Fig 1: Time series of LC3-I, LC3-II and CysLTR1 protein expression upon DEX treatment and serum shock in polarized ARPE-19 cells over a 54-hour period measured every 6 hours. Representative western blot analysis showing total protein loading, (A) LC3-I and LC3-II and (B) CysLTR1 expression upon DEX treatment and serum shock in polarized ARPE-19 cells within 54 hours. RLU levels of (C) LC3-I, (D) LC3-II and (E) CysLTR1 upon DEX treatment (green) and serum shock (red) in polarized ARPE-19 cells within 54 hours. Values are represented as the mean ± SD; n = 3–5. Sine waves with nonzero baselines + confidence bands were generated with a comparison of fits (amplitude = 0 versus amplitude unconstrained). Abbreviation: WL: wavelength. Western blot images are cropped showing areas of marked primary antibody interaction only.
Fig 2: Representative immunofluorescence images of CysLTR1 and Class III ß-tubulin in polarized ARPE-19 cells. ARPE-19 cells were cultured for 7 days in low-serum medium. (a) CysLTR1 (visualized in red, indicated by white arrowheads), (b) class III ß-tubulin (green) and (c) DAPI (blue) in polarized ARPE-19 cells. (d) Merged image of red, green and blue emission showing colocalization of CysLTR1 and class III ß-tubulin (yellow, indicated by white arrowheads). Internalized CysLTR1 located near the cell nucleus was observed in individual cells, as indicated by the white arrow. e) Secondary antibody only control + DAPI (blue). Scale bar = 50 µm; n = 5.
Fig 3: Pearson R correlation matrix of autophagosomal (MAP1LC3B, SQSTM1, BECN1 and mTOR), UPR-related (ATF4, ATF6, DDIT3, EIF2AK3, ERN1, HSPA5, PPP1R15A, XBP1 [total XBP1], XBP1u [unspliced], and XBP1s [spliced]), and leukotriene synthesis (ALOX5) genes and CysLTR1 in (a) untreated control polarized ARPE-19 cells and polarized ARPE-19 cells treated with (b) 100 nM ZK, (c) 300 µM H2O2 and (d) 300 µM H2O2 + 100 nM ZK for 3 h (n = 16).
Fig 4: mRNA and protein expression of CysLTR1 in polarized ARPE-19 cells. CysLTR1 expression upon treatment with 300 µM H2O2 for 3 h in polarized (7–9 days) ARPE-19 cells at the (a) mRNA and (b) protein levels detected by qPCR and western blot analysis, respectively. (c) Representative western blot analysis showing total protein loading and CysLTR1 expression in untreated polarized ARPE-19 cells and polarized ARPE-19 cells treated with H2O2 for 3 h. The values are represented in box and whisker plot format (min to max); n = 15 (mRNA), n = 12 (protein). Significance was calculated by paired t-test. ***p < 0.001. Cq quantification cycle, GOI gene of interest, RG reference gene.
Fig 5: Validation of the prognostic value of CysLT1 and CysLT2 protein expression in an independent cohort of primary UM patients. (A) Representative cores from the Spanish UM patient tissue microarray designated with a score of 1, 2, or 3 for CysLT1 staining intensity. (B) Representative cores from the same TMA designated with a score of 1, 2, or 3 for CysLT2 staining intensity. Expression of both CysLT1 and CysLT2 was predominantly cytoplasmic in primary UM tumours, with a mixture of membranous and cytoplasmic staining in cores designated a score of 2 and 3. (C) High expression of CysLT1 (red) is significantly associated with reduced survival from metastatic disease in patients presenting with primary UM (n = 64; Log Rank; p = 0.021; HR 2.28; 95% CI 1.08–4.78). (D) High expression of CysLT1 (red) is significantly associated with reduced overall survival in patients presenting with primary UM (n = 68; Log Rank; p = 0.015; HR 2.27; 95% CI 1.12–4.58). (E) Kaplan-Meier survival curve stratified based on high (red) or low (blue) CysLT2 expression and death by metastatic melanoma (n = 45; Log Rank; p = 0.643; HR 1.39; 95% CI 0.32–6.01). (F) Kaplan-Meier survival curve stratified based by high (red) or low (blue) CysLT2 expression and death by any cause (n = 48; Log Rank; p = 0.685; HR 1.34; 95% CI 0.31–5.75). The median was used as the cut-off point for high vs. low expression for all Kaplan-Meier survival curves. Number of events indicates the number of deaths due to metastatic melanoma (C,E). Number of events indicates the number of deaths due to any cause (D,F).
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