Table 1.
Human qPCR primers.
Table 2.
Mouse qPCR primers.
Fig 1.
L. infantum actively infects U937-derived macrophages.
A) U937-derived macrophages were infected with L. infantum promastigotes; after 6 h or 24 h cells were washed, stained with Hoechst dye and observed with a fluorescence microscope to monitor internalized parasites. Arrows indicate parasitized cells. B) Active infection was monitored by calculating infection index, considering at least 300 total macrophages per infection time (dotted line), and by a qPCR assay targeting kDNA of Leishmania (solid line). Both methods allowed to detect a significant increase in infection from 6 h to 24 h. The data are representative of three independent experiments. CTR, uninfected control. * p < 0.05.
Fig 2.
L. infantum infection induces cell survival and apoptosis inhibition.
U937-derived macrophages were infected with L. infantum promastigotes for 18 h. Infected and non-infected cells were treated with 0.5 μg/ml tunicamycin for 1 h, 2 h and 4 h. A) The Akt phosphorylation and cleavage of caspase-3 were analyzed in total cell lysates by western blotting. B) Band density quantification was performed using a Chemi-Doc System. Results are representative of three experiments. * p < 0.05, two-way ANOVA followed by Bonferroni post hoc test.
Fig 3.
Evaluation of the reference genes.
A) Log2 fold change of the four candidate reference genes (B2M, GAPDH, HPRT, GUSB) in U937-derived macrophages after 24 h infection in comparison to the non-infected controls. B) Evaluation of B2M (grey bars) and GUSB (black bars) expression in U937-derived macrophages infected for 6 h, 24 h, 48 h, 72 h, in comparison to the non-infected controls. The amount corresponding to 50 ng of total RNA used for cDNA synthesis was loaded per each PCR tube. * p < 0.05.
Fig 4.
ER stress expression markers in infected U937-derived macrophages.
A) Gene expression profiling in U937-derived macrophages 6 h and 24 h after infection with L. infantum. The graph shows the log2 fold changes in comparison to the control (non-infected). B) Gene expression in U937-derived macrophages following tunicamycin treatment (0.5 μg/ml, 6 h). The graph shows the log2 fold changes in comparison to the control (DMSO) values. Data are represented as the mean ± SEM of at least three experiments. * p < 0.05, ** p < 0.01.
Fig 5.
XBP1 induction following L. infantum infection in U937-derived macrophages.
U937-derived macrophages were infected with L. infantum promastigotes for 6 h or 24 h. As positive control for XBP1 splicing/induction, infected and non-infected cells were treated with 0.5 μg/ml tunicamycin for 6 h. A) XBP1 splicing was not detected using conventional PCR in infected cells. B) A significant induction of both uXBP1 and sXBP1 was shown after 24 h of infection by qPCR, while tunicamycin treatment induced an increase in sXBP1 and a decrease in the uXBP1. ** p < 0.01. TUN, tunicamycin; L, 100 bp DNA ladder; NTC, no template control.
Fig 6.
ER stress expression markers in infected murine macrophages.
A) Gene expression profiling in murine macrophages 24 h after infection with L. infantum. The graph shows the log2 fold changes in comparison to the control (non-infected). B) Gene expression in murine macrophages following tunicamycin treatment (2 μg/ml, 4 h). The graph shows the log2 fold changes in comparison to the control (DMSO) values. Data are represented as the mean ± SEM of two experiments, * p < 0.05.
Fig 7.
Xbp1 induction following L. infantum infection in murine macrophages.
Murine macrophages were infected with L. infantum promastigotes for 24 h. As positive control for Xbp1 splicing/induction, infected and non-infected cells were treated with 2 μg/ml tunicamycin for 4 h. A) Xbp1 splicing was not detected using conventional PCR in infected cells. B) A significant induction of sXbp1 was shown after 24 h infection or tunicamycin treatment by qPCR. ** p < 0.01. TUN, tunicamycin; L, 100 bp DNA ladder; NTC, no template control.
Fig 8.
L. infantum infection induces sXBP1 and GRP78/HSPA5 proteins.
U937-derived macrophages were infected with L. infantum promastigotes for 18 h. A) The sXBP1 protein levels were analyzed in total cell lysates by western blotting. Infected cells are shown in duplicate. NIH-3T3 cells treated with tunicamycin were used as positive control for antibody signal. B) The HSPA5/GRP78 protein levels were analyzed in total cell lysates by western blotting. Two experiments are shown. Infected cells are shown in duplicate.
Fig 9.
L. infantum inhibits tunicamycin- and DTT-induced ER stress.
U937-derived macrophages were infected with L. infantum promastigotes for 18 h. Infected and non-infected cells were treated with 0.5 μg/ml tunicamycin (A, B) or 1 mM DTT (C, D) for 1 h, 2 h and 4 h. A, C) The phosphorylation of eIF2α and DDIT3/CHOP protein levels were analyzed in total cell lysates by western blotting. B, D) Band density quantification was performed using a Chemi-Doc System. Results are representative of three experiments. * p < 0.05, two-way ANOVA followed by Bonferroni post hoc test. TUN, tunicamycin.
Fig 10.
L. infantum inhibits the expression of tunicamycin-induced ER stress marker genes.
U937-derived macrophages were infected with L. infantum promastigotes for 18 h. Infected and non-infected U937-derived macrophages were treated with 0.5 μg/ml tunicamycin for 2 h. The graph shows the log2 fold changes of ER stress marker genes induced by tunicamycin in infected cells compared to the non-infected cells. * p < 0.05.