Journal: Cancers

Article Title: HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells

doi: 10.3390/cancers16020384

Figure Lengend Snippet: Gene expression profile of Hs578T HuR-silenced cells. ( A ) Volcano plot displaying changes in the transcriptome of HuR-depleted cells. Genes with a log2 fold change of > 1.0 or < −1.0 and p < 0.05 are shown as pink (downregulated) or green (upregulated) dots, respectively. ( B ) Validation of downregulation of selected DEGs in Hs578T HuR-ablated cells using quantitative RT-PCR. Data represent the mean ± SEM of three independent experiments with triplicate assays. The p -value was calculated using a two-sided unpaired Student’s t -test (* p < 0.05; ** p < 0.01; *** p < 0.001). ( C ) GSEA plot of DEGs showing enrichment of Hallmark signatures. The y-axis displays the FDR-values (−Log10 FDR) of the enrichments.

Article Snippet: Hs578T and BT549 human basal-like triple-negative breast cancer cell derivatives were obtained from the American Type Culture Collection.

Techniques: Gene Expression, Biomarker Discovery, Quantitative RT-PCR

Journal: Cancers

Article Title: HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells

doi: 10.3390/cancers16020384

Figure Lengend Snippet: Selection of putative HuR targets in Hs578T cells. ( A ) DEGs list displaying the location of ARE and overlapping HuR binding sites in the cDNA of the selected DEGs. ( B ) Validation of downregulation of putative targets in Hs578T HuR-silenced cells using quantitative RT-PCR. Data represent the mean ± SEM of three independent experiments assayed in triplicate. The p -value was calculated using a two-sided unpaired Student’s t -test. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Hs578T and BT549 human basal-like triple-negative breast cancer cell derivatives were obtained from the American Type Culture Collection.

Techniques: Selection, Binding Assay, Biomarker Discovery, Quantitative RT-PCR

Journal: Cancers

Article Title: HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells

doi: 10.3390/cancers16020384

Figure Lengend Snippet: HuR binds to the SOX9 ARE in Hs578T cells. ( A ) Quantitative RT-PCR confirming downregulation of SOX9 in HuR-silenced cells. Data are the mean ± SEM of three independent experiments assayed in triplicate (*** p < 0.001). ( B ) RIP analysis of shSCR cells using IgG and anti-HuR antibodies. HuR immunoprecipitation efficiency was tested using Western blot (upper panel). ACTB and SOX9 mRNAs were quantified using RT-qPCR and represented as fold enrichment compared to IgG RIP analysis. Data are the mean ± SEM of three independent experiments assayed in triplicate (** p < 0.01). ( C ) HuR pull down using biotinylated synthetic RNA corresponding to the wild type SOX9 ARE (AREwt) or a mutated version (AREmut). The presence of HuR, hnRNPD (AUF1), or PABPC1 in the input and pull down fractions was detected using Western blot.

Article Snippet: Hs578T and BT549 human basal-like triple-negative breast cancer cell derivatives were obtained from the American Type Culture Collection.

Techniques: Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells

doi: 10.3390/cancers16020384

Figure Lengend Snippet: HuR silencing reduces SOX9 expression. ( A ) Relative luciferase activity in control (shSCR) or HuR-silenced Hs578T cells (shHuR-1, shHuR-2) transfected with SOX9 3′UTR reporter plasmids (3′UTR-SOX9) or control plasmid (3′UTR-control). Data are the mean ± SEM of three independent experiments (* p < 0.05; *** p < 0.001; ns, not significant). ( B ) SOX9 mRNA half-life was measured using RT-qPCR in control (shSCR) or HuR-depleted (shHuR-1, shHuR-2) Hs578T cells treated with actinomycin D. The data were normalized to 18S rRNA levels and represented as a percentage of the mRNA levels measured at time 0, before adding actinomycin D, using a semilogarithmic scale. A discontinuous horizontal line indicates a 50% decrease in mRNA abundance. Data are the mean ± SEM of three independent experiments (** p < 0.01). ( C ) Western blot analysis of HuR and SOX9 protein levels in control (shSCR) or HuR-silenced (shHuR-1, shHuR-2) Hs578T cells. GAPDH was used as a loading control.

