cell lines cultures ht29 cells  (ATCC)

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: TCN induces necroptosis in apoptosis-resistant cancer cells. Cells were pretreated with zVAD.fmk (20 μM) for 1 h, followed by different dosages of TCN (0, 1, 2 μM) treatment for 24 h. Cell viability of (A) HT29 and (B) Raji cells were analyzed by CCK-8 assay. (C) Cells were stained with SYTOX Green Nucleic Acid Stain and imaged by fluorescent microscopy. The intensity of green fluorescence was analyzed using Image J software and shown in bar graphs. Scale bar, 50 μm. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (**, ***) indicate significant differences (P < 0.01, P < 0.001, respectively). (D) Representative electron microscope photographs of HT29 and Raji cells pretreated with zVAD.fmk (20 μM) for 1 h, followed by TCN (0, 1 μM) treatment for 24 h. TCN treatment resulted in necroptotic cell death, which was featured by plasma membrane rupture, organelle swelling and vacuolation as indicated by yellow arrows in HT29 and Raji cells.

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: CCK-8 Assay, Staining, Microscopy, Fluorescence, Software, Clinical Proteomics, Membrane

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: TCN up-regulates RIP3 upon caspase inhibition. Cells were pretreated with zVAD.fmk (20 μM) for 1 h, followed by different dosages of TCN (0, 1, 2 μM) treatment for 24 h. A. The mRNA levels of RIPK3 were examined using real-time PCR in HT29 and Raji cells. B. The protein levels of RIPK3 and p-MLKL (S358) were detected by western blot assay in HT29 and Raji cells. β-actin was used as a loading control. C. HT29 cells were seeded on coverslips overnight and pretreated with zVAD.fmk (20 μM) for 1 h, followed by TCN (0, 1 μM) treatment for 24 h. The fluorescence of RIPK3 was detected by confocal microscopy and nuclei were stained blue. Quantification of the fluorescent intensity of RIPK3 imaging was shown as bar graphs. Scale bar, 25 μm. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (P < 0.05, P < 0.01, P < 0.001, respectively).

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Control, Fluorescence, Confocal Microscopy, Staining, Imaging

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: Silencing of RIP3 rescues cancer cells from TCN-induced necroptosis. Cells were pretreated with zVAD.fmk for 1 h, followed by different dosages of TCN (0 or 1 μM) treatment for 24 h. Cell viability of (A) HT29 shCON/shRIP3 (3# or 6#) and (B) Raji shCON/shRIP3 (3# or 6#) cells as designated in each group were analyzed by CCK-8 assay. (C) Cells were stained with SYTOX Green Nucleic Acid Stain and imaged by fluorescent microscopy. The intensity of green fluorescence was analyzed and shown in bar graphs. Scale bar, 50 μm. (D) Cells were stained using Annexin V PE/7-ADD apoptosis detection kit and applied to flow cytometry analysis. The sum of Q1 and Q2 quadrants was shown as bar graphs, which reflects the rate of necroptotic cell death. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (**, ***) indicate significant differences (P < 0.01, P < 0.001, respectively). (E) Representative electron microscope photographs of HT29 shCON/shRIP3 and Raji shCON/shRIP3 cells as designated in each group.

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: CCK-8 Assay, Staining, Microscopy, Fluorescence, Flow Cytometry

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: RIP3 mediates the up-regulation of PYGL and PDC-E1α induced by TCN. Cells were pretreated with zVAD.fmk for 1 h, followed by different dosages of TCN (0, 1 or 2 μM) treatment for 24 h. The mRNA levels of PYGL, GLUL, GLUD1 and PDC-E1α, E1β, E2, E3 genes were detected by real-time PCR both in (A) HT29 and (B) Raji cells. The mRNA levels of PYGL and PDC- E1α genes was examined by real-time PCR in (C) HT29 shCON/shRIP3 (3# or 6#) (D) Raji shCON/shRIP3 (3# or 6#) cells as designated in each group.

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: Real-time Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: RIP3 mediates TCN-induced necroptosis through activating mitochondria energy metabolism and ROS production. HT29 shCON/shRIP3 cells were pretreated with zVAD.fmk for 1 h, followed by different dosages of TCN (0, 1 μM) treatment for 24 h. (A) Left, seahorse extracellular flux analyzer measurements of OCR metabolic profile using the mito stress cell assay. Traces shown were representative of two independent experiments in which each data point represents replicates of five wells. Data are shown as mean values ± S.D. Right, quantitative determination of basal and maximal OCR values of each designated group. Mitochondrial ROS levels of (B) HT29 and (C) Raji cells were determined by using MitoSOX Red, a specific mitochondrial probe, and detected by flow cytometry. Cell viability of (D) HT29 and (E) Raji cells were analyzed by CCK-8 assay. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (P < 0.05, P < 0.01, P < 0.001, respectively).

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: Flow Cytometry, CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: RIP3 mediates TCN-induced necroptosis through activating mitochondrial metabolism and ROS production in chemotherapy-resistant cancers

doi:

Figure Lengend Snippet: TCN sensitizes cisplatin in tumor treatment in vivo. A. Athymic BALB/c nude mice bearing HT29 cells were randomly separated into 4 groups (n = 4) and intraperitoneal administrated with corn oil (vehicle), TCN (1 mg/kg), DDP (1 mg/kg) or DDP combined with TCN (0.5 mg/kg each) every day for 13 days. Tumor volume was examined every day and shown in the graph. B. Upper, at the end of the experiment, the mice were sacrificed and the tumors were separated. Lower, tumor mass of each group was weighed and shown in the graph. C. During the experiment, body weight of the mice in each group was monitored and shown in the graph. D. Images of tumor sections in each group stained with hematoxylin-eosin (HE) and indicated antibodies. Antibody staining is in brown and nuclear counter staining is in blue. Scatter diagram shows Histoscore for the indicated antibody staining in tumor samples.

Article Snippet: Cell culture Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics.

Techniques: In Vivo, Staining