Fig 2: PLK1 protein levels are increased in hamartin and tuberin deficient cells and are rapamycin-sensitive. (A) Lysates from exponentially growing Tsc1+/-, Tsc1-/-, Tsc2+/+/Tp53-/- and Tsc2-/-/Tp53-/- MEF were immunoblotted with the indicated antibodies. (B) Lysates from Tsc1-/- MEF transduced with vector (208-P2) or TSC1 (208-T3), and from tuberin-deficient ELT3 cells transduced with vector (ELT3-V3) or TSC2 (ELT3-T3) were immunoblotted with the indicated antibodies. (C) Lysates from Tsc1+/- and Tsc1-/- MEF treated for 24 hours with 0, 50 or 100 nM rapamycin were immunoblotted with the indicated antibodies. PLK1 protein levels normalized to ß-actin and to vehicle-treated Tsc1+/- MEF are indicated below the PLK1 immunoblots.

Fig 3: Pharmacological inhibition of PLK1 decreases the clonogenic survival of hamartin and tuberin deficient cells. (A) Tsc1+/- and Tsc1-/- MEFs were treated with 30 nM BI-2536 for 3 days, followed by clonogenic survival assay. (B) HeLa cells after shRNA-mediated silencing of TSC1 or TSC2 were treated with 30 nM BI-2536 for 24 hours, followed by clonogenic survival assay. For both panels n = 3, and *** indicates P < 0.001.

Fig 4: Affinity purification of R2TP/PFDL subunits reveals interactors.(a) Diagram of the network of high-confidence interactions formed around the R2TP/PFD complex. Solid lines denote interactions with a FDR lower than 0.1 while dashed lines are interactions of relevance in this network with FDR scores higher than 0.1, but lower than 0.2 (Supplementary Data 1). Green-coloured nodes are tagged subunits used in this experiment. (b) CoImmunoprecipitation (CoIP) of FLAG-tagged subunits of the R2TP/PFD-like complex (RPAP3 and URI1) in HeLa S3 cells. Various Western blots were made to detect endogenous or recombinant proteins, as marked to the right. (c) Outline of the splicing cycle with emphasis on the recycling steps of the U5 snRNP and its reintegration within the U4/U6.U5 tri-snRNP (inner loop). (d) Regulation of the TORC1 kinase complex by TSC1–TSC2 (annotated here as ‘TSC'). Guanine nucleotides (GTP, GDP) and phosphate groups (P) are indicated in green or red, depending on whether they have an activating or inhibitory effect on their associated proteins, respectively.

Fig 5: MicroRNA (miR)-92b-3p regulates the mTOR signaling pathway by targeting TSC1, and TSC1 is downregulated in clear cell renal cell carcinoma (ccRCC) specimens compared to adjacent normal tissues. A, Representative images of western blot analysis from 3 independent experiments showing the protein levels of TSC1 and p70S6K(Thr389) and S6K in ACHN cells transfected with miR-92b-3p mimic, or a negative control (NC) miRNA mimic, and also in Caki-2 cells transfected with miR-92b-3p inhibitor, or NC miRNA inhibitor. B, Proliferation assay of ACHN cells transfected with a miR-92b-3p mimic or NC mimic after 96 h incubation with either everolimus or vehicle control (0.1% DMSO). Values are means ± SD of 3 independent experiments. Comparison was carried out by unpaired Student’s t test. **P < .01. C, Representative images of immunohistochemical staining for TSC1 in ccRCC tissues and normal tissues. Paired tissues (30 samples of each) were examined and TSC1 staining intensity was classified into 2 levels: weak = 1 and moderate = 2. Mean scores of normal tissues and ccRCC tissues are shown in the bar graph. Scale bar = 100 µm