Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: Proteomic analysis after miR-196b overexpression in HepG2 cells. HepG2 were transfected with pre-miR negative control or pre-miR-196b (50 nM) during 24 h and 2D-DIGE analysis of cytosolic fraction was performed (n = 4). (A) Proteins were loaded on IPG strips (18 cm, 3–11 non linear), followed by the second dimension on a vertical continuous 12.5% gel. Significantly down- or up-regulated proteins (cut-off 1.2, p value < 0.05) were identified and labeled with their serial number. Arrow: spot 952/942, IGF2BP1 protein. Table shows the number of down-regulated or up-regulated spots. (B) List of proteins identified by MS/MS analysis and predicted by softwares: TargetScan, MicroCosm and miRDB. (C) Intensity of spot 952 was significantly lower when HepG2 cells were transfected with pre-miR-196b versus pre-miR negative control during 24 h. Dot plots showed the variation between pre-miR negative control and pre-miR-196b for 4 replicates.

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Over Expression, Transfection, Negative Control, Labeling, Tandem Mass Spectroscopy

Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: IGF2BP1 is a direct target of miR-196b in HepG2 cells. (A) The putative miR-196b targeted sequence in the igf2bp1 gene. TargetScan6.2 predicts three binding sites in IGF2BP1 3′-UTR. The alignment of the seed region of miR-196b with 3′UTR is shown. (B) Expression level of IGF2BP1 mRNA in the pre-miR-196b transfected cells determined by RT-qPCR was down-regulated in comparison to pre-miR negative control transfected cells (pre-miR CTL-) or untransfected cells (Cells), 24 or 48 h post-transfection (means ± 1 SD, n = 3). °, °°: significantly different from untransfected cells (p < 0.05, p < 0.01), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001), for each group (N, H, NE, HE) respectively. (C) Protein abundance of IGF2BP1 in the pre-miR-196b transfected cells determined by western blot was down-regulated in comparison to pre-miR negative control transfected cells (pre-miR CTL-) or untransfected cells (Cells), 24, 48 and 72 h after transfection. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin. (D) Schematic representation of the seed region match between miR-196b and the putative IGF2BP1 3′UTR. The mutation of five nucleotides in the seed region is shown. (E) pmiRGLO luciferase reporters containing either the wild-type or the mutant (mutated) human IGF2BP1 3′UTR were co-transfected into HepG2 cells with pre-miR negative control or pre-miR-196b (50 nM) during 72 h. 72 h post-transfection, the cells were assayed using a dual luciferase assay. Firefly luciferase values were normalized to Renilla luciferase values and plotted as relative luciferase activity (means ± 1 SD, n = 8). **: significantly different from wild-type reporter (p < 0.01), ***: significantly different from pre-miR negative control transfected cells (p < 0.001).

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Sequencing, Binding Assay, Expressing, Transfection, Quantitative RT-PCR, Comparison, Negative Control, Quantitative Proteomics, Western Blot, Mutagenesis, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: Effects of miR-196b overexpression or IGF2BP1 silencing on cell morphology, viability and proliferation. HepG2 cells were transfected either with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM) either with RISC Free negative control (RF) or IGF2BP1 siRNA (50 nM). (A) 72 h after transfection, the cell morphology was observed by phase contrast microscopy. (B) Cell viability assay (MTT assay) was performed 24, 48, 72 and 96 h after transfection of HepG2 cells (means ± 1 SD, n = 3). (C) Cell proliferation assay (BrdU ELISA assay) was performed 72 h after transfection (means ± 1 SD, n = 3). °°, °°°: significantly different from untransfected cells (Cells) (p < 0.01, p < 0.001), $$, $$$: significantly different from pre-miR negative control transfected cells or RF negative control transfected cells (p < 0.01, p < 0.001).

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Over Expression, Transfection, Negative Control, Microscopy, Viability Assay, MTT Assay, Proliferation Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: Effects of miR-196b overexpression or IGF2BP1 silencing in regulation of apoptosis under normoxia. (A) HepG2 cells were transfected with either pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM), either with RISC free negative control (RF) or siRNA IGF2BP1 for 24, 48 and 72 h. IGF2BP1, cleaved PARP and cleaved caspase 3 were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin. (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001).

