Figure 6 ( A , B ) Overexpression of hTau in HEK293 cells for 48 h increased the activity - dependent phosphorylation of JAK2 , JNK1 and ERK1 compared with the empty vector control ( Ctrl ) measured by Western blotting ( n = 3 , student ' s t test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT . Pharmacological inhibition of ERK1 ( C , D ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts ( C and the nuclear fraction ( D measured by Western blotting ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT . Pharmacological inhibition of JNK1 ( E , F ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts E ) and the nuclear fraction F ) measured by Western blotting ( n = 3 ) . The alteration of pS - STAT1 ( Ser727 ) confirms the efficacy of JNK1 inhibitors . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT . Pharmacological inhibition of JAK2 ( G , H ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts ( G and the nuclear fraction ( H ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT . knockdown JAK2 by siRNA ( I , J ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts I ) and the nuclear fraction J ) ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT . ( K , L ) The phosphorylated JAK2 level increased in the hippocampus of 12 - month - old hTau transgenic mice ( K ) , and the hippocampus of C57 mice infected with AAV - hTau ( 1 . 13 × 1013 v . g . / ml ) ( L ) ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3, student's t test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.Pharmacological inhibition of ERK1 (C, D) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C and the nuclear fraction (D measured by Western blotting (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.Pharmacological inhibition of JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts E) and the nuclear fraction F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.Pharmacological inhibition of JAK2 (G, H) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G and the nuclear fraction (H (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts I) and the nuclear fraction J) (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.(K, L) The phosphorylated JAK2 level increased in the hippocampus of 12-month-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×1013 v.g./ml) (L) (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Figure 7AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( A ) The representative fluorescence image confirms expression of AAV - hTau and AAV - Y701F - STAT1 . Scale bar , 200 μm , or 100 μm for the enlarged . AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( B ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial learning deficits shown by the decreased escape latency during water maze training ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau . AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( C - E ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial memory impairment shown by the decreased latency to reach the platform ( C ) , the increased crossing time in the platform site ( D ) and time spent in the target quadrant ( E ) measured at day 6 by removed the platform ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau . AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( F ) Expression of Y701F - STAT1 did not change the swimming speed of the mice in water maze task ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau . ( G - I ) Simultaneous expression of Y701F - STAT1 did not induced any further change on basal synaptic transmission ( I / O curve ) compared with expression of hTau alone , recorded in hippocampal CA3 ( G ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( I ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau . Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs protein expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) . Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs protein ( K ) expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau . Simultaneous expression of Y701F - STAT1 rescued the hTau - induced suppression of NMDARs mRNA expression measured by qRT - PCR in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (A) The representative fluorescence image confirms expression of AAV-hTau and AAV-Y701F-STAT1. Scale bar, 200 μm, or 100 μm for the enlarged.AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (B) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial learning deficits shown by the decreased escape latency during water maze training (n=7-10 each group). Data information: Data were presented as mean ± s.e.m (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (C-E) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial memory impairment shown by the decreased latency to reach the platform (C), the increased crossing time in the platform site (D) and time spent in the target quadrant (E) measured at day 6 by removed the platform (n=7-10 each group). Data information: Data were presented as mean ± s.e.m , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (F) Expression of Y701F-STAT1 did not change the swimming speed of the mice in water maze task (n=7-10 each group). Data information: Data were presented as mean ± s.e.m two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.(G-I) Simultaneous expression of Y701F-STAT1 did not induced any further change on basal synaptic transmission (I/O curve) compared with expression of hTau alone, recorded in hippocampal CA3 (G). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (I). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4).Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein (K) expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs mRNA expression measured by qRT-PCR in hippocampal CA3 of C57 mice (n=4). Data information: Data were presented as mean ± SD , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


