Extended Data Fig. 6: An elevated level of BAG3 increases the formation of TDP-43 skein-like structure and the sensitivity of TDP-43DNTD-Clover expressing cells to HSP70 inhibition in a HSP70-binding dependent manner. | Nature Cell Biology

Extended Data Fig. 6: An elevated level of BAG3 increases the formation of TDP-43 skein-like structure and the sensitivity of TDP-43DNTD-Clover expressing cells to HSP70 inhibition in a HSP70-binding dependent manner.

From: TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin-binding proteins

Extended Data Fig. 6: An elevated level of BAG3 increases the formation of TDP-43 skein-like structure and the sensitivity of TDP-43DNTD-Clover expressing cells to HSP70 inhibition in a HSP70-binding dependent manner.

(a) Representative images showing the localization of endogenous BAG3 in cells stained with BAG3 antibody from more than three biological replicates. (b) Western blot analysis of BAG3 in cells without the mRuby2-tagged BAG3 transgene or in cells stably expressing BAG3mRuby2 or BAG3R480A-mRuby2. (c-d) Western blot demonstrating the reduction of BAG3 (c) and HSPB1 (d) following siRNA treatment. (e) Decrease of HSPB8 following siRNA treatment detected by immunostaining and quantification of mean fluorescent intensity in cells. Volcano plots show the mean fluorescent intensities of HSPB8 and HSPB1 in cells treated with siHSPB8 or control siRNA. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. (f) Schematic illustrating the assessment of BAG3 association with TDP-43∆NTD-Clover on skein-like structures. (g-h) Representative live images of cells co-expressing TDP-43∆NTD-Clover with BAG3mRuby2 (g) or BAG3R480A-mRuby2 (h) upon 100 µM NaAsO2 treatment. (i) Measurement of HSP70 activity before, during, and after the removal of arsenite stress from more than three biological replicates. Error bar: SD. (j) The effect of expressing BAG3mRuby2 and BAG3 R480A-mRuby2 on cell resistance to HSP70 inhibition. Data are from N = 3 biological replicates. Number of quantified cells per replicate are 136, 168 and 202 (TDP-43∆NTD-Clover, no Dox); 140, 140, 156 (TDP-43∆NTD-Clover, Dox); 275, 286, 258 (TDP-43∆NTD-Clover and BAG3mRuby2, no Dox); 234, 178, 233 (TDP-43∆NTD-Clover and BAG3mRuby2, Dox); 198, 161, 210 (TDP-43∆NTD-Clover and BAG3 R480A-mRuby2, no Dox); 175, 166, 164 (TDP-43∆NTD-Clover and BAG3 R480A-mRuby2, Dox), respectively. Error bar: SD.

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