Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

Figures

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  Figure 1. LA stimulates Ca2+-independent hemolysis. (A) LA but not PA induces dose-dependent hemolysis. (B) Ca2+ sensitivity of LA-induced hemolysis. (C) Ionophoric challenge with IMC does not aggravate LA-induced hemolysis. (D) Inhibition of glucose uptake by Glut1 inhibitor WZB117 does not exasperate LA-induced hemolysis. ns indicates not significant, ** (p < 0.01) and **** (p < 0.0001) indicate significantly different values from control conditions whereas ## (p < 0.01) and ### (p < 0.001) indicate statistical significance compared to LA-treated cells.

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  Figure 2. LA elicits Ca2+-dependent PS exposure. (A) Representative histograms of Annexin V-FITC of cells exposed to 0–250 μM of LA. (B) LA-induced fold increase in mean intensity fluorescence (MFI) of Annexin V-FITC. (C) LA but not PA induces an increase in the percentage of Annexin V-positive cells. (D) LA-induced increase in ESR. (E) Representative histograms of Annexin V-FITC of cells exposed to 0–100 μM of LA with and without Ca2+. (F) LA-induced fold increase in MFI of Annexin V-FITC with and without Ca2+. (G) LA-induced fold increase in the percentage of Annexin V-positive cells with and without Ca2+. ns not significant, * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001) indicate significantly different values from control conditions whereas # (p < 0.05) indicate statistical significance compared to LA-treated cells.

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  Figure 3. LA promotes extracellular Ca2+ influx. (A) Representative histograms of Fluo4 of cells exposed to 0–250 μM of LA. (B) LA-induced increase in MFI of Fluo4. (C) LA-induced increase in the percentage of Fluo4-positive cells. (D) Representative histograms of Fluo4 of cells exposed to 0–100 μM of LA with and without Ca2+. (E) LA-induced increase in Fluo4 MFI with and without Ca2+. (F) LA-induced increase in the percentage of Fluo4-positive cells with and without Ca2+. ns indicates not significant, * (p < 0.05), ** (p < 0.01), and **** (p < 0.0001) indicate significantly different values from control conditions whereas # (p < 0.05) indicate statistical significance compared to LA-treated cells.

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  Figure 4. LA causes loss of cellular volume and increased granularity. (A) Representative histograms of FSC of cells exposed to 0–250 μM of LA. (B) LA-induced decrease in MFI of FSC. (C) LA-induced decrease in FSC MFI with and without Ca2+. (D) RDW expressed in terms of coefficient of variation (CV) and standard deviation (SD). (E) MCV. (F) Dot plot scattergrams of SSC and FSC of control and LA-treated cells in the presence and absence of Ca2+. (G) Fold increase in SSC. (H) Fold increase in SSC in the presence and absence of Ca2+. ns indicates not significant, * (p < 0.05), ** (p < 0.01), and ****(p < 0.0001) indicate significantly different values from control conditions whereas ## (p < 0.01) indicates statistical significance compared to LA-treated cells.

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  Figure 5. LA triggers Ca2+-dependent oxidative stress. (A) Representative histograms and fold increase in MFI of DCF fluorescence of cells exposed to 0–100 μM of LA. (B) Representative histograms and fold increase in MFI of DCF fluorescence of cells exposed to 0 or 100 μM of LA in presence or absence of Ca2+. (C) Intracellular content of H2O2 in cells treated with 0 or 100 μM of LA. (D) Intracellular content of SOA in cells treated with 0 or 100 μM of LA. (E) CAT activity in cells treated with 0 or 100 μM of LA. (F) SOD activity in cells treated with 0 or 100 μM of LA. ** (p < 0.01) and **** (p < 0.0001) indicate significantly different values from control conditions whereas ## (p < 0.01) indicates statistical significance compared to LA-treated cells.

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  Figure 6. LA disrupts glutathione homeostasis. (A) GPx activity in control and experimental (100 μM of LA) cells. (B) Intracellular GSH content. (C) Intracellular GSSG content. (D) GSH/GSSG ratio. (E) Representative histograms and arithmetic means of fold increase in MFI of DCF fluorescence with or without 100 μM of LA in the presence or absence of 20 μM of GSH. (F) Eryptosis and (G) hemolysis of cells treated with 100 μM of LA in presence or absence of 20 μM of GSH. (H) Hemolysis with or without 100 μM of LA in the presence or absence of 50 μM of Asp. **** (p < 0.0001) indicates significantly different values from control conditions whereas # (p < 0.05) indicates statistical significance compared to LA-treated cells.

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  Figure 7. LA triggers Ca2+-dependent lipid peroxidation and protein carbonylation. (A) Representative histograms of BODIPY fluorescence of cells exposed to 0–100 μM of LA. (B) LA-induced fold increase in MFI of BODIPY. (C) LA-induced increase in the percentage of cells with high lipid peroxides. (D) PCC content in cells exposed to 0 or 100 μM of LA. (E) Representative histograms of BODIPY fluorescence of cells exposed to 0–100 μM of LA in presence or absence of Ca2+. (F) LA-induced BODIPY fold increase with and without Ca2+. (G) LA-induced increase in the percentage of cells with high lipid peroxides with and without Ca2+. * (p < 0.05), ** (p < 0.01), and **** (p < 0.0001) indicate significantly different values from control conditions whereas # (p < 0.05) and ### (p < 0.001) indicate statistical significance compared to LA-treated cells.

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  Figure 8. LA-induced hemolysis is mitigated by D4476. Hemolytic rates under inhibition of (A) caspase by zVAD, (B) p38 MAPK by SB203580, and (C) CK1α by D4476. (D) LA-induced hemolysis under EPO supplementation. **** (p < 0.0001) indicates significantly different values from control conditions whereas # (p < 0.05) indicates statistical significance compared to LA-treated cells.

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  Figure 9. LA elevates NLR. (A) Representative dot plot scattergrams of control LA-treated peripheral WB cells. WNR channels show the distribution of WBCs in terms of side fluorescence (SFL) and SSC, while the WDF channel relates FSC and SFL. (B) Total WBC viability. (C) Differential count of WBC subsets. (D) Percentage of lymphocytes. (E) NLR. (F) Percentage of eosinophils. ns indicates not significant whereas * (p < 0.05) and ** (p < 0.01) indicate significantly different values from control conditions.