Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: RD114-pseudotyped retroviral vectors efficiently transduce primary human B cells (A) Experimental design. Human naive B cells were negatively selected from buffy coat and activated with human CD40L multimer in presence of IL-4 and IL-21 for 2 days. Simultaneously, retroviral packaging cell lines were plated for 20 h before transfection. Two days post transfection, retroviral supernatants were harvested and used for human B cell transduction. Transduced B cells were harvested on day 4 after transduction and analyzed by flow cytometry. (Created with BioRender.com ). (B) Representative FACS plots show gating strategy to identify mCherry + B cells at day 4 post transduction. (C) Representative FACS plots (top) and the graph (bottom) show the percentages of mCherry + B cells 4 days post transduction. Non-transduced cells were used to define mCherry + gate. HEK-293T was co-transfected with sgRNA plasmid, packaging plasmid (pHIT60), and envelope plasmid (GaLV WT); Platinum-A, 293Vec-GaLV, and 293Vec-RD114 were transfected with sgRNA plasmid only. Data were pooled from eight donors, shown as mean ± SEM. Statistical significance was calculated using one-way ANOVA test, only significant p values are shown: ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: Retroviral, Transfection, Transduction, Flow Cytometry, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: Establishing efficient transduction conditions for primary human B cells with RD114-and GaLV-pseudotyped retroviral vectors (A) Experimental design. Negatively selected human naive B cells were activated either with (i) human CD40L multimer in the presence of IL-4 and IL-21, or with (ii) 40LB feeder cells, in the presence of IL-2 and IL-21. 40LB feeder cells were irradiated (30 Gy) and plated 1 day prior to the B cell culture. (Created with BioRender.com ). (B) Representative FACS plots (top) show the percentage of mCherry + B cells at day 4 post transduction using different plasmid conditions as indicated. Picomole of sgRNA plasmid increased as opposed of packaging and envelope plasmids, respectively. The graphs (bottom) show frequencies of mCherry + B cells obtained from three donors for condition (i) and (ii). (C) The graph shows the percentage of mCherry + B cells activated with condition (i) compared with condition (ii) at day 4 post transduction with retrovirus produced by the 293Vec-RD114 cell line. Groups were compared using unpaired t test, only significant p values are shown: ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Data shown as mean ± SEM.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: Transduction, Retroviral, Irradiation, Cell Culture, Plasmid Preparation, Produced

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: Efficient CRISPR-Cas9-mediated knockout of ß2M housekeeping gene in primary human B cells (A) ß2M -specific targeting sequence was inserted into BbsI restriction site of sgRNA plasmid (left). The scheme shows the targeted site of ß2M exon 1 and two primer sites for amplifying the flanking region of ß2M -targeted site by PCR (right). (B) The sequence of T2A-GFP was amplified from plasmid MSCV-Cas9-2A-GFP-sgRNA and Furin-GSG sequence was included in the forward primer. HindIII restriction sites were added in both forward and reverse primers. The amplified fragment was digested with HindIII enzyme and replaced for T2A-mCherry sequence in plasmid MSCV-Cas9-T2A-mCherry. The reconstructed plasmid carries sequences of Cas9-Furin-GSG-T2A-GFP. (C) Experimental design. Negatively selected human naive B cells were activated with CD40L multimer in presence of IL-4 and IL-21 for 2 days. In parallel, 293Vec-RD114 retroviral packaging cells were plated for 20 h until transfection. Two days post transfection, retroviral supernatant was harvested for transduction. Day 4 post transduction, a half of B cells was harvested for FACS analysis or cell sorting and the other half was remained in culture with IL-10 and IL-21 for another 4 days. (Created with BioRender.com ). (D) Representative FACS plots show co-transduced B cells before (top) and after (bottom) sorting at day 4 post transduction. (E) Agarose gel from T7 endonuclease I assay shows digested bands from sg ß2M -targeted compared with non-targeted sample. sg ß2M : sgRNA plasmid with ß2M -targeted sequence; (−): non-targeted. Red asterisks highlight the digested DNA bands. (F) Sanger sequencing signal traces (left) show noise peaks at ß2M -targeted site compared with non-targeted sample. The dashed line indicates cleavage site of Cas9. The graph (right) shows the percentage of Indel mutations at ß2M -targeted site compared with non-targeted sample. Alignment and the percentage of Indel mutations were performed by ICE analysis online program (Synthego). (G) Representative FACS plots show co-transduced B cells with both sgRNA (mCherry) and Cas9 (GFP) (left) and β2M − B cells (right) at day 4 and 8 post transduction. mCherry + GFP + B cells were gated based on non-transduced sample, β2M − B cells were gated based on non-targeted sample (transduced with sgRNA and Cas9 plasmids but sgRNA has no targeted sequence). The graph (right) shows the percentage of mCherry + GFP + and surface β2M − B cells in mCherry + GFP + cells at day 0, 4, and 8 post transduction. Statistical significance was calculated using the unpaired t test: ∗∗p < 0.01; ∗∗∗∗p < 0.0001. Data were pooled from three donors (F) and six donors (G), shown as mean ± SEM.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: CRISPR, Knock-Out, Sequencing, Plasmid Preparation, Amplification, Retroviral, Transfection, Transduction, FACS, Agarose Gel Electrophoresis, T7EI Assay

