Table 1.
Main characteristics of study subjects with systemic lupus erythematosus.
Figure 1.
Association of ACSL5 mRNA expression levels with SLE.
A) ACSL5 transcript levels in PBMCs from healthy controls (0) and SLE patients (1) for ACSL5(N) (number of samples n: 0 = 49, 1 = 45), activated ACSL5(A) (n, 0 = 34, 1 = 40) and the ratio A/N (ACSL5(SI) (n, 0 = 34, 1 = 40). B) ACSL5 transcript levels in PBMCs from males (0) (n = 24) and females (1) (n = 25), all of them from the control group. C) ACSL5 transcript levels in PBMCs from prednisone treated (1) (n = 28) and untreated (0) (n = 12) SLE patients. Results are represented in box plots given medians, quartiles, outsiders (circle points) and extremes (asterisk points). P-values of differences between the groups (0) and (1) are defined by Mann-Whitney Statistics. N.S. stands for non significant.
Table 2.
Logistic regression analysis to determine the effect on diseases and the area under curve (AUC) of ACSL5 values in PBMCs.
Figure 2.
Effect of PMA+Io activation in PBMCs.
PBMCs from healthy controls were obtained and cultured in the presence or absence of PMA+Io for up to 24 h. A) Cells were collected at defined times, washed with PBS, fixed with 70% Ethanol, stained with Propidium Iodide (PI) and analyzed in a FACSCaliburTM flow cytometer to determine the apoptotic hipodiploid cell fragments (defined as percentage of PI incorporation). B) Cells were washed twice with PBS and double stained with Annexin V and PI, then analyzed by FACS to determine the percentage of apoptotic cells (Annexin V positive and double positive cells). C–I) Total RNA was extracted, cDNA synthesized and qRT-PCR implemented to determine mRNA expression. Results are given by means of three independent experiments and the bars show the standard deviation. P-value has been calculated with the paired Student t test.
Figure 3.
Effect of PMA+Io activation in Jurkat T cells.
Jurkat T cells were cultured in the presence or absence of PMA+Io for up to 24 h. A) Cells were collected at defined times, washed with PBS, fixed with 70% Ethanol, stained with Propidium Iodide (PI) and analyzed in a FACSCaliburTM flow cytometer to determine the apoptotic hipodiploid cell fragments (defined as percentage of PI incorporation). B) Cells were washed twice with PBS and double stained with Annexin V and PI, then analyzed by FACS to determine the percentage of apoptotic cells (Annexin V positive and double positive cells). C–I) Total RNA was extracted, cDNA synthesized and qRT-PCR implemented to determine mRNA expression. Results are given by means of three independent experiments and the bars show the standard deviation. P-value has been calculated with the paired Student t test.
Figure 4.
Effect of ACSL5 silencing in the apoptosis induced in Jurkat T cells by PMA+Io activation.
Jurkat T cells were electropororated with either siRNA for ACSL5 (siACSL5) or unspecific control of siRNA (siRNA), and left for 18 h in culture media. Then both siACSL5 and siRNA Jurkat T cells were cultured with or without PMA+Io for 24 h. A) ACSL5 expression at 24 h was determined in each type of culture as described in material and methods. B) Percentage of apoptosis in siACSL5 or siRNA Jurkat T cells measured by cytometry as Annexin V positive and double Annexin V and PI positive cells. C–I) Total RNA was extracted, cDNA synthesized and qRT-PCR implemented to determine mRNA expression. Results are given by means of three independent experiments and the bars show the standard deviation. P-value has been calculated with the paired Student t test.