human colon carcinoma cell line ht29  (ATCC)

Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency disrupts mitochondrial homeostasis in CRC cells. (A) Western blotting verified the reduction of PLSCR3 protein levels in HT29 cells. (B) RT‐qPCR analysis verified reduction of PLSCR3 mRNA levels normalized to GAPDH compared with negative control (NC) cells ( p < 0.0001, n = 3). (C) Representative JC‐1 staining (scale bar: 20 μ m) images showing red fluorescence (polarized mitochondria) and green fluorescence (depolarized mitochondria). (D) Quantification of the JC‐1 red/green fluorescence intensity ratio. Fluorescence intensity was quantified using ImageJ software as described in the section. (E) Real‐time oxygen consumption rate (OCR) profiles under sequential treatment with oligomycin, FCCP, and rotenone/Antimycin A. (F) Quantification of cytosolic mtDNA by qPCR using the D‐loop ratio in PLSCR3‐knockdown and control cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using an unpaired two‐tailed Student′s t ‐test for two‐group comparisons.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Quantitative RT-PCR, Negative Control, Staining, Fluorescence, Software, Knockdown, Control, Two Tailed Test

Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency activates mtDNA‐associated cGAS‐STING signaling. (A) 2 ′ ‐3 ′ ‐cGAMP quantification by ELISA in PLSCR3 knockdown (KD_PLSCR3) HT29 cells and normal controls (NCs) treated with Scramble or dideoxycytidine (DDC) ( p < 0.0001; ns = not significant; n = 3). (B, C) KD_PLSCR3 cells exhibited an elevation in IFN β and CXCL10 production measured by ELISA compared to NC ( p < 0.0001, n = 3). CRISPR‐mediated STING knockout (STING sg) completely abrogated these effects. (D) Western blot analysis showed upregulation of phosphorylated STING and innate immune effectors (IFIH1, ISG60, and ISG15 protein) in STING‐competent KD_PLSCR3 cells relative to GAPDH loading controls. (E) Normalized mRNA level quantification by qPCR demonstrated significant induction of interferon‐stimulated genes (ISGs) in STING‐competent KD_PLSCR3 cells compared with the other groups. Data in panels A–C and E are presented as mean ± SD. Statistical significance was determined using one‐way ANOVA followed by Tukey′s multiple‐comparisons post hoc test for multiple‐group comparisons.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, CRISPR, Knock-Out, Western Blot

Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency enhances the sensitivity to immune cell–mediated killing in CRC. (A) Schematic of coculture system: PLSCR3 knockdown (KD_PLSCR3) HT29 cells or control were cocultured with human cord blood–derived NK cells at an E:T ratio of 5:1. (B, C) Phase‐contrast microscopy (scale bar: 50 μ m) showing morphological changes. Morphological analysis demonstrated decreased cell number in KD_PLSCR3 cells in the coculture condition ( p < 0.0001, n = 5). (D) Mouse graft carcinoma with control or PLSCR3 knockout (PLSCR3 sg) CT26 cells were treated with or without anti‐PD‐1 antibodies ( α PD‐1). Tumor growth curves (mm 3 ) were recorded at Days 5, 10, 15, and 20 after tumor cell inoculation in immunocompetent BALB/c mice. (E) At Day 20, terminal tumor weights in the mouse graft carcinoma were measured. (F–H) Flow cytometry analysis of CD4+ and CD8+ tumor‐infiltrating T cells. The representative cell populations of CD4+ and CD8+ are shown. (I, J) Flow cytometry and ELISA analysis of Granzyme B (GZMB) expression ( n = 5). Data are presented as mean ± SD. Tumor growth curves were analyzed using two‐way repeated‐measures ANOVA with post hoc multiple‐comparison tests. For multiple‐group comparisons, including panels I and J, one‐way ANOVA followed by Tukey′s post hoc test was used.

Article Snippet: The human colon carcinoma cell line HT29 (Cat. No. ATCC‐HTB‐38) and the mouse colon carcinoma cell line CT26.WT (Cat. No. CRL‐2638, ATCC, Virginia, United States) were maintained in RPMI 1640 medium (Cat. No. BC‐M‐017, Bio‐Channel, Jiangsu, China) supplemented with 10% fetal bovine serum.

Techniques: Knockdown, Control, Derivative Assay, Microscopy, Knock-Out, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Comparison

Journal: Biomedicines

Article Title: Ribosome-Inactivating Proteins from Salsola soda L. and Saponaria officinalis L. Are Promising Candidates for Targeted Therapy of Colon Cancer

doi: 10.3390/biomedicines14050981

Figure Lengend Snippet: Effect of sodin 5 and saporin-S6 on cell viability in HT29 and Caco-2 cells. HT29 ( a ) and Caco-2 ( b ) cell viabilities were evaluated at 24, 48 and 72 h after exposure to the indicated concentrations of sodin 5 (red lines) and saporin-S6 (blue lines). Cell viability was measured using a colorimetric assay based on MTS reduction. The mean results ± S.D. are reported, representing the percentage of control values. Data were analyzed by ANOVA/Bonferroni test, followed by a comparison with Dunnett’s test (confidence range 95%; ** p < 0.01, *** p < 0.001, **** p < 0.0001, versus untreated controls). Asterisks indicate the first concentration at which a statistically significant reduction in cell viability is observed compared to controls. The morphology of HT29 ( c ) and Caco-2 ( d ) cells was evaluated at the indicated times for control (Ctrl) and for treated cells (sodin 5 or saporin-S6 at 10 −6 M concentration). Images were taken using phase-contrast microscopy (total magnification 100×).

