Hsf1 regulates cell size in response to stress. The scale bars on the images on the far right are 50 μM (images are representative of n = 2 biologically independent experiments). Images were captured with a fluorescence microscope (Zeiss, Germany). The cytoskeleton is stained with phalloidin-Alexa488 (green), and the nucleus is stained with DAPI (blue). (D) Fluorescence microscopy images of A549 WT and Hsp90α/β KO cells after 4 days of chronic HS. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests. (C) Flow cytometric quantification of cell size in chronic HS at different time points during a period of 7 days (HS = 39 ☌ for HEK and 40 ☌ for A549) (n = 4 biologically independent samples) The data are represented as mean values ± SEM for all bar graphs. Note that the X axis does not have a linear scale and that lines connecting the data points are drawn as a visual aid. (B) Flow cytometric quantification of cell viability of HEK, A549, and their respective Hsp90α/β KO cells in chronic HS at different time points during a period of 7 days (n = 5 biologically independent samples). GAPDH serves as the loading control (α KO Hsp90α KO and β KO Hsp90β KO) (representative images of n = 4 biologically independent experiments). (A) Immunoblots of Hsp90α and Hsp90β in WT HEK and A549 cells, and their respective Hsp90α/β KO cells. Cells are unable to adapt to chronic stress in the absence of one of the cytosolic Hsp90 isoforms. The data are represented as mean values ± SEM for all bar and line graphs. ( I) Flow cytometric analysis of cell cycle in chronic HS and post HS recovery (n = 3 biologically independent samples). (H) Flow cytometric quantification of cell size in chronic HS and recovery (n = 3 biologically independent samples). See scheme of the experiment on the right. The adapted cells were maintained at 40 ☌ for one week before this experimental start point and continued at 40 ☌ during the experiment. (G) Proliferation of A549 and RPE1 cells measured with a crystal violet assay (n = 3 biologically independent experiments). The numbers of live cells counted after 7 days are plotted (n = 5 biologically independent experiments). Cells were seeded at a density of 5 x 10 6 and 3 x 10 6 per 15 cm plate for HEK and A549 cells, respectively. (F) Proliferation of HEK and A549 cells at the indicated temperature for the indicated period presented as cell numbers. (E) Flow cytometric quantification of cell size during different time intervals of chronic HS (n = 4 biologically independent samples). (C and D) Flow cytometric analysis of cell cycle (n = 3 biologically independent samples). (B) Flow cytometric quantification of cell size after 7 days of chronic HS (biologically independent samples: n = 6 for HEK and A549 n = 5 for RPE1 n = 4 for HCT116). (A) Flow cytometric quantification of cell viability under chronic HS for 7 days (HS = 39☌ for HEK and HCT116 cells HS = 40 ☌ for A549 and RPE1 cells) (n = 4 biologically independent samples). Cells increase their size in response to chronic stress.
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