Figure 1A , B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG - A for 16 h at 37 ° C . Anti - BDCA2 - mediated IFNα inhibition in whole - blood assays ( A ) . IFNα levels were detected using human IFNα ELISA . Graph depicts average of duplicate wells of one representative donor ( n = 10 ) . IC50 of 24F4A - mediated IFNα inhibition ( B ) in whole blood ( circles ) ( n = 10 ) and PBMC ( n = 18 ) from healthy human donors ( squares ) or SLE patients ( triangles ) ( n = 11 ) . Horizontal bar represents the mean IC50 for each sample type . Error bars represent SD of IC50 between donors . C Anti - BDCA2 - mediated internalization in whole - blood assays . Whole blood was treated with increasing concentrations of 24F4A for 16 h . Mean fluorescence intensity ( MFI ) of BDCA2 was determined with a non - cross - blocking anti - BDCA2 mAb ( 2D6 ) . Shown is a representative plot of 10 experiments conducted .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.


Figure 2A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0 , 1 , 3 , 6 , 9 , and 24 h . FACS analysis was performed to determine BDCA2 levels on pDCs . Fluorescence minus one ( FMO ) represents background staining of BDCA2 . Shown is a representative plot of three independent experiments . B Isolated pDCs were treated with 10 μg / ml 24F4A - AF647 at 4 ° C or at 37 ° C and analyzed at the indicated time points . Subcellular localization of BDCA2 ( red ) compared to LAMP1 ( green ) was assessed using confocal microscopy . Phalloidin was used to delineate the cell membrane . Yellow represents co - localization of BDCA2 and LAMP1 . Shown is a representative image of four experiments conducted . C , D Whole blood was either treated with 24F4A for 1 h at 37 ° C ( pre - incubation ) or left untreated . PBMC were isolated from each condition . PBMC from the untreated whole blood were subsequently treated with 10 μg / ml 24F4A , and cells from all conditions were stimulated with CpG - A for 16 h . Plot represents mean of duplicate wells . Flow cytometry was used to measure BDCA2 levels ( MFI ) . Shown is a representative donor of eight donors tested . E Whole blood was pre - treated for 0 , 1 , 3 , 6 , and 9 h with 10 μg / ml of 24F4 or the isotype control and then stimulated with CpG - A for additional 16 h . IFNα levels were measured by ELISA . Shown is a representative plot of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.


Figure 3Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . A - C Prior to in vivo dosing , baseline surface levels of BDCA2 for both the vehicle ( Ai ) and 1 mg / kg ( Bi ) animals ( red , dotted line ) were established by staining with fluorescently labeled 24F4A ( direct method ) . Maximal binding of 24F4A to BDCA2 was also established pre - dose in the vehicle ( Aii ) and 1 mg / kg ( Bii ) animals ( red , solid line ) by treating whole blood with 10 μg / ml of 24F4A at 4 ° C and then detecting bound 24F4A with a fluorescently labeled anti - human IgG1 ( indirect method ) . The direct method was used to stain whole blood from both the vehicle ( Aiii ) and 1 mg / kg 24F4A ( Biii ) animals 6 h post - dose ( red , dotted line ) . In a separate stain , the indirect method was used to detect bound 24F4A in the vehicle ( Aiv ) and 1 mg / kg ( Biv ) treated animals ( black , solid line ) . ( C ) Percent BDCA2 internalization relative to pre - dose BDCA2 levels 6 h post - dose with vehicle , 10 mg / kg , or 1 mg / kg 24F4A . Graph shows mean ± standard deviation for each group ( n = 3 ) . Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . D PK / PD relationship between 24F4Aserum concentrations ( red triangle , left axis ) and BDCA2 expression on pDCs ( black squares , right axis , normalized to pre - dose levels ) from the 1 mg / kg group ( i - iii ) . Serum 24F4A was measured by ELISA . ( iv ) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested . Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . E Whole blood from vehicle - or 1 mg / kg 24F4A - treated monkeys was stimulated with CpG - A , and induction of IFN - I was measured by MxA bioassay at various time points pre - and post - treatment . Horizontal black lines represent the model - based estimates of the geometric mean of IFN - I in pre - and post - dose samples . Duplicate symbols represent independent replicates of the MxA bioassay for that time point . Statistical analysis was performed using a two - way mixed - effects analysis of variance ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).


