human crc cell lines ht29  (ATCC)

Journal: bioRxiv

Article Title: A Sequential Triple-Drug Strategy for Selective Targeting of p53-Mutant Cancers

doi: 10.1101/2025.11.06.687028

Figure Lengend Snippet: ( a-c ) Cell cycle analysis was performed on cell lines treated with vehicle control (C), 500nM TAS102 and 100nM talazoparib for 24 hours ( T0 ) followed by incubation in drug-free medium. All experiments were done in at least two independent biological replicates. ( d-g ) Immunoblotting on whole-cell extracts from HCT116-p53WT, HCT116-p53KO, HCT116-p53 R248W/- and HT29 cell lines treated as described above ( a-c ). The experiments were repeated in at least two independent biological replicates and two technical replicates. Blots were probed with antibodies to phopsho-CHK1, p53, cyclin D1, phospho-CDK1, p21, phospho-H2AX (γH2AX), phospho-histone H3, and GAPDH as a loading control.

Article Snippet: The human CRC cell lines HT29 (RRID:CVCL_0320) and DLD1 (RRID:CVCL_0248), human non-tumor breast epithelial MCF10A (RRID: CVCL_0598) and human breast cancer MDA-MB-231 (RRID:CVCL_0062) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended by ATCC.

Techniques: Cell Cycle Assay, Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: A Sequential Triple-Drug Strategy for Selective Targeting of p53-Mutant Cancers

doi: 10.1101/2025.11.06.687028

Figure Lengend Snippet: ( a-e ) Cytotoxicity curves for p53 wild-type (MCF10A and HCT116), and p53-deficient (HCT116-p53KO, HCT116-p53 R248W/- , HT29) cells. Cells were treated with increasing concentrations of TAS102 plus 100nM talazoparib for 24 hours. After a 24-hour drug washout, cells were incubated with 50, 100, 250, or 500 nM of the WEE1 inhibitor (WEE1i) for an additional 72 hours. ( f ) Combination index (CI) analysis for the tested WEE1i concentrations. Data are presented as mean ± SEM from at least two independent biological replicates, each with six technical replicates. ( g ) Flow cytometry analysis of cells treated with vehicle control or a sequential regimen of 500 nM TAS102 plus 100 nM talazoparib, followed by 250 nM WEE1i. Cells were stained with propidum iodide and annexin-V. ( h-k ) Quantification of Annexin V and propidium iodide (PI) double-positive cells from two biological replicates. Statistical significance was determined using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Article Snippet: The human CRC cell lines HT29 (RRID:CVCL_0320) and DLD1 (RRID:CVCL_0248), human non-tumor breast epithelial MCF10A (RRID: CVCL_0598) and human breast cancer MDA-MB-231 (RRID:CVCL_0062) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended by ATCC.

Techniques: Incubation, Flow Cytometry, Control, Staining

Journal: bioRxiv

Article Title: A Sequential Triple-Drug Strategy for Selective Targeting of p53-Mutant Cancers

doi: 10.1101/2025.11.06.687028

Figure Lengend Snippet: ( a-d ) SCID mice (10mice/group) were subcutaneously implanted with colon cancer HT29 cells. At tumor size of 100mm 3 , mice were randomized into four treatment groups: vehicle control, WEE1 inhibitor (WEE1i, MK1775, 60 mg/kg), a combination of TAS102 (50 mg/kg) and talazoparib (0.15 mg/kg), and a sequential regimen consisting of one week of TAS102/talazoparib followed by one week of the WEE1 inhibitor. All treatments were administered via oral gavage on a schedule of five days ON and two days OFF. Tumor growth was measured twice weekly, and statistical significance was determined using a two-way ANOVA (*p < 0.05). ( b ) Tumor growth inhibition (TGI) was calculated as TGI = 100% x [Vehicle − Treatment) / Vehicle]. Comparisons were made using a one-way ANOVA (**, P<0.01, ***, P<0.001, ****, P<0.001). ( c ) Survival was evaluated using Kaplan-Meier estimator based on time-to-arrive at 750 mm 3 of tumor size. The comparison was made using the log-rank test. ( d ) Mouse body weights were measured twice per week over the course of study. ( e-h ) Patient-derived tumor xenografts (PDXs) from a patient with PDAC (PDAC-14312-4p) were subcutaneously implanted into SCID mice (10 mice/group). At 100 mm 3 , mice were randomized and treated as described above for HT29 xenografts. ( f ) Tumor growth inhibition in the PDX model was calculated as described in ( b ). Comparisons were made using a one-way ANOVA (**, P<0.01, ***, P<0.001, ****, P<0.001). ( g ) Survival was evaluated using Kaplan-Meier estimator based on time to arrive at 750 mm3 of tumor size. Group comparisons were performed with the log-rank test. ( h ) Mouse body weights were measured twice weekly over the course of study. ( i-l ) Patient-derived tumor xenografts (PDXs) from a colorectal cancer patient (PDX-3B1-29-RP-3p) were subcutaneously implanted into SCID mice (10 mice/group). Experiments and the analysis were done essentially as described for ( e-h ). ( m ) A model of the triple-drug drug strategy acting as an inducer-amplifier-terminator trio in p53 mutant cancers.

