Journal: Nature cell biology

Article Title: HSF1 critically attunes proteotoxic-stress sensing by mTORC1 to combat stress and promote growth

doi: 10.1038/ncb3335

Figure Lengend Snippet: ( a ) HEK293T cells were stably transduced with lentiviral scramble or two combinations of JNK1/2 -targeting shRNAs, A (shJNK1_2 and shJNK2_1) and B (shJNK1_4 and shJNK2_2). Transduced cells were treated with DMSO or 500 nM MG132 for 4 hr. (b ) Three independent lines of primary MEFs were prepared for each genotype. ( c ) and ( d ) Primary Jnk1 -/- or Jnk2 -/- MEFs were treated with 200 nM MG132 for 6 hr. While JNK1 antibodies only recognized the p46 isoform, JNK2 antibodies recognized both p54 and p46 isoforms. ( e ) HEK293T cells were transfected with indicated plasmids for 48 hr. The JNK1 CA plasmid encodes a fusion protein between MKK7 and JNK1A1. ( f ) and ( g ) Following transfection with indicated siRNAs or plasmids, HEK293T cells were labeled with 6-FAM-dc-puromycin for 30 min and analyzed by flow cytometry. ( h ) - ( j ) Sizes of transfected HeLa cells (h and i) and primary Jnk -deficient MEFs (j) were measured by a Multisizer™ 3 Coulter Counter. JNK manipulation causes statistically significant changes in cell size distribution (Kolmogorov-Smirnov test, p<0.001). (k ) Freshly prepared single liver cell suspensions were immediately analyzed by flow cytometry. Results were normalized against wild-type controls (mean±SD, n=3 or 4 mice per genotype, One-way ANOVA). ( l ) mTORC1 signaling in livers of 6-week-old male mice was immunoblotted. ( m ) Two hours before harvesting tissues, mice were i.p. injected with 1mg puromycin. The levels of puromycin-labeled proteins in livers were quantitated by ELISA (mean±SD, n=3 mice per genotype, unpaired two-tailed Student's t test). ( n ) - ( p ) Whole-body weight and composition were measured in 6-week-old female mice (mean±SD, n=5, 6, or 9 mice per genotype, One-way ANOVA). Statistics source data for 2k, m can be found in .

Article Snippet: Cells were incubated with 6-FAM-dc-puromycin (50 nM, Jena Bioscience) in vitro for 1h and analyzed by flow cytometry.

Techniques: Stable Transfection, Transduction, Transfection, Plasmid Preparation, Labeling, Flow Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test