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Over Expression, Transfection, Negative Control, Western Blot, Immunodetection, Activity Assay, Fluorescence

Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: Effects of miR-196b overexpression or IGF2BP1 silencing on c-myc mRNA expression and protein level. (A) HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 24 or 48 h and the mRNA expression level of IGF2BP1 was determined by RT-qPCR (means ± 1 SD, n = 3). °, °°, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.01, p < 0.001), $$$: significantly different from RF negative control transfected cells (p < 0.001) for each group 24 or 48 h respectively. (B) HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 24, 48 and 72 h and the mRNA expression level and the protein abundance of c-myc were determined by RT-qPCR and western blot respectively. (C) HepG2 cells were transfected with pre-miR negative control (pre-miR CTL-) or pre-miR-196b (50 nM), during 24, 48 and 72 h and the mRNA expression level and the protein abundance of c-myc were determined by RT-qPCR and western blot respectively. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin.

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Over Expression, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Quantitative Proteomics, Western Blot

Journal: Molecular Cancer

Article Title: miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

doi: 10.1186/s12943-015-0349-6

Figure Lengend Snippet: Role of IGF2BP1 in the chemoresistance induced by hypoxia. HepG2 cells were transfected with RISC free negative control (RF) or siRNA IGF2BP1 (50 nM) during 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A) IGF2BP1, cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin (B) 48 h after transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). ***: (p < 0.001), °, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) for each group N, H, NE, HE respectively.

Article Snippet: For fluorescence detection, membranes were blocked 1 h at room temperature in Odyssey blocking buffer (BD Biosciences, Franklin Lakes, MJ, USA) diluted 2X in PBS and then overnight at 4°C in Odyssey blocking buffer with 0.1% Tween containing a specific antibody: mouse anti-PARP-1 monoclonal antibody (#556493 from BD Pharmingen, Franklin Lakes, MJ, USA), rabbit anti-caspase3 antibody (#9662 from Cell Signaling, Dancer, MA, USA), rabbit anti-IGF2BP1 antibody (#2852 from Cell Signaling), mouse anti-Myc (#sc-42 from SantaCruz Technologies, Dallas, TX, USA) and mouse anti-α-tubulin monoclonal antibody (#T5168 from Sigma-Aldrich).

Techniques: Transfection, Negative Control, Incubation, Western Blot, Immunodetection, Activity Assay, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endogenous tRNA‐derived small RNA (tRF3‐Thr‐AGT) inhibits ZBP1/NLRP3 pathway‐mediated cell pyroptosis to attenuate acute pancreatitis (AP)

doi: 10.1111/jcmm.16972

Figure Lengend Snippet: The regulatory mechanisms of tRF3‐Thr‐AGT and ZBP1. (A) The potential downstream targets for tRF3‐Thr‐AGT were predicted by using the bioinformatics analysis. (B) The binding sites between tRF3‐Thr‐AGT and 3′UTR of ZBP1 mRNA were predicted, which were validated by using the following (C) dual‐luciferase reporter gene system assay and (D) RNA pull‐down assay (each experiment repeated for three times). tRF3‐Thr‐AGT negatively regulated ZBP1 at both (E) mRNA and (F) protein levels (each experiment repeated for three times). (G, H) ZBP1 was upregulated in the AP rats pancreatic tissues, in contrast with the normal rats (each group had 3 rats, and individual experiment was repeated for three times). * p < 0.05

Article Snippet: The membranes were then incubated with the primary antibodies against NLRP3 (1:1500, Cell Signaling Technology, MA, USA), ASC (1:2000, Cell Signaling Technology, MA, USA), Gasdermin D (1:2000, Cell Signaling Technology, MA, USA), ZBP1 (1:1500, Cell Signaling Technology, MA, USA) and GAPDH (1:1500, Cell Signaling Technology, MA, USA) at 4℃ overnight and were subsequently probed with the secondary antibody.

Techniques: Binding Assay, Luciferase, Pull Down Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endogenous tRNA‐derived small RNA (tRF3‐Thr‐AGT) inhibits ZBP1/NLRP3 pathway‐mediated cell pyroptosis to attenuate acute pancreatitis (AP)

doi: 10.1111/jcmm.16972

Figure Lengend Snippet: Overexpression of tRF3‐Thr‐AGT suppressed STC‐induced cell pyroptosis by downregulating ZBP1. (A, B) The expression levels of NLRP3, ASC and Gasdermin D were determined by Western Blot analysis (each experiment was repeated for three times). (C) Immunofluorescence staining assay was performed to determine the expression levels and localization of caspase‐1 in AR42J cells. * p < 0.05

Article Snippet: The membranes were then incubated with the primary antibodies against NLRP3 (1:1500, Cell Signaling Technology, MA, USA), ASC (1:2000, Cell Signaling Technology, MA, USA), Gasdermin D (1:2000, Cell Signaling Technology, MA, USA), ZBP1 (1:1500, Cell Signaling Technology, MA, USA) and GAPDH (1:1500, Cell Signaling Technology, MA, USA) at 4℃ overnight and were subsequently probed with the secondary antibody.

