Fig 2: UBIAD1 loss leads to ROS increase, impaired cell proliferation and decreased survival in melanoma cells.(A) Growth curves of melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD compared to control conditions (ctrl). 2-way ANOVA with Sidak's multiple comparisons test was used to quantify statistical significance. Error bars represent SEM, n = 3.(B) Western blot analysis of cyclin A and RRM2 proteins upon UBIAD1KD in SkMel28, A375 and Mel Juso melanoma cell lines. ß-actin was used as loading control.(C) FACS assay of DHE (left) and DCFH-DA (right) staining in SkMel28 and A375 upon UBIAD1KD Low or UBIAD1KD. Data are reported as percentage of positive cells over singlets. Incubation with 50 µM of menadione and 1 mM of H2O2 for 90 min were used as positive controls for DHE and DCFH-DA, respectively. One-way ANOVA with Sidak’s multiple comparisons test was used to quantify statistical significance. Error bars represent SEM, n = 3.(D) HPLC-MS analyses of total CoQ10 level upon UBIAD1KD in SkMel28 and A375 lines, in comparison control conditions (ctrl), normalized to total protein (TP) concentration. Unpaired Student two-tailed t-test was used to quantify statistical significance between control and UBIAD1KD samples. Error bars represent SEM, n = 6.(E) HPLC-MS analyses of CoQ10 in plasma membrane fraction (PL) upon UBIAD1KD in SkMel28 and A375 lines normalized to membrane ceramide abundance (Cer16:0). Unpaired Student two-tailed t-test was used to quantify statistical significance between control conditions (ctrl) and UBIAD1KD samples. Error bars represent SEM, n = 6. Below, Western blot analysis of subcellular fractionation in SkMel28 and A375 lines. M, Mitochondria-enriched fraction; W, Whole cell; PL, Plasma membrane-enriched fraction. PMCA was used as marker of plasma membrane, Tom20 as mitochondrial marker, ERK1/2 and ß-actin as cytosolic markers.(F) Growth curves of melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD Low in presence or absence of idebenone treatment (1 µM idebenone for SkMel28, 10 µM idebenone for A375 and 50 nM idebenone for Mel Juso). 2-way ANOVA with Sidak's multiple comparisons test was used to quantify statistical significance between UBIAD1KD Low and UBIAD1KD Low + idebenone. Error bars represent SEM, n = 3.(G) Western blot analysis of cell proliferation markers (RRM2 and cyclin A) and DNA-damage marker (pH2AX) in melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD Low in presence or absence of idebenone (100 nM of idebenone for SkMel28, 200 nM of idebenone for A375 and 200 nM idebenone for Mel Juso). Three biological replicates are shown. Unpaired Student two-tailed t-test was used to quantify statistical significance between UBIAD1KD Low and UBIAD1KD Low + idebenone samples. Error bars represent SEM, n = 3.

Fig 3: Knockdown of AFAP1-AS1 decreases the tumourigenicity and chemoresistance of NSCLC cells in nude mice. Male BALB/c nude mice injected with A549 cells and treated for 4 weeks as follows: Lv-Scramble+PBS, Lv-AFAP1-AS1 KD+PBS, Lv-Scramble+DDP, and Lv-AFAP1-AS1 KD+DDP. (A–C) Tumor volumes, sizes and weights of 2 independent repeats. Data shown as means ± S.D. n = 5. #P < 0.05 indicates a significant difference between the two indicated groups. (D) Schema of the signaling pathways involved in AFAP1-AS1/miR-139-5p/RRM2-mediated chemotherapy resistance in NSCLC cells.

Fig 4: RRM2 increases the chemotherapy resistance of NSCLC cells via EGFR/AKT signaling pathway. Cancer cells were transfected with empty vector, RRM2, RRM2+DMSO and RRM2+AST1306, and then incubated with the EGFR inhibitor AST1306 at 1 μM for 24 h. (A,B) In NSCLC cells incubated with DDP for 36 h, overexpression of RRM2 enhanced chemotherapy resistance, and AST1306 reversed this effect. (C,D) In NSCLC cells incubated with 5-FU for 36 h, overexpression of RRM2 enhanced chemotherapy resistance and AST1306 reversed this effect. (E,F) In A549 and SPCA-1 cells, overexpression of RRM2 elevated the protein expression of EGFR and the level of p-AKT, and AST1306 reversed this effect. All data shown as means ± S.D. #P < 0.05 indicates a significant difference between the two indicated groups.

Fig 5: TAK-981 upregulates GR expression in primary multiple myeloma and cell lines through downregulation of miR-130b. A GR expression is associated with Dex sensitivity in primary MM cells. GR (NR3C1) mRNA expression was assessed by q-PCR and presented as relative level normalized to GAPDH. IC50 values were calculated by GraphPad Prism 8. Pearson correlation analysis showed a significant negative correlation negative correlation between GR RNA (NR3C1) and IC50 for Dex in 15 primary MM. B NR3C1 mRNA level was decreased and (C) miR-130b level was increased in dose-dependent manner in primary MM cells treated with TAK- 981. Primary MM cells from 5 relapsing patients were treated with TAK-981 at indicated concentrations for 48 h. Total RNA extracted and NR3C1 mRNA and miR-130b level was measured by q-PCR. Data presented as mean ± SD, n = 5 (D) TAK-981 treatment increases GR protein level in primary MM cells. Western blot presents GR protein of 3 primary MM samples treated with or without 0.1 μM TAK-981 for 48 h. Relative protein level was quantified using Image J, normalized to GAPDH, and labeled below each blotting band. E NR3C1 mRNA was increased and miR-130b was decreased in MM1S and H929 cell lines upon TAK-981 treatment. Estimated variation is indicated as SD. P values were derived using a two-tailed Student t test. * p < 0.05, ** p < 0.01, ***p < 0.001 (F) TAK-981 treatment increases GR protein level in MM1S and H929 cell lines. MM1S and H929 cells were treated with or without 20 nM TAK-981 for 48 h. Cell lysates were used for western blot. G GR-repressed gene RRM2 and apoptosis marker cleaved PARP (c-PARP) were measured by western blot in MM1S and H929 cell lines treated with TAK-981, Dex or both for 48 h. GAPDH was used as loading controls. Relative protein level was quantified by Image J and labeled below each blot