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. 2016 Mar 24;7:10953. doi: 10.1038/ncomms10953

Figure 3. DNAJB1 is a downstream transcriptional target of FOXE3 and is indispensable for the cell cycle.

Figure 3

(a) Schematic illustration of the 5′ UTR of DNAJB1 with golden boxes indicating potential FOXE3-binding sites predicted by ProSuite algorithms. Blue arrows, designated 1–8 F and 1–8 R, represent eight primer pairs used for amplifying regions of interest. (b) Amplification of specific segments in the 5′ UTR of DNAJB1 using pull-down DNA as the template in the amplification reaction. (c) HEK293FT cells were transiently transfected with the DNAJB1-GLuc-SEAP vector and an expression vector harbouring wild-type or mutant (p.E103K, p.N117K and p.C240*) FOXE3 alleles. Error bars represent percent deviation in each data set of luciferase activity normalized against SEAP performed in seven independent replicates for each condition. (d) HEK293FT cells were transfected with the wild-type or mutant FOXE3-expressing plasmids in a pT-Rex-DEST30 vector and expression levels of DNAJB1 were quantitated with TaqMan assays. Error bars represent s.d. in each data set of the DNAJB1 expression normalized against GAPDH, analysed in six independent replicates. (e) HLE cells were transfected with either scrambled (i–iii) or DNAJB1 shRNA (iv–vi), and the increase in apoptotic cell population in response to shRNA-targeting DNAJB1 was assessed 48 h post transfection. (f) Increased apoptosis resulting from downregulation of DNAJB1 was estimated through flow cytometry of TUNEL stained cells 48 h post transfection of DNAJB1 shRNA. The loss of DNAJB1 results in increased apoptosis in HLE cells resulting in a threefold increase in TUNEL-positive cells compared with the control cells transfected with scrambled shRNA (P<0.000005; 2-tailed student's t-test). Error bars represent s.d. in each data set analysed in four independent replicates. (g) The effect of the downregulation of DNAJB1 on the G0 population was examined through flow cytometry of propidium iodide-stained cells 48 h post transfection of DNAJB1 shRNA. The DNAJB1 shRNA-transfected cells exhibited a significant increase in the G0 population as compared with the control cells transfected with scrambled shRNA. Error bars represent s.d. in each data set analysed in four independent replicates. ‘***' indicates P<0.00001 (two-tailed student's t-test).