Fig 2: NF-κB signaling is activated by IL1–IL1R1 interaction. (A) Relative Il1a mRNA level in cumulus cells. (B) Relative Il1b mRNA level in cumulus cells. (C) Relative Il1r1 mRNA level in oocytes. (D) The expression level of IL1A protein in cumulus cells. (E) The quantified intensity of IL1A/TUBB. (F) The expression level of IL1B protein in cumulus cells. (G) The quantified intensity of IL1B/TUBB. (H) The expression level of IL1R1 protein in oocytes. (I) The quantified intensity of IL1R1/TUBB. (J) The string diagram shows the protein–protein interaction network. (K) The expression level of RELA protein in oocytes. (L) The quantified intensity of RELA/TUBB. (M) The expression level of NFKB1 protein in oocytes. (N) The quantified intensity of NFKB1/TUBB. Each lane requires 200 oocytes or corresponding cumulus cells for the protein detection mentioned above. Expression of TUBB was applied as the interior control. The data are presented as mean ± SD. p < 0.05 indicates a statistically significant difference. The number of experiments is marked in brackets in the legend.

Fig 3: TβR3 positively impacts EVT migration and invasion through a non-canonical signaling pathwayHTR cells were transfected with siCon or siTβR3. TβR3 RNA and protein levels were determined by RT-qPCR (A) and Western blot (B) analysis 48 h post-transfection. Protein molecular size markers in kDa are indicated on the right of the blots. Cell migration (C) and invasion (D) were analyzed 48 h post-transfection. Numbers are mean ± SD (n = 3). **p < 0.01. (E) HTR cells were transfected with siCon, siTβR3 or siPar6. In the left panel, at 48 h post-transfection, cells were treated with (+) or without (−)TGF-β2 for 6 h, followed by Western blot analysis to determine protein levels of RhoA (top blot) and beta-tubulin (bottom blot), which was used as a loading control. In the right panel, at 48 h post-transfection, proteins were extracted and Western blot analysis was carried out to determine Par6 protein levels. TUBB serves as a loading control. (F) Western blot quantifications using ImageJ from three independent transfection experiments are shown. One-sample t tests were performed to compare each data point with the siCon control. Numbers are mean ± SD (n = 3). *p < 0.05.

Fig 4: The atg5 knockout MEFs are more sensitive to DHA compared to wild-type MEFs. (A) The levels of ROS were measured in wild-type (WT) and atg5−/− MEFs after 3 h DHA treatment (70 and 140 µM) using the fluorescent DCF probe. The data from one representative experiment of 3 independent experiments are displayed. Each experiment was performed in triplicate wells where the mean intensity ±SD of 10,000 cells per well was measured. (B) The levels of NFE2L2 and SQSTM1 after vehicle or DHA (70 μM, 16 h) treatment in wild-type and atg5−/− MEFs (85 μg protein loaded). TUBB/β-tubulin was used as loading control. The immunoblot is representative for 3 independent experiments. (C) Wild-type and atg5−/− MEFs were exposed to DHA (75 μM) and cellular responses observed over time using the xCELLigence real-time monitoring system. The cell index was normalized to one at the start of the experiment. Mean normalized cell index ±SD of triplicate wells of vehicle or DHA treated cells are displayed. The results are representative for 5 independent growth experiments scored by cell index using xCELLigence.

Fig 5: Autophagy inhibition promotes oncogenic RAS-induced EMT. (a) Immunofluorescence staining of LC3 (green), CDH1 (green), ZEB1 (red) or F-actin in HKe3 ER:HRAS V12 cells with the indicated treatments. Rhodamine-phalloidin was used to stain F-actin. TO-PRO-3 (blue) was used to stain nucleic acids. Scale bar: 20μm. (b) Protein expression of CDH1, ZEB1, SNAI2, SNAI1, ZEB2, TWIST1, ATG12–ATG5 and phospho-MAPK/ERK (p-MAPK) in HKe3 ER:HRAS V12 cells with the indicated treatments. TUBB was used as a loading control. (c) Fold change in mRNA levels of CDH1, ZEB1 and SNAI2 in HKe3 ER:HRAS V12 cells with the indicated treatments. mRNA levels normalized to ACTB/β-actin in control cells were used to set the baseline value at unity. Data are mean ± s.d. n = 3 samples per group. n.s. P > 0.05, *** P < 0.001. (d) Protein expression of CDH1, ZEB1, SNAI2 and ATG12–ATG5 in 4-OHT-treated HKe3 ER:HRAS V12 cells with the indicated treatments. TUBB was used as a loading control.