Article Snippet: The human CRC cell lines HT29 (RRID:CVCL_0320) and DLD1 (RRID:CVCL_0248), human non-tumor breast epithelial MCF10A (RRID: CVCL_0598) and human breast cancer MDA-MB-231 (RRID:CVCL_0062) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended by ATCC.

Techniques: Control, Inhibition, Comparison, Derivative Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: Targeting the Met-RIPK1 signaling axis to enforce apoptosis and necroptosis in colorectal cancer

doi: 10.1038/s41419-025-08054-5

Figure Lengend Snippet: A HT29 cells treated with indicated doses of Cabozantinib for 48 h. Remaining fraction of adherent cells quantified via crystal violet. Mean of three independent experiments ± standard deviation (SD) ( n = 3). p < 0.05 (*), p > 0.05 (no significance, n.s.) (Student’s t test). B HT29 cells pre-treated with Cabozantinib (5 μM for two hours), followed by indicated doses of BV6 for 48 h. Fractional survival quantified via crystal violet. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) (Student’s t test). C Representative microscopy images illustrating morphological signs of cell death. D Western blot for cleaved Caspase-3 in HT29 cells treated with Cabozantinib and BV6 in the presence or absence of pan-caspase inhibitor ZVAD (25 μM, representative data of three independent experiments). E Fractional cellular survival after 48 h of treatment with BV6 (0.6 μM), Cabozantinib, or ZVAD. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*) (Student’s t test). F Western blot for pMLKL (representative data of three independent experiments).

Article Snippet: The human CRC cell line HT29 (ATCC) was maintained in RPMI-1640 medium (Gibco, 21875-034) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P0781) at 5% CO 2 and 37 °C.

Techniques: Standard Deviation, Microscopy, Western Blot

Journal: Cell Death & Disease

Article Title: Targeting the Met-RIPK1 signaling axis to enforce apoptosis and necroptosis in colorectal cancer

doi: 10.1038/s41419-025-08054-5

Figure Lengend Snippet: A HT29 cells treated with Cabozantinib (5 μM) and BV6 (0.6 μM) in the presence or absence of RIPK1 kinase inhibitor Necrostatin-1 (10 μM). Fractional survival after 48 h quantified via crystal violet. Mean of three independent experiments ± standard deviation (SD) ( n = 3). p < 0.01 (**) (Student’s t test). B Fractional survival relative to control cells shown in Fig. 2A after 48 h of indicated treatments, here in the presence of ZVAD (25 μM). Mean of three independent experiments ± SD ( n = 3). p < 0.01 (**), p < 0.001 (***) (Student’s t test). C , D Western blot for PARP and Caspase-3 in HT29 treated with indicated substances for 30 h. Relative quantification of cleaved protein normalized to proform. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**), (Student’s t test). E , F Western blot for pRIPK3 and pMLKL in HT29. Relative quantification of phosphorylated normalized to total protein. Mean of three independent experiments ± SD ( n = 3). p > 0.05 (no significance, n.s.), p < 0.01 (**), (Student’s t test). G , H Fractional survival in HT29 wild-type (CRISPR control) and CRISPR/Cas9-knockout (KO) cells after 48 h of indicated treatments. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (Student’s t test).

Article Snippet: The human CRC cell line HT29 (ATCC) was maintained in RPMI-1640 medium (Gibco, 21875-034) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P0781) at 5% CO 2 and 37 °C.

Techniques: Standard Deviation, Control, Western Blot, Quantitative Proteomics, CRISPR, Knock-Out

Journal: Cell Death & Disease

Article Title: Targeting the Met-RIPK1 signaling axis to enforce apoptosis and necroptosis in colorectal cancer

doi: 10.1038/s41419-025-08054-5

Figure Lengend Snippet: A , B Fractional survival in HT29 wild-type (CRISPR control) and CRISPR/Cas9-knockout (TNFR KO) cells after 48 h of indicated treatments (BV6: 0.6 μM, Cabozantinib: 5 μM, ZVAD: 25 μM) quantified via crystal violet. Mean of three independent experiments ± standard deviation (SD) ( n = 3). p < 0.05 (*), p < 0.01 (**) (Student’s t test). C Western blot for pMet and Met in HT29 after indicated treatments. Mean of three independent experiments ± SD ( n = 3). p < 0.01 (**), p < 0.001 (***) (Student’s t test). D–F Relative mRNA expression in HT29 after indicated treatments measured via qRT-PCR. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (Student’s t test). G Western blot for RIPK1 in HT29 treated with Cabozantinib for 24 h (representative data of three independent experiments). H Relative quantification of RIPK1 protein in Cabozantinib-treated cells in Fig. 3G. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*) (Student’s t test). I–K : Relative mRNA expression measured via qRT-PCR. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (Student’s t test).

