flag hsf1  (Addgene inc)

Journal: eLife

Article Title: Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress

doi: 10.7554/eLife.88658

Figure Lengend Snippet: ( A ) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells at 37 °C as measured by luciferase reporter assay (n=3 biologically independent samples and 2 experimental replicates each time). ( B ) Immunoblots of Hsf1 in the cytosolic and nuclear fractions of HEK WT and Hsp90α/β KO cells (α KO, Hsp90αKO; β KO, Hsp90βKO). GAPDH and lamin B1 serve as loading controls (representative blots of n=2 biologically independent experiments). ( C ) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells in chronic HS as measured by luciferase reporter assay (n=3 biologically independent samples, and 2 experimental replicates each time for HEK; n=3 biologically independent samples, and 4 experimental replicates each time for A549). ( D ) Volcano plots of the normalized fold changes in protein levels of some core Hsf1 target genes (list obtained from https://hsf1base.org/ ) in chronic HS, determined by quantitative label-free proteomic analysis of Hsp90α/β KO and WT HEK cells. Molecular chaperones, whose expression is regulated by Hsf1, are excluded from this dataset. Each genotype was compared with its respective 37 °C control (n=3 biologically independent samples). Log2 fold changes of >0.5 or <–0.5 with q-values (adjusted p-values) of <0.05 were considered significant differences for a particular protein. ( E ) Flow cytometric quantification of cell size of HEK, A549, and RPE1 cells upon overexpression of WT Hsf1 (with plasmid pcDNA-Flag HSF1 wt) or mutant Hsf1 (with plasmid pcDNA-Flag HSF1 C205; retaining only the first 205 amino acids), and with plasmid pcDNA3.1(+) as empty vector control. Transfected cells to be measured were identified on the basis of their coexpression of EGFP (n=4 biologically independent experiments). ( F ) Flow cytometric quantification of cell size in chronic HS after knockdown of Hsf1 in HEK WT, A549 WT and Hsp90αKO cells. Here the chronic HS for A549 cells is at 39 °C instead of 40 °C to reduce HS-induced damage in Hsf1 knockdown conditions (n=3 biologically independent samples). For all bar graphs, the statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests. Figure 3—source data 1. Values related to all graphs. Figure 3—source data 2. Raw and annotated immunoblots.

Article Snippet: The plasmid pcDNA-Flag HSF1 C205 allows expression of the Hsf1 mutant comprising only the N-terminal 205 amino acids. pcDNA3.1(+) (Thermo Fisher Scientific # V79020) was used as the empty vector control.

Techniques: Activity Assay, Luciferase, Reporter Assay, Western Blot, Expressing, Control, Over Expression, Plasmid Preparation, Mutagenesis, Transfection, Knockdown, Two Tailed Test

Journal: eLife

Article Title: Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress

doi: 10.7554/eLife.88658

Figure Lengend Snippet: ( A ) Volcano plots of the normalized fold changes in protein levels of some of the core Hsf1 target genes (list obtained from https://hsf1base.org/ ) in Hsp90α/β KO cells compared to WT HEK as determined by quantitative label-free proteomic analysis (extracted from ). Molecular chaperones, whose expression is regulated by Hsf1, are excluded from this dataset (n=3 biologically independent samples). Log2 fold changes of >0.5 or <–0.5 with a q-value (adjusted p-value) of <0.05 were considered significant differences for a particular protein. ( B ) Fold change of Hsf1 activity of HEK, A549, and RPE1 cells upon overexpressing WT and mutant Hsf1 in combination with EGFP, as measured with the Hsf1 luciferase reporter. Control is transfected with only Hsf1 reporter plasmid and pEGFP-C1, those are common to all the experimental conditions; (n=3 biologically independent samples). ( C ) Fold change of Hsf1 activity in A549 WT, Hsp90α KO, and Hsp90β KO cells after 4 days of capsaicin treatment as measured by luciferase reporter assay (n=3 biologically independent samples). ( D ) Flow cytometric quantification of cell size after 4 days of capsaicin treatment of Hsp90α/β KO and WT A549 cells (n=3 biologically independent samples). ( E ) Immunoblots of Hsf1 after Hsf1 knockdown in A549 WT, A549 Hsp90α KO, and HEK WT cells. β-actin serves as the loading control (representative of n=2 independent experiments). ( F ) Fold change of Hsf1 activity in HEK WT, A549 WT, and A549 Hsp90α KO cells in chronic HS after Hsf1 knockdown as measured by luciferase reporter assays. Here the chronic HS for A549 cells is 39 °C to instead of 40 °C to reduce HS-induced damage in Hsf1 knockdown conditions (n=3 biologically independent samples). ( G ) Flow cytometric quantification of cell size of mouse fibroblast. (90αKO, 90β HET), homozygous hsp90α KO, heterozygous hsp90β KO cells (n=6 biologically independent samples). ( H ) Fold change of Hsf1 activity in (90αKO, 90β HET) MAFs compared to WT at 37 °C, as measured by luciferase reporter assays (n=3 biologically independent samples). ( I ) Fold change of Hsf1 activity in MAFs subjected to chronic HS (orange bars) compared to 37 °C (blue bars), as measured by luciferase reporter assay (n=3 biologically independent samples). ( J ) Flow cytometric quantification of cell size of MAFs subjected to chronic HS (orange bars) by comparison to 37 °C (blue bars; n=4 biologically independent samples). The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests. Figure 3—figure supplement 1—source data 1. Values related to all graphs of . Figure 3—figure supplement 1—source data 2. Raw and annotated immunoblots of .

Article Snippet: The plasmid pcDNA-Flag HSF1 C205 allows expression of the Hsf1 mutant comprising only the N-terminal 205 amino acids. pcDNA3.1(+) (Thermo Fisher Scientific # V79020) was used as the empty vector control.

Techniques: Expressing, Activity Assay, Mutagenesis, Luciferase, Control, Transfection, Plasmid Preparation, Reporter Assay, Western Blot, Knockdown, Comparison, Two Tailed Test

Journal: eLife

Article Title: Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress

doi: 10.7554/eLife.88658

Figure Lengend Snippet:

Article Snippet: The plasmid pcDNA-Flag HSF1 C205 allows expression of the Hsf1 mutant comprising only the N-terminal 205 amino acids. pcDNA3.1(+) (Thermo Fisher Scientific # V79020) was used as the empty vector control.

Techniques: Generated, Recombinant, Plasmid Preparation, Over Expression, Mutagenesis, Control, Sequencing, shRNA, Flow Cytometry