Journal: The Journal of Experimental Medicine
Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation
Figure Lengend Snippet: Deletion of an ATF3 binding region 2 Mb upstream of the IL6 gene results in specific induction of IL6 expression . (A) Overview of the construction of a dual-guide RNA (gRNA)–Cas9 expression vector. A two-step PCR amplification followed by insertion into the lentiCRISPR_v2 vector backbone was used to create a dual-expression cassette with mouse (mU6) and human (hU6) U6 promoters driving expression of two gRNAs targeting the ATF3 binding region upstream of the Il6 locus. For details, see Materials and methods. (B, top) Schematic representation of ATF3 binding region 2.07 Mb upstream of the Il6 locus, with the position of the dual gRNAs (gRNA 1 and 2) chosen for CRISPR/Cas9–mediated deletion and of the two primers (P1 and P2) used for genomic PCR analysis. (B, bottom) Nucleotide sequence of the genomic locus targeted by the two gRNAs (blue and red, respectively) together with PAM motifs (bold lines); below are the nucleotide-sequencing results of 300-bp genomic PCR products derived from two HDF clones expected to harbor the deletion (as shown in the panel below). (C) Small clusters/colonies of HDFs infected with the dual gRNAs lentiCRISPR vector (▵) and empty vector control (C) were analyzed by genomic PCR analysis with the P1 and P2 primers indicated above. Clones harboring the deletion in either a heterozygous or homozygous state, on the basis of PCR products of 300 bp versus 500 bp in size, were further analyzed by RT-qPCR for levels of Il6 , Tnc , and Tead4 expression. (D) Two additional deletion-harboring clones were analyzed together with controls by IF with Ab against IL6 and TNC. Shown are representative images together with a quantification of the fluorescence intensity signal in individual cells of deletion-harboring clones (▵) versus controls (C). n = 20 cells per condition, mean ± SEM, two-tailed unpaired t test, *, P
Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.
Techniques: Binding Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, CRISPR, Sequencing, Derivative Assay, Clone Assay, Infection, Quantitative RT-PCR, Fluorescence, Two Tailed Test