Figure 4A - F Isolated human pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( black squares ) , or 10 μg / ml of the isotype control ( black triangle ) and stimulated with 5 μM of R848 ( A and B ) , 1 μM of CpG - A ( C and D ) , or immune complexes ( E and F ) for 16 h at 37 ° C . IFNα concentrations in cultured supernatants were determined using ELISA . Representative plots for each stimulation ( A , C and E ) and the average IC50 of 24F4A and 24F4A - ef for each stimulation condition ( B , D and F ) are shown ( n = 3 ) . Horizontal bars represent mean IC50 , and error bars represent SD of the mean from three independent experiments . G Isolated human pDCs were treated with 10 μg / ml of 24F4A ( black line ) , 24F4A - ef ( gray shaded histogram ) , or the isotype control ( gray dotted line ) for 10 min at 37 ° C . Flow cytometry was used to evaluate pSYK and pPLCγ2 . Shown is a representative plot of two independent experiments . H Isolated pDCs treated with 10 μg / ml of anti - CD32a ( AT10 ) and stimulated with CpG - A , R848 , or immune complexes . Error bars represent SD of percent inhibition of IFNα from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.


Figure 5A - F Isolated pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( squares ) , Chi - 6G6 ( triangle ) , or isotype control ( diamond ) for 16 h at 37 ° C and stimulated with CpG - A . Flow cytometry was performed to evaluate the MFI of ( A ) BDCA2 and ( C ) CD32a on the surface of pDCs . Percent internalization of BDCA2 ( B ) and CD32a ( D ) on pDCs treated with 10 μg / ml of 24F4A , 24F4A - ef , Chi - 6G6 , or isotype control . Error bars represent SD of percent internalization of five experiments conducted . ( E ) Percent CD32a downmodulation on isolated pDCs treated with 10 μg / ml of an anti - CD40 mAb . Error bars represent SD of percent downmodulation of two independent experiments . FMO represents background staining of CD32a ( inverted triangle ) . ( F ) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi - 6G6 . Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells . Shown is a representative plot of five independent experiments conducted .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.


Figure 1 ( A ) mH2A1 . 2 and different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , and P0 ) . ( C ) A scatter diagram shows the change trend of mH2A1 . 2 and different neurogenesis markers . n = 5 mice , independent replicates . ( E ) Electroporation of mH2A1 . 2 - shRNA results in abnormal cell distribution in the developing neocortex . Electroporation was performed at E13 . 5 , and the mouse was harvested at E16 . 5 . Scale bar represents 50 μm . ( F ) Graph shows the percentage of GFP - positive cells distributed in the VZ / SVZ , IZ and CP . n = 7 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. (F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.


Figure 2 ( D ) Abnormal cell distribution was observed in the mH2A1 . 2 silenced neocortex . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( E ) Graphs of the percentage of GFP - positive cells in the VZ / SVZ , IZ and CP . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . ( F ) Brain sections of WT and KO mice at E16 . 5 were immunostained with the mitotic marker pH3 and DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + cells of the cortex . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . ( H ) Representative images of E16 . 5 coronal brain sections were immunostained for PAX6 . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) Quantification of GFP and Pax6 double positive cells in the VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(D)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