Journal: Molecular Medicine Reports

Article Title: PPARγ attenuates hepatic inflammation and oxidative stress of non-alcoholic steatohepatitis via modulating the miR-21-5p/SFRP5 pathway

doi: 10.3892/mmr.2021.12463

Figure Lengend Snippet: Activation of PPARγ inhibits lipid droplet accumulation, hepatic inflammation and oxidative stress by downregulating miR-21-5p. (A) HepG2 cells were treated with Rosi (0.5, 5 and 10 µm) or control (0.1% DMSO) for 48 h and then the expression of miR-21-5p was detected by qPCR. (B) siRNA against PPARγ (si-PPARγ), or scrambled siRNA negative control (si-control) was transfected into HepG2 cells and the expression of PPARγ was detected by western blotting. (C) Following transfection with si-control or si-PPARγ, HepG2 cells were treated with DMSO or 10 µm Rosi for 24 h. Then the level of miR-21-5p was assayed by qPCR. Data are expressed as means ± SD. *P<0.05, **P<0.01 vs. DMSO. (D) HepG2 cells were treated with 1 mm free fatty acid for 16 h to establish a NASH cell model and then the cells were treated with DMSO, Rosi (10 µm), Rosi (10 µM) + miR-21-5p mimic (50 nm) or 10 µm Rosi + miR-21-5p (50 nm) mimic + miR-21-5p inhibitor (50 nm) for 24 h. The effect of Rosi on lipid accumulation in NASH cells was assayed by using oil Red O staining (magnification, ×200), the arrows indicate lipid particles. (E) miR-21-5p mimic (50 nm) or mimic NC (50 nm) were transfected into HepG2 cells. After 24 h, the cells were harvested and the miR-21-5p was detected by qPCR. (F) The inflammatory cytokines TNF-α, IL-6 and MCP-1 were detected by qPCR. (G) The lipid peroxidation product MDA was tested using an MDA assay kit. (H) The activity of SOD was measured using a SOD assay kit. (I) The ratio of GSH/GSSG was evaluated. Data are expressed as means ± SD. **P<0.01 vs. Rosi, ## P<0.01 vs. Rosi + miR-21-5p mimic. PPARγ, peroxisome proliferator-activated receptor γ; miR, microRNA; Rosi, rosiglitazone; qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; NC, negative control; NASH, non-alcoholic steatohepatitis; MCP, monocyte chemotactic protein; MDA, methane dicarboxylic aldehyde; SOD, superoxide dismutase; GSH, glutathione peroxidase; GSSG, oxidized glutathione.

