Fig 2: The activity of TFE3 after SCI is regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. (A) Western blot analysis of AMPK-mTOR signal pathway in the cytoplasm in spinal cords from the control mice, SCI mice, and SCI mice treated with DMSO or Compound C, at Day3. (B) Densitometric analysis of the AMPK, p-AMPK, p-mTOR and p-4EBP1 data from (A), normalized to the loading control GAPDH. (C) Western blotting of TFE3 nuclear translocation at the lesion for each group. (D) Corresponding densitometric analysis of the TFE3 bands in (C) normalized to the loading control H3. (E) Western blots of the AMPK-SKP2-CARM1 signaling pathway in the nucleus of the indicated groups at Day3 after SCI. (F) Densitometric analysis of AMPK, p-AMPK, p-FOXO3a, SKP2 and CARM1 bands from (E) normalized to control H3. (G) Nuclear CARM1-TFE3 complex was detected by IP in indicated groups at Day3 after SCI. (H) Densitometric analysis of TFE3 and CARM1 data from (G) normalized to loading control H3. (I) Western blot analysis of LC3, SQSTM1/p62 and UB in the spinal cord lesion of each group at Day3 after SCI. (J) Densitometric analysis of band data from (I) normalized to the loading control GAPDH. (K) Western blotting of LC3II in the indicated mice spinal cord slides cultured in the presence or absence of CQ at Day3. (L) Densitometric analysis of LC3II from (K) normalized to the loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Fig 3: Illustration of the interaction between miR-378i and Skp2 inducing cell senescence in PTECs in DN.

Fig 4: Skp2- and Cdt2-dependent pathways degrade p21 with different rates and timings.(a) Single-cell traces, coloured by p21-GFP intensity in G1, aligned to the G1/S transition. Four siRNA treatments are shown: n=65 cells, control; n=81, Skp2; n=37, Cdt2; n=25, Skp2 and Cdt2 co-depletion. Black circles represent mitosis. Note: only cells entering S-phase are shown. (b) Graph shows correlation between p21-GFP intensity and G1 length for each siRNA condition shown in (a): n=50 cells, control; n=54, Skp2; n=9, Cdt2; n=14, Skp2 and Cdt2 co-depletion. (c) Graph shows average p21-GFP nuclear level across FOV for four different siRNA treatments. Four FOVs are shown for control siRNA cells and two FOVs are shown for the other treatments, each curve is one FOV. Control siRNA is shown in blue, Skp2 siRNA is shown in green, Cdt2 siRNA is shown in yellow and double Skp2 and Cdt2 depletion is shown in red.

Fig 5: MLN4924-responsive cells have higher levels of SKP2. A-B. Primary mesothelioma cells of epithelioid (EPI; n= 10), biphasic (BIP; n = 5), sarcomatous (SAR; n =4) origin were incubated 72 h in fresh medium (CTRL), with 50 µM cisplatin (PT), 0.2 µM MLN4924 (MLN) or their combination (PT+MLN). Crystal violet staining was quantified spectrophotometrically. A: disaggregated data of cell viability. B: MPM cells divided in responders (samples where the combination of cisplatin+MLN4924 decreased the number of viable cells below 50%) and non-responders (samples where the combination of cisplatin+MLN4924 maintained >50% viable cell, red line). Data are presented as means + SD; each sample was analyzed in quadruplicates. * p < 0.05, **p < 0.01, ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. C-F. Top responder (the epithelioid EPI BAP1+ MPM UPN7, the biphasic BIP BAP1- MPM UPN16, the sarcomatous SAR BAP1+ MPM UPN22) and top non-responder (the epithelioid EPI BAP1+ MPM UPN6, the biphasic BIP BAP1+ MPM UPN18, the sarcomatous SAR BAP1-MPM UPN21) cells were incubated as in A-B for 24 h C. SKP2 mRNA levels were measured by RT-PCR, in triplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells, °°°p < 0.001: treated cells vs PT-treated cells; ###p < 0.001: non-responder cells vs respective responder cells. D. The expression of SCF complex proteins – cullin 1, SKP1 and SKP2 - was measured by immunoblotting. Tubulin was used as a loading control. The figure is representative of 1 out 3 experiments with similar results. E. UBE2M activity was measured spectrophotometrically, in duplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells, °°°p < 0.001: treated cells vs PT-treated cells; ###p < 0.001: non-responder cells vs respective responder cells. F. Proteasome activity was measured fluorometrically, in duplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells; °°p < 0.01: treated cells vs PT-treated cells