplasm; (j) pancreatic islet tumor. (B) SLC35D3 staining in human normal tissues: (a) cerebrum; (b) bone marrow; (c) lung; (d) heart; (e) liver; (f) kidney; (g) eye; (h) colon; (i) adrenal gland; (j) pancreas; (k) hypophysis; (l) stomach; (m) small intestine; (n) prostate. Arrowheads indicate SLC35D3-positive cells. (C) SLC35D3 staining of negative and positive control cell lines: (a) HCT 116-mock (negative control); (b) HCT 116-hSLC35D3 (engineered overexpression); (c) NCI–H1930 (endogenously SLC35D3-expressing).

Article Snippet: The human pancreatic islet cell carcinoma line QGP-1 (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan; Cat. No. JCRB0183), human small-cell lung carcinoma line NCI–H1930 (American Type Culture Collection (ATCC), Manassas, VA, USA; Cat. No. CRL-5906), human colorectal carcinoma line LoVo (ATCC; Cat. No. CCL-229), human gastric carcinoma line SNU-16 (ATCC; Cat. No. CRL-5974), and human colorectal carcinoma line HCT 116 (ATCC; Cat. No. CCL-247) were used.

Techniques: Immunohistochemical staining, Expressing, Positive Control, Staining, Negative Control, Over Expression

Journal: Biochemistry and Biophysics Reports

Article Title: Integrated transcriptomic and proteomic validation identifies SLC35D3 as a tumor-selective surface antigen for colorectal and neuroendocrine carcinomas

doi: 10.1016/j.bbrep.2026.102587

Figure Lengend Snippet: Validation of cell-surface SLC35D3 expression in cancer cell lines by flow cytometry and comparison with CCLE transcriptomic data. Flow cytometry histograms of cell-surface SLC35D3 staining in human cancer cell lines (HCT 116, LoVo, QGP-1, NCI–H1930, and SNU-16). For each cell line, the MFI ratio (anti-SLC35D3/isotype) and the corresponding mRNA expression level (log2[TPM+1]) from the CCLE are indicated below the histogram. HCT 116 served as a negative control and showed minimal surface staining, consistent with a CCLE value of log2[TPM+1] = 0.0.

Article Snippet: The human pancreatic islet cell carcinoma line QGP-1 (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan; Cat. No. JCRB0183), human small-cell lung carcinoma line NCI–H1930 (American Type Culture Collection (ATCC), Manassas, VA, USA; Cat. No. CRL-5906), human colorectal carcinoma line LoVo (ATCC; Cat. No. CCL-229), human gastric carcinoma line SNU-16 (ATCC; Cat. No. CRL-5974), and human colorectal carcinoma line HCT 116 (ATCC; Cat. No. CCL-247) were used.

Techniques: Biomarker Discovery, Expressing, Flow Cytometry, Comparison, Staining, Negative Control

Journal: Aging and Disease

Article Title: Postbiotic Parabacteroides Distasonis Supplementation Enhances Intestinal and Skeletal Muscle Function in Aged Mice

doi: 10.14336/AD.2025.0188

Figure Lengend Snippet: Postbiotic Pd modifies mitochondria in a colon cell line. (A) Right panel-representative images of HCT116 treated or not with postbiotic Pd for 24h and labeled with TMRE to determine mitochondrial morphology. Bar = 10 µm. Left panel- bar graphs of the mitochondrial length analysis. Data are expressed as MEAN ± SEM of five independent experiments ****p≤0.0001. Mann-Whitney test . (B) Upper panel- representative Western blot of PGC1α and GADPH as a loading control in HCT116 cells treated or not with postbiotic Pd. Bottom panel- Bar graphs represent quantification of PGC1α/GADPH expressed as MEAN ± SEM of five independent experiments. ∗p < 0.05. Mann-Whitney test . (C) Upper panel- representative Seahorse trace of HCT116 cells treated or not with postbiotic Pd. A; oligomycin (1 µM), B;FCCP (250 µM), C; rotenone plus antimycin A (1 µM each). Bottom panel- basal and maximum OCR of HCT116 cells treated or not with postbiotic Pd. MEAN ± SEM of three independent experiments with 10 replicates each. ***P < 0.001 compared to control. Mann-Whitney test. (D) Upper panel- representative Western blot of CHOP and β-actin as a loading control in HCT116 cells treated or not with postbiotic Pd. Bottom panel- bar graphs represent quantification of CHOP/β-actin expressed as MEAN ± SEM of 3 independent experiments. ∗p < 0.05. Mann-Whitney test . (E) Upper panel- representative Western blot of VDAC1 and β-actin as a loading control in colon mucosa samples from 26-months-old mice treated or not with postbiotic Pd. Bottom panel- bar graphs represent quantification of VDAC1/β-actin expressed as MEAN ± SEM. Note that β-actin blot in is the same as in this figure, as the same membrane was stripped and re-probed for VDAC1. N = 3 in the control group and 4 for the Pd treated group. ∗p < 0.05. Mann-Whitney test .

Article Snippet: Human colorectal cell line HCT116 (ATCC) was maintained at 37oC (95%/5% air/CO 2 ) in DMEM media (GIBCO) supplemented with 10% (v/v) FBS.

Techniques: Labeling, MANN-WHITNEY, Western Blot, Control, Membrane