Techniques: Over Expression, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endogenous tRNA‐derived small RNA (tRF3‐Thr‐AGT) inhibits ZBP1/NLRP3 pathway‐mediated cell pyroptosis to attenuate acute pancreatitis (AP)

doi: 10.1111/jcmm.16972

Figure Lengend Snippet: tRF3‐Thr‐AGT suppressed STC‐induced cell death and inflammation via modulating the ZBP1/NLRP3 pathway. (A, B) Cell proliferation and viability were measured by MTT assay and trypan blue staining assay (each experiment was repeated for three times). (C–F) The mRNA and protein levels of IL‐1β and IL‐18 in AT42J cells and its supernatants were measured by using Real‐Time qPCR and ELISA (each group had 6 rats, and individual experiment was repeated for three times). * p < 0.05

Article Snippet: The membranes were then incubated with the primary antibodies against NLRP3 (1:1500, Cell Signaling Technology, MA, USA), ASC (1:2000, Cell Signaling Technology, MA, USA), Gasdermin D (1:2000, Cell Signaling Technology, MA, USA), ZBP1 (1:1500, Cell Signaling Technology, MA, USA) and GAPDH (1:1500, Cell Signaling Technology, MA, USA) at 4℃ overnight and were subsequently probed with the secondary antibody.

Techniques: MTT Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver

doi: 10.1038/s41467-024-51109-y

Figure Lengend Snippet: a qPCR analysis of the gene expression of nucleic acid sensing pathway target genes in total mRNA isolated from 12-week-old WT and COX14 M19I mice liver samples. Means ± SEM, n = 4. b Western blot analysis and quantification of tissue lysates from 12-week-old WT and COX14 M19I mice livers with indicated antibodies. Means ± SEM, n = 4, Unpaired t test, ISG15 p = 0.0005; OASL1a p < 0.0001; IFIT1 p = 0.0003. c Graphical presentation on the generation of the COA3 Y72C mouse line using CRISPR/CAS9-mediated double stranded cut in the Coa3 allele and subsequent single stranded Oligonucleotide DNA (ssODN) mediated repair. d Enzyme activity of cytochrome c oxidase from 60-week-old WT and COA3 Y72C brain, heart, liver and muscle plotted as percentage of average of WT. Means ± SEM, n = 3, One-way ANOVA, ns=non-significant, Brain p = 0.0028; Liver p < 0.0001; Muscle p = 0.0004 e qPCR analysis of gene expression of nucleic acid sensing pathway target genes in total mRNA isolated from 22-week-old WT and COA3 Y72C mice liver samples. Means ± SEM, n = 4. f Measurement of relative mRNA abundance in the cytosolic fractions of 12-week-old WT and COX14 M19I mice liver samples. Means ± SEM, n = 4. g Immunoblot analysis and quantification of tissue lysates from 24-week-old WT and COX14 M19I mice liver for indicated proteins. Means ± SEM, n = 4, Unpaired t test, RIG1 p = 0.0002; MDA5 p = 0.0051; STING p = 0.0416; ZBP1 p = 0.0015. Source data are provided as a Source Data file.

Article Snippet: Antibodies used: Rabbit polyclonal Anti-IFIT1 antibody (ab111821) Abcam, dilution 1:100; Mouse monoclonal Anti-Oas1a Antibody (E-2): sc-365072 Santa Cruz, dilution: 1:1000; Mouse monoclonal Anti-ISG15 Antibody (F-9): sc-166755 Santa Cruz, dilution: 1:500; Rabbit polyclonal anti-VDAC Home-made antibody, dilution: 1:1000 Rabbit polyclonal anti-ATP5 Home-made antibody, dilution: 1:2500; Rabbit polyclonal anti-RIESKE Home-made antibody, dilution: 1:2000 Anti-Rig-I (D14G6) Rabbit mAb#3743 Cell Signaling Technologies, dilution: 1:1000; Anti- MDA-5 (D74E4) Rabbit mAb#5321 Cell Signaling Technologies, dilution 1:1000; Anti- STING (D2P2F) Rabbit mAb #13647 Cell Signaling Technologies, dilution 1:100; Mouse monoclonal ZBP1 Antibody (H-9): sc-271483 Santa Cruz, dilution 1:500; Rabbit polyclonal Anti-Lamin B1 antibody Abcam, dilution 1:500; Mouse monoclonal Anti-MTCO1 antibody [1D6E1A8] (ab14705) Abcam, dilution 1:1000;

Techniques: Gene Expression, Isolation, Western Blot, CRISPR, Activity Assay