Article Snippet: Hs578T and BT549 human basal-like triple-negative breast cancer cell derivatives were obtained from the American Type Culture Collection.

Techniques: Expressing, Luciferase, Activity Assay, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells

doi: 10.3390/cancers16020384

Figure Lengend Snippet: HuR silencing decreases proliferation migration and invasion in Hs578T cells. ( A ) Cell viability analyses of control (shSCR) or HuR-silenced (shHuR-1, shHuR-2) cells. MTT assays were performed at the indicated time points after seeding. Data were normalized to the value detected at 24 h. Error bars represent the mean ± SEM of three independent experiments assayed in quintuplicate ( B ) Cell motility of the indicated clones was analyzed using wound-healing assay. Images were taken at 0, 8, 24, and 48 h (h) after culture scratch (upper panel) and area closure was quantified using ImageJ (lower panel). Results represent the mean ± SEM of three independent experiments. ( C ) Cells were seeded in the upper chambers of Transwell, allowed to migrate for 18 h, membrane stained with crystal violet, and as described in Methods section were photographed. Left panel: representative images of the lower membrane (invading cells). Right panel: percentage of invasiveness by direct measurement with ImageJ. Values represent the mean ± SEM from three independent experiments performed in triplicate. (** p < 0.01, *** p < 0.001; ns, not significant). The images in ( B ) and ( C ) were taken using an inverted microscope with the 10× objective.

Article Snippet: Hs578T and BT549 human basal-like triple-negative breast cancer cell derivatives were obtained from the American Type Culture Collection.

Techniques: Migration, Control, Clone Assay, Wound Healing Assay, Membrane, Staining, Inverted Microscopy

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: CD133 expression in the human gallbladder cancer cell line GBC-SD following cell sorting using immunomagnetic beads. (A) Transmembrane CD133 expression in CD133 + and CD133 − groups. CD133 was labeled with red fluorescence as indicated by arrows (magnification, ×200). (B) Proportion of the CD133 + subset in the CD133 + and CD133 − groups in GBC-SD cells, determined using flow cytometry. (C) Detection of CD133 mRNA expression using polymerase chain reaction. GADPH was used as the control. (D) Detection of CD133 protein expression using western blot analysis. GADPH was used as the loading control. CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, FACS, Labeling, Fluorescence, Flow Cytometry, Polymerase Chain Reaction, Control, Western Blot

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Colony formation assay and in vivo tumor formation assay. (A) Shape of the colony formed by the CD133 + subset (magnification, ×10). (B) Colony formation efficiency in CD133 + and CD133 − groups. (C) Representative images of the xenograft tumors formed by CD133 + cells, with representative HE staining and CD133 IHC staining, as observed under an inverted microscope (magnification, ×200). The arrow indicates CD133 + cells. CD, cluster of differentiation; H&E, hematoxylin and eosin; IHC, immunohistochemistry.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Colony Assay, In Vivo, Tube Formation Assay, Staining, Immunohistochemistry, Inverted Microscopy

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Cell biology characteristics of cells in the CD133 + and CD133 − groups. (A) Comparison of the proliferative ability between the CD133 + and CD133 − groups. (B) Inhibiting rate of cell growth in the CD133 + and CD133 − groups 72 h after treatment with 5-fluorouracil or gemcitabine. CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Comparison

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Cell invasion and EMT. (A) Number of transmembrane cells in the CD133 + and CD133 − groups, observed under an inverted microscope (magnification, ×200). (B) Quantitative analysis of the number of invasive cells. (C) Expression of EMT-associated proteins in the CD133 + and CD133 − groups. (D) Quantitative analysis of mRNA expression of EMT-associated genes in the CD133 + and CD133 − groups. EMT, epithelial-mesenchymal transition; CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Inverted Microscopy, Expressing

