Fig 2: SPAG5 reverses miR-133a-3p-inactivated PI3K/AKT pathway in PC9/GR cell. (A) Protein expression of phosphorylated PI3K, total PI3K, phosphorylated AKT, total AKT in PC9/GR cells co-transfected with SPAG5 or vector plasmids and miR-101-3p mimics was determined by western blotting. (B) Protein expression of phosphorylated PI3K, total PI3K, phosphorylated AKT, total AKT in PC9 cells co-transfected with si-SPAG5 or si-NC plasmids and miR-101-3p inhibitor was determined by western blotting.

Fig 3: DNMT3B/miR-451a/EPHA2/PI3K/AKT axis modulates BCa cell growth in vivo. T24 cells (2 × 106 cells/mL) with stable poor expression of DNMT3B or miR-451a were injected into the right armpit of NOD/SCID mice. a, from the fifth day, the growth rate of T24 cells was evaluated by measuring the volume every 5 days in vivo; b, tumor weight; c, KI67 staining intensity in tumors evaluated by immunohistochemistry; d, EPHA2 staining intensity in tumors evaluated by immunohistochemistry; e, phos-PI3K staining intensity in tumors evaluated by immunohistochemistry; f, phos-AKT1 staining intensity in tumors evaluated by immunohistochemistry. Data are represented as the means ± SD from three independent assays. Statistically significant differences were calculated using one-way or two-way ANOVA, followed by Tukey’s multiple comparison test. **p < 0.01 vs mice injected with cells transfected with sh-NC or sh-DNMT3B + inhibitor NC

Fig 4: Ovarian cancer ascites stimulate MUC16 expression and release through Akt. a Meso 7 cells were incubated with FBS 10% (control), OV401 10% or 3 different ovarian cancer ascites at 10%. The expression of Akt and phosphorylated Akt were determined by immunoblot using anti-Akt and anti-phospho-Akt antibodies. Representative images from two independent experiments. Densitometric quantification of phosphorylated Akt normalized to total Akt is represented between the two blots. b OVC346 ascites was heat-inactivated by heating at 100 °C for 10 min followed by centrifugation at 13,000 rpm for 15 min. Meso7 cells were incubated with OVC346 (10%) or heat-inactivated OVC346 ascites (10%). Akt and phosphorylated Akt expression were assessed by immunoblot after 24 h. Expression of phosphorylated Akt was normalized to total Akt and densitometric quantification is shown between the blots. Representative images from two independent experiments. c Meso 7 cells were incubated with increasing concentrations of OVC346 ascites (0 to 10% v/v) and Akt and phosphorylated Akt expression were determined by immunoblot. d Meso7 cells were incubated with OV401 (10%) or ovarian cancer ascites (10%) in the presence or absence of Akt1/2 inhibitor (5 µM). MUC16, Akt and phosphorylated Akt expression were determined by immunoblot. Representative images from two independent experiments. Expression of phosphorylated Akt was normalized to total Akt and MUC16 expression was normalized to housekeeping gene GAPDH. e MUC16 release was measured in supernatants of Meso7 cells incubated with three different ovarian cancer ascites in the presence or absence of Akt1/2 inhibitor (5 µM). Bars represent standard deviation from two independent experiments conducted in duplicates. f Meso7 cells were incubated with OVC346 or OVC690 (10%) in the presence or absence of Akt1/2 inhibitor. MUC16 mRNA levels were quantified by ddPCR. MUC16 concentration data from ddPCR experiments are expressed as ratio against reference genes for each sample. Results are from two independent experiments conducted in duplicates. g Meso7 cells were incubated with benign fluid OV401 or ovarian cancer ascites OVC346 or OVC690 (10%). Expression of phosphorylated FAK, phosphorylated and total c-Src was determined by immunoblot. Representative images from two independent experiments. Densitometric quantification of phosphorylated FAK normalized to GAPDH is shown between the blots. h Cells were incubated with ß1 or ß5 integrin-blocking antibodies (5 µg/ml) for 30 min before adding 10% OVC508 ascites overnight. Cells were then washed 3 times and fresh FBS and hormone-free medium was added. MUC16 release was then measured in supernatants after 24 h. Data are from a single experiment

Fig 5: TOM1L1 regulates EGF-stimulated Akt phosphorylation. (A–D) ARPE-19 (A–C) or MCF10A (D) cells were transfected with siRNA targeting Fyn, TOM1L1, or nontargeting siRNA (control), as indicated, followed by stimulation with 5 ng/ml EGF for the time indicated. (A) Western blotting using anti-phospho-Akt (pY473, pan isoform). Also shown are the mean ± SEM phospho-Akt with points representing individual experiment measurements; n = 4; *, P < 0.05, relative to the control siRNA-treated EGF-stimulated condition at each time point. (B) After siRNA transfection, intact cells were subjected to immunofluorescence microscopy with antibodies that selectively recognize the EGFR ectodomain (Alexa Fluor 488 labeling). Representative fluorescence microscopy micrographs of cell surface EGFR immunostaining are shown. Scale = 10 µm. Also shown are mean fluorescence intensity of cell-surface EGFR ± SEM with points representing individual experiment measurements; n = 3. con., control. *, P < 0.05 relative to EGF-stimulated, control siRNA condition. (C) Western blotting using anti-phospho-EGFR (pY1068) or anti-phospho-Erk; also shown are the mean ± SEM phospho-EGFR or phospho-Erk levels with points representing individual experiment measurements; n = 5. (D) MCF10A cells: Western blotting using anti-phospho-Akt (pY473, pan isoform), as well as TOM1L1 or Fyn. Also shown are the mean ± SEM phospho-Akt at 3 min of EGF stimulation with points representing individual experiment measurements; n = 3; *, P < 0.05, relative to the control siRNA-treated EGF-stimulated conditions at each time point. Source data are available for this figure: SourceData F2.