hct116 human colorectal cancer cells  (ATCC)

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: Platelet activation and binding to colorectal cancer cells. Whole blood obtained from healthy individuals ( n = 5) was treated with DPBS or 1mM aspirin before incubation with different concentrations of HCT15 or HCT116 colorectal cancer cells. Platelet activation was measured using flow cytometry for the platelet activation markers ( a ) P-selectin, ( b ) LAMP-3 and ( c ) activated GPIIb/IIIa. Representative images of platelets binding to d) HCT15 and e) HCT116 cells. Platelets were stained using an anti-CD41 antibody (green) and CRC cells were stained using an anti-EpCAM antibody (red) and DAPI (blue) and imaged using immunofluorescence microscopy. Data presented as box whisker plots for n = 5 experiments. * P < 0.05, ** P < 0.01

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Activation Assay, Binding Assay, Incubation, Flow Cytometry, Staining, Immunofluorescence, Microscopy, Whisker Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: The effect of platelets and platelet releasate on colorectal cancer cell migration and invasion. Inserts in transwell assays were ( a ) uncoated to measure migration or ( b ) Matrigel coated to measure invasion of HCT15 and HCT116 cells co-incubated with platelets or platelet-releasate from healthy individuals ( n = 5) for 24 h. Platelets were pretreated with DPBS or 1mM aspirin and platelet releasate was generated from platelets pretreated with DPBS or aspirin. Data is presented box whisker plots for n = 5 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Incubation, Generated, Whisker Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: The effect of platelets and platelet releasate on colorectal cancer cell proliferation. a ) HCT15 and b ) HCT116 cells stained with CFSE were co-incubated with platelets or platelet releasate from healthy individuals ( n = 6) for 48 h. Platelets were pretreated with DPBS or 1mM aspirin and platelet releasate was generated from platelets pretreated with DPBS or aspirin. The cells were analysed using flow cytometry and ModFit LT software which calculated a proliferation index. Data is presented box whisker plots for n = 6 experiments. ( c ) HCT15 and ( d ) HCT116 cells were co-incubated for 48 h with platelets from healthy individuals ( n = 5) pretreated with DPBS or 1mM aspirin, with proliferation determined using MTS. Data is presented as the mean with error bars representing the standard error of the mean for n = 5 experiments. ( e ) HCT15 and ( g ) HCT116 cells were incubated with 1mM aspirin for 48 h ( n = 10) and the effect it had on proliferation was determined using MTS proliferation assays. Data is presented box whisker plots for n = 10 experiments. ** P < 0.01

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Staining, Incubation, Generated, Flow Cytometry, Software, Whisker Assay

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal inhibit the formation of endogenous TopBP1 condensates. ( A ) Representative immunofluorescence images of TopBP1 foci in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h, and ( B ) the corresponding quantification. ( C ) Representative immunofluorescence images of TopBP1 foci in HCT116 cells incubated with thimerosal (20 µM) and/or SN-38 (300 nM) for 2 h, and ( D ) the corresponding quantification. The experiment was repeated three times with similar results. Data from the three independent replicates were pooled and displayed as superplots in panels (B) and (D), error bars represent the standard error of the mean. ( E ) Representative immunofluorescence images of PML nuclear bodies in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h and ( F ) the corresponding quantification. “−” denotes the absence of the indicated compound (control condition). CellProfiler 4.2.8 was used to quantify TopBP1 and PML nuclear foci (>300 cells analyzed per condition). Statistical significance was assessed using one-way ANOVA followed by Šídák’s post hoc test. ns, non-significant; * P -value <.05; **P-value <.01; and ****P-value <.0001. Scale bars, 10 µm for TopBP1 images and 20 µm for PML images.

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Immunofluorescence, Incubation, Control

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal inhibit ATR/Chk1 signaling activation in cell-free extracts and in living cells. ( A ) Immunoblot showing phosphorylated Chk1 (pChk1) levels after incubation of nuclear extracts from optoTopBP1-HEK293 cells with increasing concentrations of quinacrine (QC) or ( B ) thimerosal (TH) for 30 min before induction of TopBP1 condensation at 37°C. The experiments were reproduced three times for QC in panel (A) and once for TH in panel (B). ( C ) Fractionation experiment to monitor TopBP1 association with chromatin in HCT116 cells incubated with quinacrine (40 µM) and/or SN-38 (300 nM) for 2 h. The experiments were reproduced three times. ( D ) Immunoblot showing the effect of SN-38 and/or quinacrine on histone marks in HCT116 cells. H3K4me3, H3K9Ac, and H3K9me3 levels were assessed to evaluate changes in chromatin state. Representative immunofluorescence images of pChk1 signals obtained with a Celigo Imaging Cytometer in HCT116 cells incubated with SN-38 (300 nM) and increasing concentrations of quinacrine (4.4, 13.3, and 40 µM) ( E ) and the corresponding quantification ( F ) or increasing concentrations of thimerosal (2.2, 6.7, and 20 µM) for 2 h ( G ) and the corresponding quantification ( H ). Error bars represent the standard deviation from three independent experiments; ns, non-significant; * P -value <.05; ** P- value <.01; and **** P -value <.0001 (one-way ANOVA; SN-38 alone versus SN-38 with increasing doses of quinacrine or thimerosal).

