Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids

doi: 10.3389/fcell.2021.764725

Figure Lengend Snippet: Directed differentiationdependence of SIX6 reporter PSCs into 3D retinas to validate reporter expression. (A) Schematic of stem cells modified with a SIX6-GFP reporter. (B) CRISPR/Cas9 targeting with a guide RNA spanning the stop site of the SIX6 gene. Donor fragment the H2B-GFP expression cassette with homology arms to facilitate homology directed repair (HDR). (C) Verification of the GFP cassette insertion by PCR amplification of genomic DNA with oligonucleotides that 1- flank the GFP insert (detects homo- and heterozygosity), and 2- flank the left arm and within the insert (detects insert). A 1Kb + DNA ladder (Invitrogen) was used for DNA band size resolution. (D) The experimental approach used for reporter validation. (E) Bright-field images and (F) reporter expression in some, but not all, vesicles at day 12. Large arrows (E,F) indicate GFP + vesicles while smaller arrows indicate GFP - vesicles. (G) Neural vesicles excised at D12 were live imaged for GFP fluorescence. μm (H) A magnified image from panel (G) (squared box) with GFP fluorescence overlaid onto a bright-field image. (I) Percentage of vesicles that exhibit GFP +, GFP – or mixed expression patterns following manual excision. Scale bars = (E) 175 μm, (G) 300 μm.

Article Snippet: For transfection, PSCs were Accutase treated for 10 min, and 200,000 cells pelleted at 80 × g . For the SIX6-GFP single reporter line, electroporation was carried out in 10 μl’s of R-buffer containing 300 ng gRNA vector, 500 ng Cas9 vector (Addgene #41815) and 1 μg SIX6-GFP DONOR vector using a Neon transfection system (Invitrogen) with the following settings (1300 V, 20 ms, 1 pulse).

Techniques: Expressing, Modification, CRISPR, Amplification, Fluorescence

Journal: Scientific Reports

Article Title: Opposing effects of acute versus chronic inhibition of p53 on decitabine’s efficacy in myeloid neoplasms

doi: 10.1038/s41598-019-44496-6

Figure Lengend Snippet: p53-depleted MLL-AF9 cells were less sensitive to decitabine. ( a ) Experimental scheme used in ( c – f ). Mouse bone marrow (BM) cells were transduced with MLL-AF9 (coexpress GFP) and were transplanted into recipient mice. MLL-AF9-expressing leukemia cells were harvested from spleens of moribund mice, and were serially transplanted into recipient mice. These MLL-AF9 leukemia cells were then transduced with Cas9 together with non-targeting (NT) or Trp53 -targeting sgRNAs [sgTrp53-(1) and (2)], and were used in subsequent assays. ( b ) Schematic representation of Trp53 and sgRNA-targeting regions. ( c ) Levels of p53 protein in MLL-AF9 cells transduced with NT or Trp53 -targeting sgRNAs. Note that p53 protein in sgTrp53-(1)-transduced cells migrated faster than that in NT-transduced cells, indicating that it is a nonfunctional mutant protein. ( d ) Cell Viability Assays were performed using Cell Counting Kit-8. The control and p53-depleted MLL-AF9 cells were treated with DS-5272 (left) or decitabine (right) at indicated concentrations for 72 h in triplicate. Data are normalized to vehicle control (0 μM group), and are shown as mean ± s.d. ( e , f ) Cell-cycle status ( e ) and apoptosis ( f ) were assessed after 6 or 24 hours culture with decitabine (50 nM), respectively. ( e ) FACS profiles of DMSO- or decitabine-treated MLL-AF9 cells are shown. The numbers indicate the percentages of cells in the G0/G1, S and G2/M phases. ( f ) Shown are FACS profiles of Annexin V and DAPI expression of vehicle- or decitabine-treated MLL-AF9 cells. The numbers indicate the percentages of Annexin V + and Annexin V + DAPI + cells. ( g ) MLL-AF9/Cas9 cells were transduced with NT or sgTrp53-(1) coexpressing tRFP, following transplantation into recipient mice that were treated with vehicle or decitabine (0.6 mg/kg, every ohter day). Relative ratios of the tRFP + (sgRNA-transduced) fraction in GFP + MLL-AF9 leukemia cells after transplantation compared with that before transplantation are shown as mean ± s.d. The frequency of sgTrp53-(1)-transduced tRFP + cells was increased in mice treated with decitabine. (NT_Vehicle: n = 4, NT_Decitabine: n = 5, sgTrp53-(1)_Vehicle: n = 6, sgTrp53-(1)_Decitabine: n = 6) *p = 0.0041, **p < 0.0001. Sidak’s multiple comparisons test.

Article Snippet: The expression vector for Cas9 (lentiCas9-Blast #52962) was obtained from Addgene.

Techniques: Transduction, Expressing, Mutagenesis, Cell Counting, Control, Transplantation Assay

Journal: Scientific Reports

Article Title: Opposing effects of acute versus chronic inhibition of p53 on decitabine’s efficacy in myeloid neoplasms

doi: 10.1038/s41598-019-44496-6

Figure Lengend Snippet: p53 inhibition conferred resistance to decitabine in cSAM, cRAM and CB cells. ( a ) Experimental scheme used in ( b–d ). cSAM cells and cRAM cells were transduced with Cas9 together with non-targeting (NT) or Trp53 -targeting sgRNAs [sgTrp53-(1) and (2)], and were used in subsequent assays. ( b ) Levels of p53 protein in cSAM and cRAM cells transduced with NT or Trp53 -targeting sgRNAs. ( c , d ) Cell Viability Assay using control and p53-depleted cSAM ( c ) or cRAM ( d ) cells, treated with DS-5272 or decitabine at indicated concentrations for 72 h in triplicate. Data are normalized to vehicle control (0 μM group), and are shown as mean ± s.d. ( e ) Human cord blood (CB) CD34 + cells were transduced with a vector control or p53DD (coexpress NGFR). The cells were cultured in cytokine containing media to monitor the changes of NGFR frequency. p53DD showed the growth-promoting effect in CB cells only in the presence of decitabine in culture.

Article Snippet: The expression vector for Cas9 (lentiCas9-Blast #52962) was obtained from Addgene.

Techniques: Inhibition, Transduction, Viability Assay, Control, Plasmid Preparation, Cell Culture