Fig 2: Exogenous VEGF-A exacerbated high glucose (HG)-induced activation of ERK/PLA2 axis in human retinal endothelial cells (HRECs). Panel (A): immunoblot analysis of HREC whole-cell lysates, using antibodies against phospho-ERK1/2 (p-ERK1/2), total ERK1/2, phospho-cPLA2 (p-cPLA2), total cPLA2 and iPLA2. The blot was probed with anti ß-actin antibody to verify equal loading of 30 µg proteins per lane. Panel (B): densitometric analysis of immunoblot indicating protein quantification of each band (in arbitrary densitometry unit, a.d.u.), carried out with the Image J program. Panel (C): immunocytochemical staining for p-ERK 1/2 (green fluorescence) and ERK 1/2 (red fluorescence) in HRECs grown in normal glucose (NG, 5 mM; a, b, c, d), in normal glucose plus 80 ng/mL of VEGF-A (a’, b’, c’, d’), in high glucose (HG, 25 mM; a’’, b’’, c’’, d’’) or in high glucose supplemented with 80 ng/mL of VEGF-A (HG + VEGF-A; a’’’, b’’’, c’’’, d’’’). Blue fluorescence indicates DAPI staining of cell nuclei. Merged pictures are shown in the fourth column (d, d’, d’’, and d’’’). In control HRECs, green fluorescence was almost undetectable, either in the cytoplasm or in the nucleus. HG-culture conditions induced an increase in green fluorescence (indicating p-ERK1/2 activation), particularly in the nuclear region (b’’’ vs. b’ and b’’ vs. b, respectively). Panel (D): immunocytochemical staining for p-cPLA2 (green fluorescence) and cPLA2 (red fluorescence) in HRECs grown in normal glucose (NG, 5 mM; a, b, c, d), in normal glucose plus 80 ng/mL of VEGF-A (a’, b’, c’, d’), in high glucose (HG, 25 mM; a’’, b’’, c’’, d’’) or in HG supplemented with 80 ng/mL of VEGF-A (HG + VEGF-A; a’’’, b’’’, c’’’, d’’’). Blue fluorescence indicates DAPI staining of cell nuclei. Merged pictures are shown in the fourth column (d, d’, d’’, and d’’’). In control HRECs, green fluorescence is detectable only at nuclear level; an increase (indicating cPLA2 activation) can be observed in both cytoplasm and nuclei in HG-culture conditions, (b’’’ vs. b’ and b’’ vs. b, respectively). Panel (E): immunocytochemical staining for iPLA2 (green fluorescence) and ß-actin (red fluorescence) in HRECs grown in normal glucose (NG, 5 mM; a, b, c, d), in normal glucose plus 80 ng/mL of VEGF-A (a’, b’, c’, d’), in high glucose (HG, 25 mM; a’’, b’’, c’’, d’’) or in high glucose supplemented with 80 ng/mL of VEGF-A (HG + VEGF-A; a’’’, b’’’, c’’’, d’’’). Blue fluorescence indicates DAPI staining of cell nuclei. Merged pictures are shown in the fourth column (d, d’, d’’, and d’’’). Immunostaining for iPLA2 is increased in HRECs cultured in high glucose supplemented with 80 ng/mL of VEGF-A (b’’’ vs. b’’, b’, and b). Magnification: ×40; scale bars: 100 µm.

Fig 3: PACOCF3 prevents HuPrP106-126-induced cPLA2 activation. (A) Primary cortical neurons were treated with 40 µM HuPrP106-126 for 1 hour or 24 hours, or pre-treated with 1 µM PACOCF3 before the addition of peptide. Samples were analysed by immunoblotting and the amount of p-cPLA2 and total PLA2 was calculated by densitometry. (B) Neurons were further analysed by confocal microscopy. Samples were treated with HuPrP106-126 for 30 minutes, 1 hour or 24 hours, or pre-treated with PACOCF3 prior to HuPrP106-126. p-cPLA2 was labelled (red) and nuclei were stained with DAPI. Fluorescence intensity of p-cPLA2 was measured and normalised to cell number, shown in the accompanying graph. Data expressed as mean ± S.D. of three experiments *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: LPS modulated cPLA2 gene expression via activation of JAK2. Serum-starved 3T3-L1 cells were pretreated with 100 nM or different concentrations of AG490 for 1 h. Or cells were transfected with 100 nM of scramble (Scr) or JAK2 siRNA for 24 h. Inhibitor or siRNA-treated cells were then incubated 20 µg/mL of LPS for the indicated time points (A, B), 16 h (C, D) or 6 h (E). At the end of incubation, cells were harvested and cell lysates or mRNA were extracted. (A, B, C, D) Western blot was used to evaluate the expression of phosphorylated JAK2, total JAK2, cPLA2 or GAPDH protein. (E) RT-PCR was used to analyze the expression of cPLA2 mRNA. Data are expressed as means ± SEM of at least 3 independent experiments (n=3). &P < 0.05, as compared with the 0 point or indicated group. #P < 0.05, as compared with the same time points or LPS treated alone.

Fig 5: The relationship between PLA2G4A expression and the prognosis of patients with colorectal cancer.(A) Kaplan-Meier overall survival curves showed high-level expression of PLA2G4A correlated with poor prognosis in stage III–IV RSCRC. n = 20. (B) Patients with stage III–IV LSCRC with high-level of PLA2G4A had a better outcome in overall survival. n = 18. (C) Kaplan-Meier overall survival curves of patients with CRC. Log-rank test were used for analysis. n = 38. PLA2G4A median level was used as cut-off. (D) The representative IHC staining of PLA2G4A, CD8, PD-1, and ?d TCR expression in RSCRC samples (Patient 1 survival: 77 months, alive; Patient 2 survival: 14 months, deceased). Scale bars: 200 µm. (E and F) The infiltration of ?d T cells in COAD (E) and READ (F). (G and H) The infiltration of CD8+ T cells in COAD (G) and READ (H). (I and J) The infiltration of CD4+ T cells in COAD (I) and READ (J). COAD, n = 288, and normal, n = 40; READ, n = 95, and normal, n = 10. COAD, colon adenocarcinoma; READ, rectum adenocarcinoma. Data are shown as mean ± SEM. Unpaired 2-tailed t test was used to compare variables. ** P < 0.01, ***P < 0.001, ****P < 0.0001.