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Expression of stem cell-associated genes. (A) DNA electrophoresis of stem cell-associated genes in the CD133 + and CD133 − groups, determined using polymerase chain reaction. (B) Quantitative analysis of mRNA expression level of stem cell-associated genes. GADPH was used as the loading control. CD, cluster of differentiation; CXCR4, C-X-C motif chemokine receptor 4; p-, phosphorylated; Akt, protein kinase B; Erk, extracellular signal-regulated kinase; SDF-1α, stromal cell-derived factor 1α.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Control, Derivative Assay

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Regulation of the CXCR4/Akt/CD133 signaling pathway. (A) Differences in CXCR4/Akt/CD133 protein expression between the CD133 + and CD133 − group. (B) Protein expression of CXCR4/Akt/CD133 as induced by treatment with SDF-1α at different concentrations (25, 50, 100, and 200 ng/ml) and for different durations (15, 30 min, 1, 2, and 24 h).

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: (A) CD133 and CXCR4 mRNA expression profiles in the CD133 + and CD133 − groups following treatment with SDF-1α/AMD3100/LY294002. (B) CXCR4, p-Akt, Akt, p-Erk, Erk, and CD133 protein expression profiles in the CD133 + and CD133 − groups following treatment with SDF-1α/AMD3100/LY294002. The CXCR4/Akt/CD133 axis and its potential function. CXCR4, C-X-C motif chemokine receptor 4; Akt, protein kinase B; CD, cluster of differentiation; SDF-1α, stromal cell-derived factor 1α; p-, phosphorylated; Erk, extracellular signal-regulated kinase.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, Derivative Assay

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: L23 inhibits HDM2-mediated p53 polyubiquitination and degradation. (A) Ectopic expression of L23 stabilizes HDM2 and p53. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Plasmid DNA expressing GFP was cotransfected as a control. +, present; −, absent; α-HDM2, anti-HDM2; α-p53, anti-p53; α-myc, anti-myc; α-GFP, anti-GFP. (B) Ectopic expression of L23 stabilizes HDM2 and p53 in normal human fibroblast cells. WI38 cells were infected with virus expressing HDM2 for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Virus expressing GFP was coinfected as a control. α-actin, anti-actin. (C) L23 inhibits HDM2-mediated p53 polyubiquitination. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and the cells were treated with MG132 (25 μM) for 5 h before lysing. Cell extracts were analyzed by Western blotting with antibodies to p53 (D01) and myc (9E10) as indicated. (D) L23 stabilizes HDM2 independent of p53. WI38-E6 cells were infected with viruses expressing GFP, HDM2, and myc-L23 as indicated. Cells were lysed 2 days after infection, and the cell lysates were blotted as described above. Endog, endogenous; α-L23, anti-L23.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Expressing, Transfection, Plasmid Preparation, SDS Page, Infection, Western Blot

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: L23 induces a p53-dependent cell cycle arrest. (A and B) L23 overexpression stabilizes and activates p53. Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were infected with the indicated Ad for 2 days. Western blotting was performed as described above. α-myc, anti-myc; α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-actin, anti-actin. (C and D) L23 induces a p53-dependent cell cycle arrest. WI38 and WI38-E6 cells were infected with the indicated viruses. Cells were harvested 2 days after infection, fixed with 70% ethanol for 2 h, and stained with propidium iodide for 1 h, and the cell cycle distribution was determined by flow cytometry. Cell populations in the S phase are indicated as percentages of total cells. (E) L23 interacts with HDM2 in the nucleoplasm. U2OS cells were singly infected with Ad expressing myc-L23 for 2 days. Cells were then fixed with 3% paraformaldehyde for 10 min and immunostained with a rabbit anti-myc antibody (9E10) and a mouse anti-HDM2 antibody (N20). Nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Over Expression, Mutagenesis, Infection, Western Blot, Staining, Flow Cytometry, Expressing, Fluorescence, Diagnostic Assay, Inverted Microscopy