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Activation Assay, Western Blot, Incubation, Fractionation, Immunofluorescence, Imaging, Cytometry, Standard Deviation

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine and thimerosal effects on RPA and H2AX phosphorylation. Immunoblots to assess the phosphorylation (p) level of the indicated ATR substrates in HCT116 cells incubated with SN-38 (300 nM) and/or quinacrine (4.4, 13.3, and 40 µM) ( A ) or thimerosal (2.2, 6.7, and 20 µM) ( B ) for 2 h and the corresponding quantifications ( C, D ) displayed as fold change over vinculin, normalized to non-treated cells. Bars represent mean ± standard error of the mean from two biological replicates. Two-dimensional flow cytometry analysis of pRPA32 (Ser33)/DAPI ( E ) and yH2AX (Ser139)/DAPI ( F ) fluorescence signals by flow cytometry after the extraction of soluble proteins from HCT116 cells, incubated with SN-38 (300 nM) and/or quinacrine (40 µM) or thimerosal (20 µM), as indicated.

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Phospho-proteomics, Western Blot, Incubation, Flow Cytometry, Fluorescence, Extraction

Journal: NAR Cancer

Article Title: Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors

doi: 10.1093/narcan/zcaf041

Figure Lengend Snippet: Quinacrine in combination with FOLFIRI displays additive and synergistic effects in 2D and 3D spheroid cell culture models (next page). Viability matrix (blue) and synergy matrix (red) of human HCT116 ( A ) and murine CT26 cells ( B ) incubated with increasing concentrations of quinacrine and FOLFIRI. FOLFIRI concentration for HCT116 cells: 5FU (from 0.009 to 0.148 µM), SN-38 (from 0.077 to 1.235 nM); and for CT26 cells: 5FU (0.083 to 1.333 µM), SN-38 (from 0.694 to 11.11 nM). Quinacrine concentration from 0.188 to 3 µM. Cell viability was assessed with the SRB assay (2D). Viability matrix (green) and cytotoxicity matrix (orange) of HCT116 ( C ) and CT26 ( D ) cells incubated with the same quinacrine and FOLIFRI concentrations; values were obtained with the “Dead + Total” cell viability application of the Celigo Imaging Cytometer (Nexcelom) using Hoechst/propidium iodide staining. Viability and synergy matrices of 3D spheroid models of HCT116 ( E ) and CT26 ( F ) cells (same quinacrine and FOLIFRI concentrations as before); viability was measured with the 3D-CellTiterGlo assay. Viability and synergy matrices of the SN-38-resistant HCT116-SN6 (SN6) ( G ) and HCT116-SN50 (SN50) ( H ) cell lines in 2D cultures; FOLFIRI: 5FU (from 0.083 to 1.133 µM and from 0.75 to 12 µM), SN-38 (from 0.694 to 11.11 nM and from 6.25 to 100 nM); quinacrine: from 0.188 to 3 µM (for both cell lines). Viability and synergy matrices of the SN-38-resistant HCT116-SN6 (SN6) ( I ) and HCT116-SN50 (SN50) ( J ) cell lines in 3D spheroid cultures; FOLFIRI: 5FU (from 0.0625 to 0.08 µM and from 0.25 to 4 µM), SN-38 (from 0.52 to 8.33 nM and from 2.08 to 33.33 nM); quinacrine: from 0.188 to 3 µM (for both cell lines). Cell viability was quantified with the 3D-CellTiterGlo assay. Representative brightfield images of spheroids were obtained with a Celigo imaging cytometer (Nexcelom). The synergy matrix was calculated as described in the “Materials and methods” section. Cells were incubated for 96 h (2D culture) and for 7 days (3D cultures).

Article Snippet: The HCT116 human colorectal cancer cell line (ATCC, #CCL247), LNCaP human prostate cancer cell line (CRL-1740), and CT26 murine colorectal cancer cell line were cultured in RPMI-1640 (Sigma, #R8758; 500 ml) with 10% heat-inactivated FBS.

Techniques: Cell Culture, Incubation, Concentration Assay, Sulforhodamine B Assay, Imaging, Cytometry, Staining