Fig 2: Targeted silencing of PLK3 expression reduces H/R-induced apoptosis in TECs. (a) qRT–PCR analysis of PLK3 mRNA expression after transfection with 100 nM PLK3-siRNA or scrambled oligonucleotide (si-NC). (b) Immunoblotting analysis of PLK3 silencing efficiency. (c) The viability of HK-2 cells was measured by CCK-8 assays. (d) Flow cytometry was performed to examine HK-2 cell apoptosis. (e) Representative images showing TUNEL-positive HK-2 cells after PLK3 silencing (bar = 50 μM; magnification, 400x). The data are presented as the mean ± SD. n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Fig 3: Restoration of Plk3 induction rescues ATF2 phosphorylation and ER stress resolution in Lrh-1LKO mice, and loss or gain of ATF2 transcriptional activity also alters ER stress resolution capacity.(A) Primary hepatocytes were prepared from Lrh-1f/f and Lrh-1LKO mice and transduced with Ad-Plk3 or Ad-control at a MOI of 100. Cells were treated 36 hr later with vehicle or tunicamycin (TM) (0.01 µg/ml) and doxycycline (1 µg/ml) to induce Plk3 or LacZ control. Nuclear protein was obtained at timepoints indicated and immunoblotted for UPR transcription factors, with TBP as a loading control. Results are representative of three independent experiments. (B) Primary hepatocytes were prepared from Lrh-1LKO mice and transduced with Ad-Plk3 or Ad-control at a MOI of 100. Cells were treated 36 hr later with vehicle or TM (1 µg/ml) and doxycycline (1 µg/ml) to induce Plk3 or LacZ control. Nuclear protein was collected at indicated timepoints and immunoblotted for pATF2 (69/71) and total ATF2. Samples are from same gel and brightness/contrast was adjusted equally prior to cropping. Results are representative of three independent experiments. (C) Primary hepatocytes were prepared from Lrh-1f/f and Lrh-1LKO mice and transduced with Ad-Plk3 or Ad-control at a MOI of 100. Cells were treated 36 hr later with vehicle or TM (0.01 µg/ml) and doxycycline (1 µg/ml) to induce Plk3 or LacZ control. Cells were fixed at various timepoints and stained for lipids (red) using Lipidtox dye and counterstained with DAPI. Magnification is 40X (objective). Results are representative of three independent experiments. (D) Relative expression of Plk3 by qPCR. Primary hepatocytes from control and Lrh-1LKO mice were transduced with Ad-Plk3 or Ad-control at a MOI of 100 and treated with 1 µg/ml doxycycline and vehicle or 0.01 µg/ml TM. Data were normalized to Tbp expression. Significance at p<0.01 between genotypes (n = cells from 3–4 mice). (E) Primary hepatocytes from control mice were transduced with Ad-DN Atf2 (Atf2 T51A/T53A to serve as a dominant negative) or Ad-control at a MOI of 100 and treated with 5 ng/ml TM. Primary hepatocytes from control and Lrh-1LKO mice were transduced with Ad-C2/Atf2 (expressing a constitutively active Atf2) or Ad-control at a MOI of 100 and treated with 5 ng/ml TM. Nuclear protein was obtained at timepoints indicated and immunoblotted for UPR transcription factors, with TBP as a loading control. Results are representative of samples from three mice. (F) Atf2 expression by qPCR to quantify viral Atf2 overexpression. Primers were chosen to amplify regions identical between wildtype Atf2, C2/Atf2, and DN Atf2. Primary hepatocytes from control mice (n = 3–4) were transduced at a MOI of 100 and treated with vehicle for 24 hr prior to RNA collection. (G) Spliced Xbp-1 expression by qPCR for primary hepatocytes from control and Lrh-1LKO mice (n = 3–4) transduced with Ad-control, Ad-DN Atf2, or Ad-C2/Atf2 and treated with vehicle or 5 ng/µl TM. Data were normalized to Tbp expression.DOI: http://dx.doi.org/10.7554/eLife.01694.010

Fig 4: Clinical significance of PLK3 in CRC patients. a PLK3 expression level in tumor tissues and the paired normal tissues was evaluated by IHC in the tissue microarray. b CRC patients with negative expression of PLK3 presented worse overall survival, and disease-free survival compared with that of positive expression of PLK3

Fig 5: PLK3 overexpression reverses the effect of ATM inhibition in alleviating TEC apoptosis. (a) p-ATM protein levels, PLK3 protein levels, and apoptosis-related protein levels were measured by Western blot after the application of KU-60019 and PLK3 plasmid. (b) Flow cytometry was performed to examine the apoptosis of HK-2 cells after the administration of KU-60019 and PLK3 plasmids. (c) TUNEL assays showed the number of apoptotic HK-2 cells. The data are presented as the mean ± SD. n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.