Fig. 5

The 4SC-202-induced G2/M arrest is independent of LSD1. a–c MyLa cells were infected with a lentiviral vector allowing doxycyclin (Dox)-inducible expression of an shRNA targeting LSD1. Cells were treated for 5 days with 1 μg/ml Dox and a expression of LSD1 was assessed by immunoblot. b and c Following 5 days in the absence or presence of Dox cells were additionally treated with 1 μM 4SC-202, and cellular DNA content was analyzed after 24 h by propidium iodide staining. (Note that MyLa cells had acquired a substantial proportion of tetraploid cells following infection and selection.) b Representative cell cycle profiles of the partially tetraploid cells are depicted. G2/M arrest is most clearly verifiable by the increase in 8-N cells. c Mean values (± SD) of the percentage of 8-N cells derived from three independent experiments are given. d–f HeLa cells were infected with two lentiviral vectors allowing expression of Cas9 and a single guide RNA targeting LSD1. Two single cell clones with complete knockout of both LSD1 alleles (as confirmed by sequencing) were established and d lack of LSD1 expression was demonstrated by immunoblot. e and f After 24 h in the presence or absence of 1 μM 4SC-202 cellular DNA content was analyzed by propidium iodide staining. e Representative cell cycle profiles and f mean values (± SD) of the percentage of 4-N cells derived from at least five independent experiments are depicted. g and h MyLa cells were engineered to express dCas-VP64 (inactivated Cas9 fused to the VP64 transcriptional transactivator domain) and the activation helper protein MS2-p65-HSF1. To achieve specific gene activation these cells were transduced with lentiviral vectors coding for guideRNAs targeting either the HDAC1, the HDAC3, or the LSD1 promoter. A scrambled (src) guideRNA served as control. g Expression levels of the indicated mRNAs were determined by SybrGreen real time PCR. h MyLa cells expressing the indicated guideRNA were treated with either 0.3 μM 4SC-202 or 2 nM FK228 for 48 h. Then cellular DNA content was analyzed by propidium iodide staining, and the increase of sub-G1 cells compared to the respective untreated control cells was determined. Mean values (± SD) of three independent experiments are displayed. Paired t test was performed (*p < 0.05; **p < 0.01)