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Knocking down L23, but not L11, activates p53 and induces a cell cycle arrest. (A and B) U2OS cells were either untreated (Buffer) or transfected with a control scrambled RNA duplex (siScr), L23 siRNA (siL23), or L11 siRNA (siL11) for 2 days. Cell extracts were collected and analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) U2OS cells were transfected with siRNA as described for panels A and B. Cells were harvested 2 days after transfection, fixed with ethanol, and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. The averages of the results from two independent experiments are shown as bar graphs.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Transfection, Western Blot, Staining, Flow Cytometry

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Down-regulation of L23-induced cell cycle arrest is dependent on the function of p53. (A and B) Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were transfected with either a control scrambled RNA duplex (siScr) or L23 siRNA (siL23) for 2 days, and cell extracts were analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) WI38 and WI38-E6 cells were transfected siRNA as described for panels A and B. Cells were harvested 2 days after infection and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. (E) Down-regulation of L23 releases nucleolar B23. U2OS cells were transfected with the indicated siRNA for 2 days. The cells were then fixed and stained with a mouse anti-B23 (α-B23) antibody (Zymed) and an fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch). Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Mutagenesis, Transfection, Western Blot, Infection, Staining, Flow Cytometry, Fluorescence, Diagnostic Assay, Inverted Microscopy

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Inhibition of ribosomal biogenesis decreases the protein level of L23. (A) Low concentrations of actinomycin D induce p53-dependent cell cycle arrest. U2OS cells were treated with the indicated concentrations of actinomycin D (Act D) for 24 h, and the cell lysates were analyzed by Western blotting as described above. α-HDM2, anti-HDM2; α-p53, anti-p53; α-L23, anti-L23; α-actin, anti-actin. (B) Time required for actinomycin D treatment to suppress L23. U2OS cells were treated with 5 nM actinomycin D for the indicated times, and the protein levels were analyzed as described above. (C) Inhibition of ribosomal biogenesis by 5 nM actinomycin D down-regulates L23 but not L11. U2OS cells were treated with 5 nM actinomycin D for 24 h before lysing, the cell lysates were resolved by SDS-PAGE, and Western blotting was performed as described above. α-L11, anti-L11. (D) Ectopic expression of L23 suppresses endogenous L23. U2OS cells were infected with the indicated viruses for 2 days, and cell extracts were harvested and resolved by SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and blotted with the indicated antibodies. α-myc, anti-myc. (E) Suppression of endogenous L23 by ectopically expressed myc-L23 was independent of HDM2 and p53. Normal human fibroblast WI38 cells were infected with the indicated viruses for 2 days. Cell extracts were harvested and resolved by SDS-PAGE, and the proteins were analyzed as described above. +, present; −, absent.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Inhibition, Western Blot, SDS Page, Expressing, Infection

Journal: Cells

Article Title: MicroRNA-27b-3p Targets the Myostatin Gene to Regulate Myoblast Proliferation and Is Involved in Myoblast Differentiation

doi: 10.3390/cells10020423

Figure Lengend Snippet: Gene primers used for qRT-PCR.

Article Snippet: The antibody and its dilution ratio were as follows: MYHC rabbit polyclonal antibody (Proteintech, Wuhan, China, 1:500), MSTN rabbit polyclonal antibody (Bioss, Beijing, China, 1:500), GAPDH rabbit polyclonal antibody (HUABIO, Hangzhou, China, 1:500), and HRP binding Goat anti-rabbit IgG (BBI, Shanghai, China, 1:5000).

