pten knockout  (Santa Cruz Biotechnology)

Journal: Parasites & Vectors

Article Title: csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis

doi: 10.1186/s13071-023-06075-7

Figure Lengend Snippet: PTEN inhibited ICC proliferation and migration via a ferroptosis mechanism. a Stable PTEN overexpression (PTEN-EXO) or CRISPR/Cas9-based knockout (PTEN-KO) HuCCT1 cell lines were established; the scale shown is 100 um. b Cell proliferation was measured by CCK-8 assay in vitro. c Cell proliferation was measured by tumor xenograft in vivo. d Cell migration was measured by transwell assay. e Cell GSH/GSSG radio assay. f Cell Fe 2+ assay. g Cell MDA assay. h , i Cell WB assay of SLC7A11 and GPX4. ICC , intrahepatic cholangiocarcinoma; WB , western blotting; GSH , glutathione; GSSG , GSH disulfide; MDA , malondialdehyde. * P < 0.05, ** P < 0.01

Article Snippet: To create stable cell lines, recombinant lentivirus (LV) vectors containing csi-miR-96-5p mimics (Shanghai Genchem Co., Ltd.), a PTEN overexpression plasmid (PTEN-EXO) (Sino Biological, China), and a PTEN CRISPR/Cas9 based knockout (PTEN-KO) plasmid (Shanghai Genchem Co., Ltd.) were constructed.

Techniques: Migration, Over Expression, CRISPR, Knock-Out, CCK-8 Assay, In Vitro, In Vivo, Transwell Assay, Multiple Displacement Amplification, Western Blot

Journal: Cell reports

Article Title: MCK2-mediated MCMV infection of macrophages and virus dissemination to the salivary gland depends on MHC class I molecules.

doi: 10.1016/j.celrep.2023.112597

Figure Lengend Snippet: Figure 2. Neuropilin 1 (Nrp1) and CX3CR1 do not mediate MCK2-dependent MCMV infection of macrophages (A) Representative flow cytometry histograms plots of Nrp1 levels on NIH/3T3 fibroblasts or RAW 264.7 monocyte/macrophage cells without nucleofection or nucleofected with CRISPR-Cas9 ribonucleoparticles targeting Nrp1 (Nrp1 RNPs). (B) Quantification of mCherry signal at 20 hpi with indicated MCMV strains at MOI of 1 from cells treated with control (Ctrl.) RNPs or Nrp1 RNPs. (C) Representative flow cytometry histograms plots of CX3CR1 levels on NIH/3T3 fibroblasts or RAW 264.7 monocyte/macrophage cells that were nucleofected with Ctrl. or CX3CR1 RNPs. (D) Quantification of mCherry signal at 20 hpi with MCMV-3DR at MOI of 1 from cells treated with Ctrl. RNPs or CX3CR1 RNPs. (E) Quantification of mCherry signal at 20 hpi with MCMV-3D or -3DR at MOI of 1 from alveolar macrophages collected by bronchoalveolar lavage from wild-type (WT) or Cx3cr1/ mice. (B and D) Data are from 3–4 independent experiments. One dot equals a mean of the triplicates from one experiment, line at mean value per group. (E) Data are from 2 experiments with 5–6 animals per group (dots), and line represents mean value per group. (C–E) Statistical analysis: one-way ANOVA test followed by Sidak’s multiple comparison test; ns, not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Liberase Roche Cat#: 11988409001 Trypsin Biochrom AG Cat#: L2103-20 Critical commercial assays SF Cell Line 4D-NucleofectorTM X Kit L Lonza Inc. Cat#: V4XC-2012 SG Cell Line 4D-NucleofectorTM X Kit L Lonza Inc. Cat#: V4XC-3024 QIAamp DNA Mini Kit Qiagen Cat#: 51304 QIAquick PCR Purification Kit Qiagen Cat#: 28104 Mouse Brie CRISPR knockout lentiviral prep Addgene Cat#: 73633-LV Experimental models: Cell lines RAW 264.7 monocytes/macrophages ATCC ATCC TIB-71 NIH/3T3 fibroblasts ATCC ATCC CRL-1658 Primary mouse embryonal fibroblasts (MEF) from BALB/c mice In house N/A Hoxb8-immortalized Cas9-progenitor cells Hammerschmidt et al.43 N/A RMA T cell leukemia cells In house N/A RMA-S T cell leukemia cells In house N/A Experimental models: Organisms/strains Mouse: C57BL/6N Charles River Strain Code: 027 Mouse: BALB/c Charles River Strain Code: 028 Mouse: C3H Charles River Strain Code: 025 Mouse: B6.129P2-B2mtm1Unc/J mice The Jackson Laboratory Strain #:002070; RRID:IMSR_JAX:002070 Mouse: B6.129P2(Cg)-Cx3cr1tm1Litt/J The Jackson Laboratory Strain #:005582; RRID:IMSR_JAX:005582 Oligonucleotides For a list of used crRNAs please see Table S1.

Techniques: Infection, Cytometry, CRISPR, Control, Comparison