Fig. 2

HOXA1 variants induce proteasome-dependent protein degradation and reduce transcriptional activity of the protein.

a–d HEK293T cells were transfected with expression vectors coding for FLAG-HOXA1 (WT^A, + 1His, +1His^Arg, −1His, −1His^Arg and −3His^Arg). To determine the half-life of HOXA1, cells were treated with cycloheximide (CHX; 200 μg/ml) for the indicated time. a Cell lysates were collected every 6 h and then subjected to immunoblot analysis with antibodies against FLAG and β-ACTIN. b Intensity of the HOXA1 bands observed upon cycloheximide treatment was quantified and the relative HOXA1/β-ACTIN ratios were plotted (n = 6; n indicates number of biologically independent experiments (cell transfected independently in different weeks). Error bars indicate standard deviation (SD). c Involvement of the proteasome in the HOXA1 degradation was assayed by treating cells with proteasome inhibitor (MG132; 7 μM) for 20 h prior inhibition of protein translation with CHX (n = 3). Cell lysates were collected every 6 or 12 h and then subjected to immunoblot analysis with antibodies against FLAG and β-ACTIN. d The intensity of the HOXA1 bands observed upon cycloheximide treatment was quantified and the relative HOXA1/β-ACTIN ratios were plotted (n = 6). Error bars indicate standard deviation (SD). e Luciferase assay showing transcriptional activity of wild-type (WT^A) and mutated hHOXA1 (+1His, +1His^Arg, −1His, −1His^Arg, and −3His^Arg). HEK293T cells were transfected with the TSEII-luciferase reporter construct, alone (TSEII-Luc) or together with expression plasmids for PREP1, PBX1a, and FLAG-HOXA1 (WT^A, + 1His, +1His^Arg, −1His, −1His^Arg, and −3His^Arg). Same quantity of DNA was transfected for each plasmid (n = 6). **p = 0.035. f, In order to characterize the dominant negative effect of mutated human HOXA1 proteins, cells were transfected with the TSEII-luciferase reporter together with both wild-type and HOXA1 variant coding vectors (n = 6). ***p = 0.00000012. Boxes and whiskers (min to max) show the values of lower and upper quartile. Statistical values were obtained using Pairwise t test with Holm P value adjustment method test. Statistical test used was two-sided. ‘n’ represents number of biologically independent experiments (cell transfected independently in different weeks) in duplicate. Source data are provided as a Source data file.