Article Snippet: The human colon adenocarcinoma cell lines HT29 (lot number 300215-921) and Caco-2 (lot number 300137-220424) were obtained from Cytion (Eppelheim, Germany).

Techniques: Colorimetric Assay, Control, Comparison, Concentration Assay, Microscopy

Journal: Biomedicines

Article Title: Ribosome-Inactivating Proteins from Salsola soda L. and Saponaria officinalis L. Are Promising Candidates for Targeted Therapy of Colon Cancer

doi: 10.3390/biomedicines14050981

Figure Lengend Snippet: Time-dependent TEER changes across Caco-2 monoculture, Caco-2/HT29 and Caco-2/3T3/collagen co-culture models. Caco-2 monoculture ( a ), Caco-2/HT29 ( b ) and Caco-2/3T3/collagen ( c ) co-culture models were established by seeding cells (6.25 × 10 4 Caco-2 cells/cm 2 for monoculture experiments, 6.25 × 10 4 Caco-2/HT29 cells/cm 2 , in a 9:1 ratio, for co-culture experiments) on 24-well Transwell inserts with 0.4 µm transparent polyester membrane. In the Caco-2/3T3/collagen co-culture model, 3T3 cells (0.78 × 10 4 cells/cm 2 ) were seeded on the apical compartment with 0.1 µg/mL of type 1 collagen in complete medium. After 1 h at 37 °C, Caco-2 cells (6.25 × 10 4 /cm 2 ) were seeded. In all experiments, complete medium (900 µL) was added to the basolateral compartment. All cultures were maintained about 10 days, replacing culture medium every 2–3 days. When TEER reached values ≥ 500 Ω × cm 2 (about 10 days), cell monolayers were treated with sodin 5 or saporin-S6 at 10 −6 M concentration. TEER values were recorded at 0, 8, 24, 32, 48 and 72 h after sodin 5 or saporin-S6 intoxication. Results are expressed as mean ± SD of three independent experiments, each conducted in triplicate. Data were analyzed by ANOVA/Bonferroni test, followed by a comparison with Dunnett’s test (confidence range 95%; ** p < 0.01, *** p < 0.001 versus untreated controls). Asterisks indicate the first timepoint at which a statistically significant reduction in TEER is observed compared to controls.

Article Snippet: The human colon adenocarcinoma cell lines HT29 (lot number 300215-921) and Caco-2 (lot number 300137-220424) were obtained from Cytion (Eppelheim, Germany).

Techniques: Co-Culture Assay, Membrane, Concentration Assay, Comparison

Journal: Biomedicines

Article Title: Ribosome-Inactivating Proteins from Salsola soda L. and Saponaria officinalis L. Are Promising Candidates for Targeted Therapy of Colon Cancer

doi: 10.3390/biomedicines14050981

Figure Lengend Snippet: Evaluation of cell death mechanisms triggered by sodin 5 and saporin-S6. HT29 ( a ) and Caco-2 ( b ) cells (4 × 10 5 /well) were seeded in 6-well plates and cultured for 24 h in the absence or presence of sodin 5 or saporin-S6 at 10 −6 M concentration. Apoptosis, necroptosis and necrosis were evaluated through flow cytometry analysis. Representative plots of Annexin V-EGFP (FITC channel)/PI (PE channel) staining of HT29 and Caco-2 cells are shown. Cell populations were characterized based on staining: necrotic cells (PI-positive and EGFP-negative) are in the upper left quadrant; late apoptotic and/or necroptotic cells (PI-positive and EGFP-positive) are in the upper right quadrant; early apoptotic cells (PI-negative and EGFP-positive) are in the lower right quadrant. The plots are representative of two independent experiments, each conducted in triplicate. The tables report the percentages of live, early apoptotic, late apoptotic and/or necroptotic and necrotic HT29 ( c ) and Caco-2 ( d ) cells after sodin 5 or saporin-S6 treatment. The protective effect of cell death inhibitors was evaluated on HT29 ( e ) and Caco-2 ( f ) cells (3 × 10 3 /well), treated with 10 −6 M sodin 5 or saporin-S6. The inhibitors Z-VAD (apoptosis) or necrostatin-1 (NEC, necroptosis) were added at 100 μM concentration 3 h before the RIP treatment. Cells were then treated for 2 h with sodin 5 or saporin-S6 and further incubated for 24 h in complete medium. Cell viability was evaluated using a colorimetric assay based on MTS reduction. The results are expressed as means ± S.D. of three independent experiments, each conducted in triplicate. Data were analyzed by the Mann–Whitney U test (confidence range 95%; ** p < 0.01, *** p < 0.001 versus RIP-treated samples).

Article Snippet: The human colon adenocarcinoma cell lines HT29 (lot number 300215-921) and Caco-2 (lot number 300137-220424) were obtained from Cytion (Eppelheim, Germany).

Techniques: Cell Culture, Concentration Assay, Flow Cytometry, Staining, Incubation, Colorimetric Assay, MANN-WHITNEY