Figure 3. hTDP1H²⁶³A and hTDP1H⁴⁹³N mutants induce toxicity in human cells in absence of genotoxic stress.

A. Stably transfected HEK293 cell lines of hTDP1 with and without Doxycycline (Dox) were incubated at 37°C/5% CO₂ for 10 days and stained with crystal violet. Colonies were quantified using LI-COR Odyssey Infrared Imager at a fluorescence of 700 nm. Relative cell viability was determined for each cell line induced with Dox relative to its isogenic cells without Dox. Results shown of n=4. A representative photo is shown taken just before quantification of one well of each cell line/condition. Statistics determined by an unpaired student t-test: Vector-hTDP1: no significance. Vector-mutant: *p>0.0007; **p>0.0002. hTDP1-mutant: ***p>0.031; ****(p>0.0087). B. Total extracts of 48 hours +/-Dox induction of each stable cell line were resolved on 12% SDS-PAGE and stained with anti-human TDP1. Blot is subsequently stripped and re-stained with anti-αTubulin. For the -Dox 2x more total extract was loaded than for the +Dox total extracts.