Fig. 5

The expression of PTX3 is required for efferocytosis in male ECs. a Calcein-loaded M-ECs (blue bars) and F-ECs (deep red bars) were overlaid on a RAW 264.7 cell monolayer after an overnight incubation in 20% (solid bars) or 2% (horizontal bars) FBS. Two hours later, RAW-associated fluorescence was measured. Data are expressed as percent (%) of engulfed cells, that is the ratio between the RAW-associated fluorescence and the total added fluorescence (53,866 ± 9369 and 53,345 ± 7761 A.U. for 20%- and 2%-treated cells, respectively) multiplied by 100. No significant differences between the groups, n = 5/7 for 20% FBS-treated M-ECs and F-ECs, respectively; n = 10 for 2% FBS-starved M-ECs and F-ECs. b M-ECs (blue bars) and F-ECs (deep red bars) treated as described in a were plated on 0.1% gelatin-coated well, and 2 h later, well-associated fluorescence was measured. Data are expressed as percent (%) of adherent cells, that is the ratio between the well-associated fluorescence and the total added fluorescence (27,717 ± 3412 and 32,078 ± 4324 A.U. for 20%- and 2%-treated cells, respectively) multiplied by 100. No significant differences between the groups, n = 3 for 20% FBS-treated M-ECs and F-ECs; n = 5 for 2% FBS-starved M-ECs and F-ECs. c, d M-ECs (blue bars) and F-ECs (deep red bars) were transfected for 48 h with control (ctrl siRNA, solid bars) or PTX3 siRNA (diagonal bars). In c, PTX3 mRNA was measured by RT-qPCR and normalized to the housekeeping gene GAPDH. ****p < 0.0001 vs ctrl siRNA, n = 3. In d, secreted PTX3 protein was measured. ***p < 0.001 vs ctrl siRNA, **p < 0.01 between PTX3 levels in ctrl siRNA-transfected M-ECs and F-ECs, n = 2. Interaction between sexes is significant, *p < 0.05.  2-way ANOVA followed by Sidak’s multiple comparison test. e M-ECs (blue bars) and F-ECs (deep red bars) transfected with control (ctrl siRNA, solid bars) or PTX3 siRNAs (diagonal bars) were loaded with calcein-AM, and after an overnight serum deprivation, the percent of engulfed cells was measured as described in a. ***p < 0.001, *p < 0.05 vs ctrl siRNA in M-ECs and F-ECs, respectively; ****p < 0.0001 between PTX3 siRNA in M-ECs and F-ECs, n = 6. Interaction between sexes is significant, ***p < 0.001.  2-way ANOVA followed by Sidak’s multiple comparison test. f M-ECs (blue bars) and F-ECs (deep red bars) were transfected with control (ctrl siRNA, solid bars) or PTX3 (diagonal bars) siRNAs, and after an overnight serum deprivation, the percent of gated cells was measured by FACS as described in Fig. 4a, b. Data are expressed as the sum of the cells gated in the UL (early apoptotic) and UR (late apoptotic) quadrants. *p < 0.05 between ctrl siRNA-transfected M-ECs and F-ECs, n = 3