scr control aav plasmid constructs  (Addgene inc)

Journal: eLife

Article Title: mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex

doi: 10.7554/elife.58737

Figure Lengend Snippet: Figure 3. Manipulation of mTOR signaling results in migration defects. (A) shRNAs were delivered along and electroporated onto the ventricular surface of primary cortical tissue which was then acutely sectioned and cultured for six days prior to collection. (B) After electroporation of RPTOR or TSC2 shRNAs, GFP+ HOPX+ oRGs migrate less from the ventricular edge (n = 37 control, n = 33 Raptor and n = 27 TSC2-shRNA electroporated GFP +Hopx+ cells from three independent experiments; D’Agostino Pearson Normality Test: normally distributed; one-way ANOVA with multiple comparisons: Raptor shRNA: ****p<0.0001, TSC2 shRNA: ****p<0.0001, error bars represent SD). The distance of each HOPX+ GFP+ cell away from the VZ edge was measured as indicated by white brackets. (C) For dynamic imaging studies, primary cortical tissue was collected, dissociated, infected with a CMV::GFP adenovirus, and plated on glass-bottom 12 well plates. Small molecules were added one day later, two hours before the start of dynamic imaging. (D) GFP+ oRG cells undergo division via MST. Yellow arrowheads indicate cell body, white dot indicates initial position of cell body and pink arrowheads indicate two cell bodies after division. After inhibition of mTOR signaling oRG cells have shorter MSTs (n = 10 control and n = 8 rapamycin cells across two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0082, error bars represent SD). (E) oRGs migrate less from their original position after mTOR inhibition (n = 9 vehicle treated and n = 12 rapamycin treated cells from two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0058 error bars represent SD). White, yellow, and pink arrowheads indicate cell bodies at starting time-point. Multiple arrowheads of the same color over time indicate daughter cells from the same parent cell. Figure 3 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) H1/WA01 embryonic stem cell line WiCell RRID:CVCL_9771 Male Cell line (Homo sapiens) H28126 induced pluripotent stem cell line Pollen et al., 2019 Male Cell line (Homo sapiens) 13234 induced pluripotent stem cell line Bhaduri et al., 2020 RRID:CVCL_0G84 Female Transfected construct (Homo sapiens) CMV::GFP Adenovirus Vector Biolabs, 1060 1:200 dilution Transfected construct (Aequorea victoria) pCAG-EGFP Subramanian et al., 2011 Electroporation Transfected construct (Homo sapiens) Scramble shRNA Addgene plasmid # 1864 RRID:Addgene_1864 Electroporation (1 mg/ul) Transfected construct (Homo sapiens) Raptor_1 shRNA Addgene plasmid # 1857 RRID:Addgene_1857 Electroporation (1 mg/ul) Transfected construct (Homo sapiens) pLKO.1-TSC2 Addgene plasmid # 15478 RRID:Addgene_15478 Electroporation (1 mg/ul) Biological sample (Homo sapiens) Primary Cortex Tissue Samples UCSF Gamete, Embryo and Stem Cell Research Committee (GESCR) approval GW16-19 Antibody anti-Sox2 (Mouse monoclonal) Santa Cruz RRID:AB_10842165; Cat# sc-365823 (1:500) Antibody anti-Hopx (Mouse monoclonal) Santa Cruz RRID:AB_2687966; Cat# sc-398703 (1:250) Antibody anti-Cofilin (Mouse monoclonal) Santa Cruz RRID:AB_11150468; Cat# sc-376476 (1:100) Antibody anti-Cdc42 (Mouse monoclonal) Santa Cruz RRID:AB_627233; Cat#, sc-8401 (1:200) Antibody anti-Cux1 (Mouse monoclonal) Abcam RRID:AB_941209; Cat#AB54583 (1:500) Antibody anti-pVim (Mouse monoclonal) MBL International RRID:AB_592969; Cat# D095-3 (1:500) Antibody anti-pHistone H3 (Mouse monoclonal) Abcam RRID:AB_443110; Cat# ab14955 (1:500) Antibody anti-Cryab (Mouse monoclonal) Abcam RRID:AB_300400; Cat# ab13496 (1:500) Antibody anti-Hopx (Rabbit polyclonal) Proteintech RRID:AB_10693525; Cat# 11419–1-AP (1:500) Antibody anti-pS6 (Rabbit polyclonal) Cell Signaling RRID:AB_331679; Cat# 2211S (1:500) Continued on next page Andrews et al. eLife 2020;9:e58737.

Techniques: Migration, Cell Culture, Electroporation, Control, shRNA, Imaging, Infection, Inhibition, Two Tailed Test

Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Inhibition of mTORC2 RICTOR decreases CTC proliferation markers. ( A ) High-definition immunofluorescence on MCF-10A cells showing differential expression of Ki67 proliferation marker, along with high pNDRG1 expression, indicative of active mTORC2 signaling (scale bar = 10μm). ( B ) Western blotting analyses of ShRNA RICTOR knockdowns of MCF10A cells showing that RICTOR knockdown resulted in decreased pNDRG1 expression, while p4EBP1 status remained unchanged (red boxes). Control denotes non-targeting scrambled control, and numbers 1 and 2 (below sh-RICTOR) denote two distinct lentiviral shRNA constructs against RICTOR. ( C ) (Top) Significant decrease of total BMRCs collected from ex vivo experiments using MCF-10A-shRICTOR knockdowns injected in NSG mice (1.0 × 10 5 cells/mouse; N = 11). Conversely, no change of PanCK+ or CD44+/CD24− BMRC cell populations was detected. ( D ) (Bottom) Real-time PCR of ex vivo MCF-10A shRICTOR BMRCs exhibit increased gene expression for PCNA (proliferation marker), along with decreased CDKN1A (quiescence status) and increased BBC3 (PUMA) expression, consistent with a pro-apoptotic response ( N = 11).

Article Snippet: Lentiviral pLKO.1 shRICTOR (plasmid #1853 and #1854) and scrambled shRNA (plasmid #1864) constructs were obtained from AddGene (Cambridge, MA, USA), packaged in HEK293T cells using 3 rd generation lentiviral packaging system, and transduced into MCF10A cells [ ].

Techniques: Inhibition, Immunofluorescence, Quantitative Proteomics, Marker, Expressing, Western Blot, shRNA, Knockdown, Control, Construct, Ex Vivo, Injection, Real-time Polymerase Chain Reaction, Gene Expression