Fig 2: p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O2) or hypoxia (hypox, 1% O2) for 48 h prior to RNA extraction or western blot analysis. (A) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results (n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. (B) Western blot analysis with cellular lysates from SGHPL-4 cells (n = 3). GAPDH served as the loading control. (C) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results (n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. (D) Western blot analysis with cellular lysates from HTR cells (n = 3). GAPDH served as the loading control. (E) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. (F) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results (n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. (G) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated (n = 5). GAPDH was used as the housekeeping gene control. Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Fig 3: Aspirin reduces ß-catenin and EMT markers in vivo. (A) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) ApcMin/+ mouse tissue stained for ß-catenin expression. (B) Transcript expression of Tcf7 in ApcMin/+ mouse crypts treated with 400 mg/kg aspirin for 4 weeks and Apcflox/flox mouse organoids treated with 2 mmol/L aspirin for 12 days. Tcf7 transcript levels are normalized to GAPDH transcripts and expressed as fold-change compared with untreated control sample. Data represent 1 mouse per experimental condition. (C) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) ApcMin/+ mouse tissue stained for E-cadherin expression. (D) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) ApcMin/+ mouse tissue stained for vimentin expression. Quantification of ß-catenin (21 control and 30 aspirin-treated adenomas), E-cadherin (37 control and 23 aspirin-treated adenomas), and vimentin (29 control and 16 aspirin-treated adenomas) staining in adenoma tissue from cohort of 5 ApcMin/+ control and 4 ApcMin/+ aspirin-treated mice. Microscope objective magnification noted in bottom left corner of image. Scale bar = 50 µm. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by unpaired Student t test. Asterisks denote P value (*<.05, **<.01, ***<.001, ****<.0001).

Fig 4: Phagocytosis and fatty acid metabolism in siPNPLA2 cells. ARPE-19 cells were transfected with Scr or siRNAs targeting PNPLA2. At 72 hours after transfection, ARPE-19 cells were incubated with POSs (1 × 107 units/mL) in 24-well tissue culture plates for pulse–chase experiments. (A) Representative immunoblot of total lysates of ARPE-19 cells at 0.5 hour, 1 hour, and 2.5 hours of POS pulse and at 16-hour and 24-hour chase periods, as indicated at the top of the blot. Proteins in cell lysates were subjected to immunoblotting with anti-rhodopsin followed by reprobing with anti-GAPDH as the loading control. (B) Quantification of rhodopsin from duplicate samples and three blots of cell lysates from pulse–chase experiments and time periods (indicated in the x-axis) as from panel. Data are presented as mean ± S.D; **P < 0.01; ns, not significant. Intensities of the immunoreactive bands were determined, and the percentages of the remaining rhodopsin after 16-hour and 24-hour chase periods relative to rhodopsin at 2.5-hour pulse are plotted (y-axis). (C, D) Levels of secreted free fatty acids (C) and ß-HB (D) were measured in culture media of cells transfected with Scr or siPNPLA2 following incubation with POS for the indicated periods of times (x-axis). Data are presented as mean ± SD. *P < 0.05, **P < 0.01 (n = 3). Duplex siPNPLA2 C was used to generate the data (see Table 3 for sequences of duplexes).

Fig 5: Study of cellular SIL1 immunoreactivity and presence of ER-stress in Hek293 cells overexpressing SIL1 or its variant forms. (A) Immunofluorescence-based investigation of the SIL1-immunoreactivity pattern in Hek293 cell revealed a reticular distribution for WT-SIL1, p.R92W and p.K132Q variant forms of SIL1 but a non-reticular rather “clumpy” immunoreactivity for p.G312R and p.L457P mutant forms of SIL1. For p.V231_I232del mutant SIL1, a reticular distribution with focal accumulations was observed. (B) EM revealed widened and proliferated ER in cells overexpressing the mutant forms p.G312R and p.L457P, but not in cells overexpressing WT-SIL1, p.R92W, p.K132Q, and p.V231_I232del (6 h of induced overexpression) forms of the proteins. In addition, accumulation of electron dense material could occasionally be seen in cells overexpressing p.G312R and p.L457P muatnt SIL1 (black arrows). Along this line, overexpression of these two known missense mutants of SIL1 resulted in the build-up of electron dense material (most likely corresponding to aggregated protein) in the cytoplasm as well as in (autophagic) vacuoles. The white arrow highlights an abnormal centrosome in a Hek293 cell overexpressing p.G312R mutant SIL1. (C) Immunoblot-based studies aimed to investigate the presence of ER-Stress by focussing on marker proteins for activation of the unfolded protein response (GRP170, IRE1, PERK, GRP94, ERj3 & pEif2a), the ER-associated degradation pathway (VCP, DNAJB6) and proteolysis (Beclin). Results revealed a considerable presence of ER-stress in Hek293 cells overexpressing p.L457P mutant SIL1 than in cells overexpressing p.G312R mutant SIL1 where solely pEif2a and Beclin are increased compared to cells overexpressing WT-SIL1. p.R92W, p.K132Q variant SIL1, and p.V231_I232del mutant SIL1 (6 h of induced overexpression) do not show remarkable changes in abundances of the stress markers compared to cells overexpressing WT-SIL1. GAPDH was investigated to demonstrate equal protein loading. The differences in expression of these markers between C-TAP and WT-SIL1 overexpressing Hek293 cells have been described previously (12).