Article Snippet: The human CRC cell line HT29 (ATCC) was maintained in RPMI-1640 medium (Gibco, 21875-034) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P0781) at 5% CO 2 and 37 °C.

Techniques: CRISPR, Control, Knock-Out, Standard Deviation, Western Blot, Expressing, Quantitative RT-PCR, Quantitative Proteomics

Journal: Cell Death & Disease

Article Title: Targeting the Met-RIPK1 signaling axis to enforce apoptosis and necroptosis in colorectal cancer

doi: 10.1038/s41419-025-08054-5

Figure Lengend Snippet: A HT29 cells were subcutaneously injected into left and right flank of CD1 nude mice. When tumors were palpable (day 0), mice were divided into eight groups with five animals per group to apply apoptotic (left) or necroptotic treatments (right). Every 3 or 4 days, mice received drugs and tumor volumes were measured. Data represent average tumor sizes per group ( n = 10). B Tumor volumes on day 15 for apoptotic (left) and necroptotic treatment groups (right). Each circle represents one individual tumor. Bars are group average, dashed line is average of control group ( n = 10). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (one-way ANOVA with Bonferroni’s correction). C Scatter plot to illustrate inverse correlation between tumor sizes and the percentage of tumor area with high RIPK1 expression (see Supplementary Fig. , and Methods section). Each circle represents one individual tumor (total of n = 24, including n = 3 per treatment group) (r, Spearman correlation analysis). D Immunohistochemical stainings of cleaved Caspase-3, pRIPK3 and pMLKL, or Van Gieson’s Elastica (EVG) staining to quantify fibrotic tumor area in apoptotic (left, n = 4) or necroptotic treatment groups (right, n = 3). Quantification depicts percentage of positively stained tumor area; each circle represents one individual tumor, while bars represent mean ± standard error of the mean (SEM). p > 0.05 (no statistical significance, n.s.), p < 0.05 (*), p < 0.01 (**) (Student’s t test). E HMGB1 released by necroptotic HT29 cells after 24 h of treatment. Mean of three independent experiments ± standard deviation (SD) ( n = 3). p < 0.001 (***) (Student’s t test). F Time course of ATP release by necroptotic HT29 cells. Cycloheximide/TNF was included as an inducer of non-inflammatory cell death. Mean of three independent experiments ± SD ( n = 3).

Article Snippet: The human CRC cell line HT29 (ATCC) was maintained in RPMI-1640 medium (Gibco, 21875-034) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P0781) at 5% CO 2 and 37 °C.

Techniques: Injection, Control, Expressing, Immunohistochemical staining, Staining, Standard Deviation

Journal: Cell Death & Disease

Article Title: Targeting the Met-RIPK1 signaling axis to enforce apoptosis and necroptosis in colorectal cancer

doi: 10.1038/s41419-025-08054-5

Figure Lengend Snippet: A Patient-derived HROC cell lines treated with indicated doses of BV6 for 48 h. Fractional survival quantified via crystal violet assay. Mean of three independent experiments ± standard deviation ( n = 3) (SD). p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) (Student’s t test). B Western blot for PARP and Caspase-3 in HROC cells treated with BV6 (HROC24, 87: 0.6 μM; HROC46, 69: 5 μM) and ZVAD (25 μM) for 30 h. C Relative mRNA expression measured via qRT-PCR normalized to HT29 as a control. Mean of three independent experiments ± SD ( n = 3). D Fold induction of TNF mRNA expression by BV6 as measured via qRT-PCR. Mean of three independent experiments ± SD ( n = 3). E HROC cell lines treated with BV6 (HROC24, 87: 0.6 μM; HROC39, 40, 46, 69: 5 μM), ZVAD (25 μM) and Necrostatin-1 (10 μM) for 48 h. Fractional survival quantified via crystal violet assay. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.001 (**) or p < 0.001 (***). (Student’s t test). F Western blot for pMLKL in HROC cells treated as indicated in Fig. 5B. G Relative expression of RIPK3 mRNA measured via qRT-PCR and normalized to HT29. Mean of three independent experiments ± SD ( n = 3). H Cells treated with BV6 and ZVAD as specified in Fig. 5E. TNF secretion quantified in cell culture supernatants via ELISA, and normalized to HROC46. Means of three independent experiments ± SD ( n = 3). I Cells treated as specified in Fig. 5E, in the presence or absence of TNF (25 ng/ml). Fractional survival quantified via crystal violet assay. Mean of three independent experiments ± SD ( n = 3). p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) (Student’s t test).

Article Snippet: The human CRC cell line HT29 (ATCC) was maintained in RPMI-1640 medium (Gibco, 21875-034) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P0781) at 5% CO 2 and 37 °C.

Techniques: Derivative Assay, Crystal Violet Assay, Standard Deviation, Western Blot, Expressing, Quantitative RT-PCR, Control, Cell Culture, Enzyme-linked Immunosorbent Assay