Figure 3 ( A ) Representative images of E16 . 5 coronal brain sections immune stained for Tbr2 and BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of pulse labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + cells per 100μm2 surface of VZ / SVZ . The number of TBR2 + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + cells per 100μm2 surface of VZ / SVZ . The number of BrdU + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant . ( E ) Cell cycle exit was decreased in KO mice . E16 . 5 brain sections were stained with anti - BrdU and anti - Ki67 in WT mice and KO mice that were administered BrdU ( 100 mg / kg ) for 24 h and harvested at E16 . 5 . Scale bar represents 20 μm . ( F ) Percentage of cell cycle exit ( BrdU + Ki67 − / BrdU + ) in WT mice and KO mice . n = 7 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant . ( G ) Western blot analysis showed that the protein expression levels of neural stem cell marker , including TBR2 , PCNA , and PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of the normalized density of TBR2 , PCNA , and PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant . ( K ) Coronal brain slices of E16 . 5 WT mice and KO mice were immunostained using anti - TUJ1 . Scale bar represents 20 μm . ( L ) Relative TUJ1 + cells in the VZ of WT mice and KO mice . TUJ1 was reduced in cortical . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant . ( M ) The apoptosis levels between WT and KO brains showed no significant difference . E16 . 5 brain sections of WT and KO mice were subjected to TUNEL assay . Scale bar represents 50 μm . ( N ) Graph shows the numbers of apoptotic cells per field . n = 4mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT. (H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm. (L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm. (N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


Figure 4 ( A ) Representative images of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into the E13 . 5 cerebral cortices of WT and KO mice to label neural progenitor cells . After 24 h , the GFP + cells were isolated and cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that the total dendritic length of neurons is decreased when mH2A1 . 2 is deleted . n = 25 cells from four samples . ( C ) Statistics show that the number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 cells from four samples . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . ( F ) The brain sections were immunostained with CUX1 and CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + cells in the upper layer . CUX1 + cells were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + cells in the deep layer . CTIP2 + cells were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 (

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Figure 4(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


Figure 5 ( A ) Representative tracing path of WT mice and KO mice in the open field test . ( B ) The total traveling distance was not different between WT and KO mice . ( C ) KO mice spent less time in the center than WT mice . Date information : All mice were male , 8 - 12 weeks old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . Analysis was performed by researcher blinded to the experimental conditions .

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Figure 5(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.


Figure 6 ( E ) Graph shows the percentage of GFP + cells in the VZ / SVZ , IZ and CP . The number of GFP + cells in the VZ / SVZ region increased significantly , and the number of GFP + in the CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant . ( I ) Representative images of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT and KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of the percentage of GFP + cells in the VZ / SVZ , IZ and CP . Overexpression of NKX2 - 2 restored the distribution of GFP + cells to the normal state . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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Figure 6(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


Figure 1J , Ridge graph comparisons of cyclin expression across the U5 - hNSC defined cell cycle phases . The x - axis is relative expression of the cyclin , and the y - axis is counts of cells per phase . The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot .

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Figure 1J, Ridge graph comparisons of cyclin expression across the U5-hNSC defined cell cycle phases. The x-axis is relative expression of the cyclin, and the y-axis is counts of cells per phase. The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot.


Figure 4C , Number of putative stem - like cells in each ccAF cell cycle phase , and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem - like cells in Bhaduri et al . , 2020 . D , Putative stem - like cells and expression of the genes used to identify the putative stem - like cells projected onto Neftel et al . , 2019 . Expression of genes are overlaid as the color of each cell ( white = 0 , dark blue = max expression ) . Quadrants are broken up by dashed grey lines : OPC = upper left ; NPC = upper right ; AC = lower left ; and MES = lower right . Percentages of gene expression greater than 0 are shown in the corners of the plot .

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Figure 4C, Number of putative stem-like cells in each ccAF cell cycle phase, and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem-like cells in Bhaduri et al., 2020.D, Putative stem-like cells and expression of the genes used to identify the putative stem-like cells projected onto Neftel et al., 2019. Expression of genes are overlaid as the color of each cell (white = 0, dark blue = max expression). Quadrants are broken up by dashed grey lines: OPC = upper left; NPC = upper right; AC = lower left; and MES = lower right. Percentages of gene expression greater than 0 are shown in the corners of the plot.


Figure 5C , Violin plot showing PLP1 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of PLP1 in single cells is much higher in oligodendrocytes ( Oligo ) than in Neural G0 or oligodendrocyte progenitor cells ( OPC ) . D , Violin plot showing GFAP expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types . E , Violin plot showing AQP4 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit .

