Journal: Cell Death Discovery

Article Title: Ferroptosis contributes to ethanol-induced hepatic cell death via labile iron accumulation and GPx4 inactivation

doi: 10.1038/s41420-023-01608-6

Figure Lengend Snippet: A Schematic illustration of RIP analysis of the interaction between miR-214 and AGO1 or AGO2 proteins in the cytoplasm and nucleus of mouse primary hepatocytes. B RIP analysis of miR-214 enrichment in the cytoplasm and nucleus using AGO1 and AGO2 antibody. Immunoblotting analysis of the expressions of ACSL4 and PRKAA2 protein in primary hepatocytes co-transfected with miR-214 mimics (20 nM) and/or AGO1 siRNAs ( C ) or AGO2 siRNAs ( D ). Data are expressed as means ± SD from at least three independent experiments. ns not significant; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following chemicals and reagents were from commercial sources: DMEM, and penicillin–streptomycin, BasalMedia (Shanghai, China); FBS, GlutaMAX and William’s E Medium, Gibco (Grand Island, NY, USA); ethanol, Sigma-Aldrich (St. Louis, MO, USA); RSL3, Fer-1, Lip-1, Nec-1, Z-VAD, and CCK8 reagent, MedChem Express (Monmouth Junction, NJ, USA); miR-214-3p mimics, control mimics (miR-NC), miR-214 inhibitors (IH-214), and control inhibitors (IH-NC), GE Healthcare (Little Chalfont, Buckinghamshire, UK); siRNAs against GPX4 (si- GPX4 ), siRNAs against AGO1 ( si-AGO1 ), siRNAs against AGO2 ( si-AGO2 ) and the negative control siRNAs (si-NC), GenePharma (Shanghai, China); primers, Tsingke (Beijing, China); ACSL4 antibody (Cat no: ab155282), PRKAA2 antibody (Cat no: ab97275), RNA plolymerase II ChIP-grade antibody (Cat no: ab264350), and H3K27ac ChIP-grade antibody (Cat no: ab4729), Abcam (Cambridge, MA, USA); β-Actin antibody (Cat no: 81115-1-RR) and goat-anti-mouse HRP antibody, Proteintech (Wuhan, Hubei, China); Rabbit IgG, Beyotime (Shanghai, China); AGO1 (Cat no: 03-249) and AGO2 (Cat no: 03-110) RIP antibody, Merck Millipore (Billerica, MA, USA).

Techniques: Western Blot, Transfection

Journal: Cells

Article Title: Nucleolin Regulates Pulmonary Artery Smooth Muscle Cell Proliferation under Hypoxia by Modulating miRNA Expression.

doi: 10.3390/cells12050817

Figure Lengend Snippet: Figure 4. NCL expression affects specific miRNA levels. (A,B) Levels of the indicated miRNAs relative to U6 snRNA measured by qRT−PCR in PASMCs transfected with control or si−NCL for 24 h (A) or nucleofected with the control or NCL−expressing plasmid for 24 h (B). The data represent the mean ± S.E. of triplicate measurements of three independent experiments. (C,D) Immunoprecipi- tation assays from PASMC lysates were performed using an anti-NCL antibody or IgG. The presence of DGCR8, Ago2, and NCL in the pull-down and input (2%) samples was monitored by immunoblot- ting. Input: immunoblots showing the abundance of DGCR8, Ago2, or NCL in the total protein extracts. IP: IP products precipitated by the anti−NCL, anti−DGCR8, anti−Ago2 antibody, or IgG. (E,F) Immunoprecipitation assays from PASMC lysates were carried out using an anti-DGCR8 antibody, anti−Ago2 antibody, or IgG. The presence of NCL, DGCR8, and Ago2 in the immunoprecip- itates and input (2%) samples was monitored by immunoblotting. Statistical analyses were performed using two−way ANOVA Sidak’s multiple comparisons test (A,B) and two−tailed unpaired Student’s t-test (C,F). **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001.

Article Snippet: For DGCR8 IP and Ago2 IP, 5 μg of mouse anti-DGCR8 antibody (#60084-1-Ig, Proteintech, Rosemont, IL, USA) and mouse anti-Ago2 antibody (#ab57113, Abcam) were used, respectively.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells

doi: 10.3390/ijms20194933

Figure Lengend Snippet: TGF-β1 selectively recruited miRNAs validated or predicted as CFTR inhibitors to RISC in WT-and F508del-CFBE cells ( A ). Representative WB images showing that Ago2 was specifically immunoprecipitated (IP) with the anti-Ago2 antibody RN003M. Non-immune IgG was used as a negative control. Ago2 protein was detected in the IP and whole cell lysate (WCL) samples with the anti-Ago2 antibody. IP Ago2 was normalized for WCL Ago2. The non-specific band of the IP antibody is marked with an asterisk. Representative WB images ( B ) and summary of data ( C ) showing that TGF-β1 had no effect on Ago2 abundance in WCL and the Ago2 IP efficiency in WT- and F508del-CFBE cells. ( D – F ). The co-IP of miRNAs with Ago2 IP was detected by qRT-PCR in the IP samples. Threshold cycle (Ct) values of miR-145 in vehicle-treated cells were subtracted from Ct values of miR-145 in TGF-β1-treated cells to generate ΔCts. FC in miR-145 level between samples was determined using the equation 2 −ΔC t and expressed as Log2 FC versus vehicle. TGF-β1 increased the miR-145 ( D ) and miR-143 ( E ) co-IP with Ago2 in WT-CFBE and F508del-CFBE cells and did not affect the co-IP of miR-154 ( F ). Error bars, S.E.M. N = 10/group. * p < 0.05.

Article Snippet: The IP antibody complexes were prepared by conjugating 15 μg of RIP-certified anti-EIF2C2 (Ago2) mouse monoclonal antibody (MBL, RN003M) with 30 μL of 50% Pierce TM Protein G sepharose bead slurry (Thermo Scientific, Rockford, IL) overnight at 4 °C.

Techniques: Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells

doi: 10.3390/ijms20194933

Figure Lengend Snippet: Summary of data showing TGF-β1 recruitment of miR-143 and miR-145 to RISC to destabilize CFTR mRNA and impair the corrector/potentiator-mediated rescue of F508del-CFTR. miR-143 and miR-145 are transcriptionally regulated by the canonical Smad2/3 TGF-β1 signaling pathway. Modulating the total cellular miRNA levels is not sufficient to regulate expression of CFTR. Selective RISC recruitment of the experimentally validated CFTR inhibitors, miR-143 and miR-145, by TGF-β1 destabilizes CFTR mRNA, decreases CFTR protein translation, and impairs the corrector/potentiator-mediated rescue of CFTR protein and channel function in F508del HBE cells. Predicted CFTR inhibitor, miR-154 was upregulated but not recruited to RISC by TGF-β1, and thus it is not predicted to play a role in TGF-β1-induced degradation of CFTR mRNA. ER, endoplasmic reticulum; TβR-I, TGF-β receptor I; Ago2, Argonaute2; RISC, RNA-induced silencing complex.

Article Snippet: The IP antibody complexes were prepared by conjugating 15 μg of RIP-certified anti-EIF2C2 (Ago2) mouse monoclonal antibody (MBL, RN003M) with 30 μL of 50% Pierce TM Protein G sepharose bead slurry (Thermo Scientific, Rockford, IL) overnight at 4 °C.

Techniques: Expressing