Journal: Nature Cell Biology

Article Title: Aggresome assembly at the centrosome is driven by CP110–CEP97–CEP290 and centriolar satellites

doi: 10.1038/s41556-022-00869-0

Figure Lengend Snippet: ( a ) RPE-1 cells were transiently transfected with GFP-HTT97Q and stained for pHSP27 or Ub + . DNA was stained with DAPI. Scale bar 10 μm. ( b ) Immunoblot of soluble and insoluble fractions from WT and PCM1 KO cells transfected with GFP-HTT25Q. GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( c ) Cells were transfected with GFP-HTT97Q for 5 hours, then treated with DMSO or nocodazole for 5 hours before being fixed and stained for GFP and α-tubulin. Scale bar 10 μm. ( d ) Immunoblot of soluble and insoluble fractions from cells prepared as in c . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( e ) WT and STIL KO U-2 OS cells were transiently transfected with GFP-HTT97Q then stained for GFP, CEP135 and α-tubulin. Scale bar 10 μm. ( f ) Immunoblot of soluble and insoluble fractions from cells prepared as in e . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( g ) HEK293T-FLAG-miniTurbo-CP110 cells were treated and stained as indicated. Scale bar 10 μm, 2 μm. ( h ) Immunoblot of HEK293T-FLAG-miniTurbo-CP110 cells treated and probed as indicated. α-tubulin was used as a loading control. ( i ) The number of preys from DMSO- or MG132-treated groups. Preys were defined as detailed in the Methods. ( j ) Functional enrichment analysis was performed with the preys from i . using g:Profiler and the KEGG database. ( k ) Spectral counts from the genes which are implicated in Parkinson’s disease, proteasome function or Huntington’s disease are shown. Genes which are related to the respective pathways are marked in green. Unprocessed immunoblots are provided as source data. For i , j , k : the raw mass spectrometry data and analysis giving rise to these panels is available in Supplementary Table .

Article Snippet: To produce lentivirus, 4 × 10 6 HEK293T (female, human kidney; ATCC, ACS-4500) cells were seeded in a T-75 flask and transfected with the relevant lentiviral transfer vector, 3 μg psPAX2 and 2 μg pCMV-VSV-G using Lipofectamine 3000 (Invitrogen).

Techniques: Transfection, Staining, Western Blot, Control, Functional Assay, Mass Spectrometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

doi: 10.1016/j.omtm.2020.10.007

Figure Lengend Snippet: Design and Use of CRISPR gRNAs in HEK293T Cell Line (A) Exon 9 of the human PKLR gene is represented. Vertical lines and arrows are reported mutations, corresponding to one or more than 1 nt affected, respectively. Horizontal dashed lines represent the two gRNAs used. Vertical dashed lines represent predicted cut sites of gRNAs selected. (B) Sorting of green fluorescence based on the ZsGreen present in the electroporated plasmids. A 530/40- versus 580/30-nm dot plot was used to distinguish ZsGreen fluorescence from autofluorescence. The figure represents the ZsGreen fluorescence of the cell electroporated with the plasmid DNA containing only the Cas9 and the ZsGreen cDNAs (C), or containing the Cas9, the ZsGreen cDNAs and the sequences of the guide I (I) or the guide II (II). (C) Surveyor assay of the DNA obtained from the cells electroporated with the plasmids containing no guide, guide I, guide II, or both guides together (I+II). The gene editing generates a characteristic band pattern for guide I (160 bp + 135 bp), for guide II (191 bp + 104 bp), and a mixed pattern when both are used at the same time (160 bp + 104 bp). (D) Scheme of the band sizes that appear in (C) showing the 31-bp deletion resulting after the simultaneous electroporation of guides I and II.

Article Snippet: The HEK293T cell line (human embryonic kidney cell line competent to replicate vectors carrying the SV40 T antigen; ATCC, CRL-3219) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco), HyClone fetal bovine serum (10%; GE Healthcare), and penicillin/streptomycin (1%; Gibco).

Techniques: CRISPR, Fluorescence, Plasmid Preparation, Electroporation