Article Snippet: Rosiglitazone (Rosi) was purchased from Cayman Chemical Company. miR-21-5p mimic, inhibitor and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. Superoxide dismutase (SOD) assay kit, methane dicarboxylic aldehyde (MDA) assay kit, glutathione peroxidase (GSH) assay kit and oxidized glutathione (GSSG) assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. miRNA Detection kit was purchased from GeneCopoeia, Inc. ChIP kit was from Merck KGaA.

Techniques: Activation Assay, Control, Expressing, Negative Control, Transfection, Western Blot, Staining, Multiple Displacement Amplification, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: PPARγ attenuates hepatic inflammation and oxidative stress of non-alcoholic steatohepatitis via modulating the miR-21-5p/SFRP5 pathway

doi: 10.3892/mmr.2021.12463

Figure Lengend Snippet: PPARγ suppresses transcriptional activity of miR-21-5p and upregulates SFRP5. (A) Diagram of the putative wild-type PPARE ER4-WT and mutant ER4 (ER4-Mut, the mutated bases are underlined) in miR-21-5p gene promoter region. (B) HepG2 cells were transfected with luciferase reporter plasmids pGL3-PPARE-WT, or pGL3-PPARE-Mut or pGL3-Basic and pRL-TK and cultured for 12 h and then treated with control (0.1% DMSO) or 5 µm Rosi for 24 h. Dual-luciferase reporter assays were performed. The firefly luciferase activity was normalized to Renilla luciferase activity and the result was shown as relative luciferase activity. Data are expressed as mean ± SD of three assays performed in triplicate. **P<0.01. (C) HepG2 cells were treated with 0.1% DMSO or 5 µm Rosi for 24 h and the cells harvested for chromatin immunoprecipitation assay. PPARγ, peroxisome proliferator-activated receptor γ; miR, microRNA; SFRP5, secreted frizzled-related protein 5; PPARE, PPARγ response element; PPARγ, peroxisome proliferator-activated receptor γ; WT, wild-type; Mut, mutant; Rosi, rosiglitazone.

Article Snippet: Rosiglitazone (Rosi) was purchased from Cayman Chemical Company. miR-21-5p mimic, inhibitor and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. Superoxide dismutase (SOD) assay kit, methane dicarboxylic aldehyde (MDA) assay kit, glutathione peroxidase (GSH) assay kit and oxidized glutathione (GSSG) assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. miRNA Detection kit was purchased from GeneCopoeia, Inc. ChIP kit was from Merck KGaA.

Techniques: Activity Assay, Mutagenesis, Transfection, Luciferase, Cell Culture, Control, Chromatin Immunoprecipitation

Journal: Molecular Medicine Reports

Article Title: PPARγ attenuates hepatic inflammation and oxidative stress of non-alcoholic steatohepatitis via modulating the miR-21-5p/SFRP5 pathway

doi: 10.3892/mmr.2021.12463

Figure Lengend Snippet: PPARγ downregulates miR-21-5p but upregulates SFRP5. (A) Diagrams of the predicted potential binding sites of miR-21-5p in 3′-UTR of SFRP5 mRNA. HepG2 cells were transfected with miR-21-5p mimic, inhibitor, or NC and cultured for 24 h and the (B) mRNA and (C) protein levels of SFRP5 were examined by qPCR or western blotting. (D) The correlation between the miR-21-5p and SFRP5 mRNA levels in 20 liver tissue samples from NASH patients was analyzed using Pearson's correlation coefficient (r=−0.629, P<0.01). (E) HepG2 cells were transfected with the luciferase reporter plasmid in the presence of miR-21-5p mimic, inhibitor, or mimic NC and inhibitor NC and cultured for 24 h. The luciferase activities were measured by dual-luciferase reporter assays. The firefly luciferase activity was normalized to Renilla luciferase activity. HepG2 cells were transfected with miR-21-5p mimic or mimic NC and cultured for 12 h and then cells were treated with control (0.1% DMSO) or 5 µm Rosi. After treatment for 24 h, the (F) mRNA and (G) protein levels of SFRP5 were examined by qPCR or western blotting. Data are expressed as means ± SD. **P<0.01 vs. Rosi. PPARγ, peroxisome proliferator-activated receptor γ; miR, microRNA; SFRP5, secreted frizzled-related protein 5; NC, negative control; qPCR, reverse transcription-quantitative PCR; NASH, non-alcoholic steatohepatitis; Rosi, rosiglitazone.