Techniques: Sequencing

Journal: Cells

Article Title: MicroRNA-27b-3p Targets the Myostatin Gene to Regulate Myoblast Proliferation and Is Involved in Myoblast Differentiation

doi: 10.3390/cells10020423

Figure Lengend Snippet: CPMs’ differentiation is inhibited by miR-27b-3p. ( a ) The relative expression of miR-27b-3p during CPMs’ proliferation and differentiation (GM 50% and GM 100% represent the proliferation density of 50% and 100%; DM1-DM 4 represents differentiation from one to four days). ( b , c ) The mRNA expression levels of three differentiation marker genes after transfection with miR-27b-3p mimic and inhibitor in CPMs. ( d , e ) The protein expression level of MyHC after transfection with miR-27b-3p mimic and inhibitor in CPMs. ( f , g ) MyHC staining of CPMs at 72 h after transfection with miR-27b-3p mimic and inhibitor in CPMs. Microscopic images were obtained by a fluorescence inverted microscope (dmi8, Leica, Germany). ( h , i ) Myotube area (%) of CPMs 72 h after overexpression and inhibition of miR-27b-3p. In all graphs, the results are shown as mean ± SEM (standard error of the mean) (n = 3). Statistical significance of differences between means was assessed, using the unpaired Student’s t -test (* p < 0.05; ** p < 0.01) vs. NC (negative control).

Article Snippet: The antibody and its dilution ratio were as follows: MYHC rabbit polyclonal antibody (Proteintech, Wuhan, China, 1:500), MSTN rabbit polyclonal antibody (Bioss, Beijing, China, 1:500), GAPDH rabbit polyclonal antibody (HUABIO, Hangzhou, China, 1:500), and HRP binding Goat anti-rabbit IgG (BBI, Shanghai, China, 1:5000).

Techniques: Expressing, Marker, Transfection, Staining, Fluorescence, Inverted Microscopy, Over Expression, Inhibition, Negative Control

Journal: Cells

Article Title: MicroRNA-27b-3p Targets the Myostatin Gene to Regulate Myoblast Proliferation and Is Involved in Myoblast Differentiation

doi: 10.3390/cells10020423

Figure Lengend Snippet: The inhibition of miR-27b-3p on MSTN is different between CPMs proliferation and differentiation. ( a ) The relative expression of MSTN during CPMs’ proliferation and differentiation (GM 50% and GM 100% represent the proliferation density of 50% and 100%; DM1-DM 4 represents differentiation from one to four days. ( b , c ) The mRNA expression levels of differentiation marker genes after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. ( d , e ) The protein expression levels of MyHC after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. ( f , g ) MyHC staining of CPMs at 72 h after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. Microscopic images were obtained by a fluorescence inverted microscope (dmi8, Leica, Germany). ( h , i ) Myotube area (%) of CPMs 72 h after overexpression and inhibition of MSTN . ( j , k ) The proliferating (GM) and differentiating (DM) CPMs were transfected with miR-27b-3p mimic, respectively, and the relative MSTN mRNA expression levels were then analyzed. In all graphs, the results are shown as mean ± SEM (standard error of the mean) ( n = 3). Statistical significance of differences between means was assessed, using the unpaired Student’s t -test (* p < 0.05; ** p < 0.01) vs. NC (negative control).

Article Snippet: The antibody and its dilution ratio were as follows: MYHC rabbit polyclonal antibody (Proteintech, Wuhan, China, 1:500), MSTN rabbit polyclonal antibody (Bioss, Beijing, China, 1:500), GAPDH rabbit polyclonal antibody (HUABIO, Hangzhou, China, 1:500), and HRP binding Goat anti-rabbit IgG (BBI, Shanghai, China, 1:5000).

