Fig. 2

24 h of FTD-mutant-hTau expression reduces protein synthesis and impairs ribosomal biogenesis in HEK293 cells. a FTD-mutant tau expression reduces protein synthesis and abundance of RPS14 over a period of 24 h. HEK293 cells were transfected with either 2N4R hTau-EGFP, 2N4R P301L-hTau-EGFP, 1N4R K369I-EGFP or EGFP control and then treated with 4 mM AHA at 8 h post-transfection. At 24 h, AHA labelled de novo synthesised was quantified via FUNCAT-WB, with both P301L-hTau and K369I-hTau expressing cells showing reduced protein synthesis compared to EGFP and non-mutant hTau expressing cells. The abundance of RPS14 was changed in FTD-mutant tau expressing cells; the abundance of other RPs, such as RPL5 and RPS6, was unchanged at 24 h. Protein abundance was normalised to the total protein stain REVERT. n = 3 wells, one-way ANOVA, Tukey’s MCT. b Polysome profiling reveals that FTD-mutant tau expression reduces polysome, monosome, and 60S ribosomal subunit abundance after 24 h. HEK293 cells transfected with EGFP, 2N4R hTau-EGFP, 2N4R P301L-hTau-EGFP or 1N4R K369I-EGFP were treated with 100 ug/mL CHX for 5 min in order to prevent the dissociation of bound ribosomes from mRNA. Following lysis, samples were separated on a 10–50% linear sucrose gradient via ultracentrifugation. Absorbance at 260 nm was used to detect the presence of the 40S and 60S ribosomal subunits, along with monosome and polysomes, with the area under the curve (AUC) being used for quantification. Both P301L-hTau and K369I-hTau expressing cells showed reduced levels of polysomes, monosomes, and the 60S ribosomal subunit compared to EGFP and non-mutant hTau expressing cells. n = 3 wells, one-way ANOVA, Tukey’s MCT