Article Snippet: Rosiglitazone (Rosi) was purchased from Cayman Chemical Company. miR-21-5p mimic, inhibitor and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. Superoxide dismutase (SOD) assay kit, methane dicarboxylic aldehyde (MDA) assay kit, glutathione peroxidase (GSH) assay kit and oxidized glutathione (GSSG) assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. miRNA Detection kit was purchased from GeneCopoeia, Inc. ChIP kit was from Merck KGaA.

Techniques: Binding Assay, Transfection, Cell Culture, Western Blot, Luciferase, Plasmid Preparation, Activity Assay, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: PPARγ attenuates hepatic inflammation and oxidative stress of non-alcoholic steatohepatitis via modulating the miR-21-5p/SFRP5 pathway

doi: 10.3892/mmr.2021.12463

Figure Lengend Snippet: Activation of PPARγ represses the progression of NASH by regulating the miR-21-5p/SFRP5 axis in mice. Mice were fed for 8 weeks either a methionine- and choline-sufficient diet (Normal) or a methionine- and choline-deficient (MCD) diet (NASH model). Model group mice were treated with vehicle (saline containing 0.1% DMSO), Rosi (10 mg/kg), Rosi (10 mg/kg) + miR-21-5p NC (8 mg/kg), or Rosi (10 mg/kg) + miR-21-5p mimic (8 mg/kg) daily for 3 weeks (n=5). (A) The level of miR-21-5p in liver tissues of mice which treated with miR-21-5p NC or miR-21-5p mimic were detected by qPCR. (B) The photomicrographs of liver sections stained with routine hematoxylin and eosin (magnification, ×200). (C) The inflammatory cytokines TNF-α, IL-6 and MCP-1 in the liver tissue were detected by qPCR. (D) The lipid peroxidation product MDA was tested using an MDA assay kit. (E) The activity of SOD was measured using a SOD assay kit. (F) The ratio of GSH/GSSG was evaluated. Data are expressed as means ± SD. **P<0.01 vs. DMSO, ## P<0.01 vs. miR-21-5p NC. (G) The expression of miR-21-5p and SFRP5 in the liver tissues was detected by qPCR. **P<0.01 vs. DMSO, ## P<0.01 vs. miR-21-5p NC. (H) The protein level of SFRP5 in the liver tissue was detected by western blotting. PPARγ, peroxisome proliferator-activated receptor γ; NASH, non-alcoholic steatohepatitis; miR, microRNA; SFRP5, secreted frizzled-related protein 5; Rosi, rosiglitazone; NC, negative control; qPCR, reverse transcription-quantitative PCR; MCP, monocyte chemotactic protein; MDA, methane dicarboxylic aldehyde; SOD, superoxide dismutase; GSH, glutathione peroxidase; GSSG, oxidized glutathione; SFRP5, secreted frizzled-related protein 5; NASH, non-alcoholic steatohepatitis; Rosi, rosiglitazone.

Article Snippet: Rosiglitazone (Rosi) was purchased from Cayman Chemical Company. miR-21-5p mimic, inhibitor and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. Superoxide dismutase (SOD) assay kit, methane dicarboxylic aldehyde (MDA) assay kit, glutathione peroxidase (GSH) assay kit and oxidized glutathione (GSSG) assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. miRNA Detection kit was purchased from GeneCopoeia, Inc. ChIP kit was from Merck KGaA.

Techniques: Activation Assay, Saline, Staining, Multiple Displacement Amplification, Activity Assay, Expressing, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction