neisseria sicca atcc  (ATCC)

Journal: Cell death & disease

Article Title: A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

doi: 10.1038/s41419-023-05772-6

Figure Lengend Snippet: Fig. 2 Knockdown of IMP3 triggers cell death in CRC cell lines. A Left, IMP3 levels in representative images of Western blot from DLD-1, HCT- 116 and HT-29 CRC cell lines and the healthy colon epithelial cell line HCEC-1ct. β-actin was used as loading control. Right, quantitative analysis of IMP3/β-actin protein ratio as measured by densitometry scanning of Western blots (values are expressed in arbitrary units a.u.; *p = 0.04, **p = 0.03; mean ± SD, n = 3). B Representative Western blot showing IMP3 expression in HCT-116 cells untreated (Unst) or transfected with control or IMP3 siRNA. C Upper panel, representative dot plot of Annexin V (AnnV) and propidium iodide (PI)-positive HCT- 116 cells treated as indicated above for 48 h. Lower panel, quantification of the percentage of AnnV and/or PI-positive HCT-116 cells (mean ± SD; AnnV+PI + unst and CTR siRNA-treated cells versus IMP3 siRNA 10/25nM-transfected cells, *p ≤0.05, mean ± SD, n = 3).

Article Snippet: To assess effective IMP3 downregulation in vitro, cells were transfected for 24 h with 2 different commercial IMP3 siRNAs (with final concentration from 5 nmol/L to 25 nmol/L) and CTR siRNA (final concentration: 25 nmol/ L) (all from Santa Cruz Biotechnology and Thermo Fisher Scientific) in presence or absence of 5-fluoruracil or Oxaliplatin (both at 100 nM final concentration) using Opti-MEM medium and lipofectamine 3000 reagent (both from Thermo Fisher Scientific), according to the manufacturer’s instructions. siRNA-mediated silencing of AIF was also performed with a commercial AIF-specific siRNA (Santa Cruz Biotechnology, final concentration: 25 nmol/L) for 24 h. As a nonspecific control, a scrambled siRNA was used (Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Control, Expressing, Transfection

Journal: Cell death & disease

Article Title: A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

doi: 10.1038/s41419-023-05772-6

Figure Lengend Snippet: Fig. 3 IMP3 knockdown enhances the toxicity of chemotherapeutic drugs. Representative dot-plots (upper panels) and quatification percentage (lower panel) showing the AnnexinV- and/or propridiun iodide (PI)-positive HCT-116 cells pre-incubated with control or IMP3 siRNA (both final concentration 25 nM) for 12 h and then stimulated or not with 5-fluorouracil (5-flu, 10uM final concentration) or Oxaliplatin (oxa, 10uM final concentration) for 36 h. Data indicate mean ± SEM of 2 separate experiments (CTR siRNA-treated cells versus all, *p ≤0.01).

Article Snippet: To assess effective IMP3 downregulation in vitro, cells were transfected for 24 h with 2 different commercial IMP3 siRNAs (with final concentration from 5 nmol/L to 25 nmol/L) and CTR siRNA (final concentration: 25 nmol/ L) (all from Santa Cruz Biotechnology and Thermo Fisher Scientific) in presence or absence of 5-fluoruracil or Oxaliplatin (both at 100 nM final concentration) using Opti-MEM medium and lipofectamine 3000 reagent (both from Thermo Fisher Scientific), according to the manufacturer’s instructions. siRNA-mediated silencing of AIF was also performed with a commercial AIF-specific siRNA (Santa Cruz Biotechnology, final concentration: 25 nmol/L) for 24 h. As a nonspecific control, a scrambled siRNA was used (Santa Cruz Biotechnology).

Techniques: Knockdown, Incubation, Control, Concentration Assay

Journal: Cell death & disease

Article Title: A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

doi: 10.1038/s41419-023-05772-6

Figure Lengend Snippet: Fig. 4 IMP3-triggered CRC cell death is independent by caspases activation and does not affect necroptosis, pyroptosis or ferroptosis pathways. A Upper panel, representative dot plot showing Annexin V (AnnV) and propidium iodide (PI)-positive HCT-116 cells pre-incubated with pan-caspase inhibitor (Z-VAD) and left untreated (Unst) or transfected with control or IMP3 siRNA for 48 h. Lower panel shows the percentage of AnnV and/or PI-positive HCT-116 treated as above (mean ± SEM; AnnV+PI + unst and CTR siRNA-treated cells versus IMP3 siRNA-transfected cells, **p ≤0.01; Z-VAD-cells and Z-VAD CTR siRNA-treated cells versus Z-VAD IMP3 siRNA-treated cells, *p ≤0.05, n = 4). B Representative Western blot of activated form of RIP3 (p-RIP), Gasdermin D (GSDMD), Glutathione peroxidase 4 (GPX4) and cleaved capsase 3 in HCT-116 cells left untreated (Unst) or transfected with control or IMP3 siRNA for 48 h. β-actin was used as loading control (n = 3).

Article Snippet: To assess effective IMP3 downregulation in vitro, cells were transfected for 24 h with 2 different commercial IMP3 siRNAs (with final concentration from 5 nmol/L to 25 nmol/L) and CTR siRNA (final concentration: 25 nmol/ L) (all from Santa Cruz Biotechnology and Thermo Fisher Scientific) in presence or absence of 5-fluoruracil or Oxaliplatin (both at 100 nM final concentration) using Opti-MEM medium and lipofectamine 3000 reagent (both from Thermo Fisher Scientific), according to the manufacturer’s instructions. siRNA-mediated silencing of AIF was also performed with a commercial AIF-specific siRNA (Santa Cruz Biotechnology, final concentration: 25 nmol/L) for 24 h. As a nonspecific control, a scrambled siRNA was used (Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Transfection, Control, Western Blot

Journal: Cell death & disease

Article Title: A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

doi: 10.1038/s41419-023-05772-6

Figure Lengend Snippet: Fig. 5 Bcl-2 and Bcl-xL mRNA are part of the IMP3 complex. A Left, representative Western blot of IMP3 immunoprecipitation from HCT-116 cells, β-actin is used as a negative control. Input (1:10) of the total protein extracts, IMP3 immunoprecipitation (IP: IMP3), and mock immunoprecipitation (IP: IgG). Right, relative quantification of IMP3-mRNAs enrichment by RT-qPCR in IMP3 immunoprecipitation/total protein extracted from HCT-116 cells. β-actin mRNAs were used as control (mean ± SEM; IP: IMP3 versus IP: IgG *P ≤0.05; n = 3). B Bcl-xL and Bcl-2 mRNA levels detected by Real time PCR in HCT-116 cells left untreated (Unst) or transfected with CTR or IMP3 siRNA for 30 h. Unst and CTR siRNA-treated cells versus IMP3 siRNA-transfected cells, **p ≤0.01; n = 3). C Representative Western blot showing IMP3, Bcl-xL, Bcl-2 and β-actin in HCT-116 cells were left untreated (Unst) or transfected with control or IMP3 siRNA for 36 h. Right, quantification of IMP3, Bcl-xL and Bcl-2 proteins in HCT-116 cells as measured by densitometry of Western blot (values are expressed in arbitrary units (a.u.), mean ± SD, n = 4; IMP3: Unst and CTR siRNA-treated cells versus IMP3 siRNA-transfected cells, **p ≤0.01; n = 3).

Article Snippet: To assess effective IMP3 downregulation in vitro, cells were transfected for 24 h with 2 different commercial IMP3 siRNAs (with final concentration from 5 nmol/L to 25 nmol/L) and CTR siRNA (final concentration: 25 nmol/ L) (all from Santa Cruz Biotechnology and Thermo Fisher Scientific) in presence or absence of 5-fluoruracil or Oxaliplatin (both at 100 nM final concentration) using Opti-MEM medium and lipofectamine 3000 reagent (both from Thermo Fisher Scientific), according to the manufacturer’s instructions. siRNA-mediated silencing of AIF was also performed with a commercial AIF-specific siRNA (Santa Cruz Biotechnology, final concentration: 25 nmol/L) for 24 h. As a nonspecific control, a scrambled siRNA was used (Santa Cruz Biotechnology).

Techniques: Western Blot, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: Cell death & disease

Article Title: A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

doi: 10.1038/s41419-023-05772-6

Figure Lengend Snippet: Fig. 6 IMP3 knockdown affects mitochondrial membrane potential and AIF nuclear accumulation. (A) HCT-116 cells were left untreated (Unst) or transfected with CTR or IMP3 siRNA. Vanilomycin was used as positive control. HCT-116 were stained with JC-1 and analyzed by flow cytometry. Mitochondrial membrane potential loss was evaluated as a decrease in JC-1 red fluorescence and an increase in JC-1 green fluorescence. Representative dot plot (Unst and CTR siRNA-treated cells versus IMP3 siRNA-transfected cells and Unst cells versus vanilomycin- treated cells, mean ± SD, **P ≤0.01; n = 3). B Representative Western blot (left panel) and quantitative analysis (right panel) for AIF, OCT-1 and β-actin in nuclear protein extracted from HCT-116 cells untreated (Unst) or transfected with CTR or IMP3 siRNA for 48 h. (Unst and CTR siRNA- treated cells versus IMP3 siRNA-transfected cells, mean ± SD, **p ≤0.01; n = 3). C Percentage of AnnV and/or PI-positive HCT-116 cells untreated (Unst) or transfected with CTR or IMP3 siRNA in presence/absence of AIF siRNA for 48 h (CTR siRNA-treated cells versus IMP3 siRNA- transfected cells, *p = 0.04, **p ≤0.01;and IMP3 siRNA-transfected versus IMP3 siRNA + AIF siRNA transfected cells *p = 0.04, **p ≤0.01; mean ± SD, n = 3).

Article Snippet: To assess effective IMP3 downregulation in vitro, cells were transfected for 24 h with 2 different commercial IMP3 siRNAs (with final concentration from 5 nmol/L to 25 nmol/L) and CTR siRNA (final concentration: 25 nmol/ L) (all from Santa Cruz Biotechnology and Thermo Fisher Scientific) in presence or absence of 5-fluoruracil or Oxaliplatin (both at 100 nM final concentration) using Opti-MEM medium and lipofectamine 3000 reagent (both from Thermo Fisher Scientific), according to the manufacturer’s instructions. siRNA-mediated silencing of AIF was also performed with a commercial AIF-specific siRNA (Santa Cruz Biotechnology, final concentration: 25 nmol/L) for 24 h. As a nonspecific control, a scrambled siRNA was used (Santa Cruz Biotechnology).

Techniques: Knockdown, Membrane, Transfection, Positive Control, Staining, Cytometry, Western Blot

Journal: Cytotechnology

Article Title: Melilotus indicus extract induces apoptosis in hepatocellular carcinoma cells via a mechanism involving mitochondria-mediated pathways

doi: 10.1007/s10616-018-0195-7

Figure Lengend Snippet: Effect of AIF knockdown on MIE-induced apoptosis in HepG2 and SNU-182 cells. a AIF knockdown by siRNA specific to AIF decreases the nuclear AIF protein expression in MIE-treated cells determined by Western blotting. β-actin served as a loading control. b The relative protein expression levels of AIF normalized by those of β-actin were quantified by Image J. c, d MIE-induced apoptosis is substantially abrogated upon siRNA knockdown of AIF. Apoptotic cells in AIF-silenced and MIE-stimulated HepG2 or SNU-182 cells were analyzed by flow cytometry, and total annexin V-positive cells were quantified. The values are the mean ± SD of three different experiments. *p < 0.05 and **p < 0.005 indicate a significant difference compared with negative siRNA transfectants

Article Snippet: Cultured HepG2 or SNU-182 cells (2 × 10 5 cells/well) in a 6-well plate were transfected with AIF siRNA or negative control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, Control, Flow Cytometry