Techniques: Inhibition, Expressing, Marker, Transfection, Staining, Fluorescence, Inverted Microscopy, Over Expression, Negative Control

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: Representative images of three cirrhotic (A) and two control livers (C) generated with PhenoCycler (formerly Codex) with a selection of markers such as CD56, CD94, aSMA, CD36, and CD32, as well as DAPI for nucleus staining (A, C). An overview (left) and magnification (right) are shown side by side with scale bar and indicated diameters. Fibrotic regions were recognized and delineated with a white dashed line using aSMA-staining. The red arrows indicate LT-ILC1 cells automatically detected by the HALO image analysis software by CD45 + CD3 − CD56 + CD94 + EOMES − KLRG1 + definition (see staining in ). Images in (B) and (D) were artificially generated as cell contours using HALO image analysis and correspond to the respective section from (A) and (C). Only the identified LT-ILC1 cells were highlighted for enhanced visibility (surface is green, nucleus is blue and indicated with red arrows) with visualization of the non-fibrotic (NF) and fibrotic (F) regions.

Article Snippet: KLRG1 , Miltenyi , RRID: AB_2889702; Cat#130-126-458.

Techniques: Control, Generated, Selection, Staining, Software

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: (A) Sorted peripheral blood ILCPs ( ; Lin − CD45 + CD127 + CD94 − NKp80 − CD45RA + CD117 + CD294 − KLRG1 − NKP44 − ) from four healthy donors were cultured for 2 weeks on OP9-DL4 feeder cells or LSECs with IL-7 +/− TGF-β1 as shown to the left. The dot plots to the right show the expression patterns of CD56 and CD94 among Lin − lymphocytes derived in the indicated culture conditions. (B) Frequencies of CD200R1 + CD49a + EOMES − cells among total Lin − CD45 + CD94 + CD56 + cells derived in vitro following culture of ILCPs (n = 8) as in (A). (C) Representative flow cytometry analyses of EOMES, T-BET, perforin, CXCR6, NKp80, CD200R1, and CD49a expression by Lin − CD45 + CD94 + CD56 + cells derived from ILCPs in the indicated culture conditions. Fluorescence minus one (FMO) control with the respective lacking specific antibody is shown as black dotted lines in each histogram. (D) Intracellular flow cytometry analysis of IFN-γ, IL-2, GM-CSF, TNF-α, and IL-22 production by in vitro derived Lin − CD45 + CD94 + CD56 + cells generated from ILCPs as in (A) and following stimulation for 5 h with PMA and ionomycin. Unstimulated cells were set as controls (dotted black line). (E) Representative flow cytometry analyses showing expression of the indicated markers by total Lin − CD45 + cells obtained after 2 weeks culture of primary sorted liver-type hepatic cNK, CD49a + trNK, or CD49a − trNK cells on OP9-DL4 feeder cells or LSECs with IL-7 +/− TGF-β1 as shown to the left. Data are representative of at least three independent experiments. (F) Representative flow cytometry analyses showing expression of the indicated markers by total Lin − CD45 + cells obtained after 2-week culture of primary sorted liver-type ILC1s (LT-ILC1s) or hepatic NK cells (sorted as Lin − CD56 + CD94 + NKp80 + ) with OP9-DL4 cells and IL-7. Data are representative of at least three independent experiments. (G) Frequencies of CD200R1 + CD49a + EOMES − cells among total Lin − CD45 + CD94 + lymphocytes obtained from cultures initiated with either total hepatic NK cells or liver-type ILC1s (F). *p < 0.05, **p < 0.01, ***p < 0.001, error bars represent SEM. See .

Article Snippet: KLRG1 , Miltenyi , RRID: AB_2889702; Cat#130-126-458.

Techniques: Cell Culture, Expressing, Derivative Assay, In Vitro, Flow Cytometry, Fluorescence, Control, Generated

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: KLRG1 , Miltenyi , RRID: AB_2889702; Cat#130-126-458.

Techniques: Blocking Assay, Recombinant, Clinical Proteomics, Nucleic Acid Electrophoresis, Software, Inverted Microscopy