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Figure 5C, Violin plot showing PLP1 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of PLP1 in single cells is much higher in oligodendrocytes (Oligo) than in Neural G0 or oligodendrocyte progenitor cells (OPC). D, Violin plot showing GFAP expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types. E, Violin plot showing AQP4 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit.


Figure 6B , Representative contour maps of flow cytometry for FUCCI following the loss of NF2 , PTPN14 , TAOK1 , CREBBP , and TP53 . D , G0 / G1 and S / G2 / M transit times using time - lapse microscopy and FUCCI . Differences in G0 / G1 are statistically significant with p < 0 . 001 for targeted U5 - NSCs and p = 0 . 0006 for GSC - 131 compared to NTC .

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Figure 6B, Representative contour maps of flow cytometry for FUCCI following the loss of NF2, PTPN14, TAOK1, CREBBP, and TP53.D, G0/G1 and S/G2/M transit times using time-lapse microscopy and FUCCI. Differences in G0/G1 are statistically significant with p<0.001 for targeted U5-NSCs and p=0.0006 for GSC-131 compared to NTC.


Figure 2 ( A ) After 4 h of starvation in 1 % K ‐ acetate medium in the presence of PMSF , autophagic vesicles can be visualized in vacuoles of wild ‐ type and pep4prb1 cells using Normaski optics . In aut2Δ and aut7Δ cells no autophagic vesicles can be seen . Bar represents 20 μm . ( B ) Total protein breakdown of cells starved on nitrogen ‐ free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild ‐ type cells . pep4Δ ( pra1Δ ) cells are almost completely impaired in starvation ‐ induced vacuolar protein breakdown . Cells were pulse ‐ labeled with [ 35S ] methionine and chased on non ‐ radioactive starvation medium . Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al . ( 1997 ) . ( C ) Survival during starvation . Like pep4Δ ( pra1Δ ) cells , which are almost completely impaired in vacuolar proteolysis , aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen ‐ free medium as compared with wild ‐ type cells . Aliquots of cells incubated in 1 % K ‐ acetate were plated and growing colonies were determined according to Straub et al . ( 1997 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).


Figure 3Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) and aut7Δ ( C ) cells . Further details are outlined in the text . Crude extracts were prepared from cells starved 4 h on 1 % K ‐ acetate . Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) and aut7Δ ( C ) cells . Further details are outlined in the text . Crude extracts were prepared from cells starved 4 h on 1 % K ‐ acetate . Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) and aut7Δ ( C ) cells . Further details are outlined in the text . Crude extracts were prepared from cells starved 4 h on 1 % K ‐ acetate .

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Figure 3Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.


Figure 5 ( A ) Detection of protein - protein interactions using the two ‐ hybrid system . Intense staining indicates an interaction between Aut2p and Aut7p , Aut2p and Tub1p , and Aut2p and Tub2p . AUT7 , TUB1 and TUB2 are cloned in ‐ frame with the GAL4 ‐ activator domain . AUT2 and AUT7 were fused to the GAL4 ‐ binding domain ( see Materials and methods ) . ( B ) Affinity chromatography confirms protein - protein interaction between Aut2p and Aut7p . A fusion protein of Aut2p with the 26 kDa glutathione ‐ S ‐ transferase ( GST ) domain from Schistosoma japonicum ( see Materials and methods ) was bound to glutathione - Sepharose 4B . This GST - Aut2p column was incubated with a crude extract from aut7Δ cen Met25 : : AUT7 - GFP cells . The non ‐ bound supernatant ( S ) fraction ( lane 1 ) was taken away . Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non ‐ specifically bound proteins . After the final 500 mM NaCl wash ( W , lane 2 ) , bound proteins were eluted with Laemmli buffer ( E , lane 3 ) . Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP . Using Aut2p - GST , a clear band corresponding to Aut7p - GFP is seen in the eluate fraction ( lane 3 , arrowhead ) . As a control , GST alone was attached to the column and no band was detectable ( lane 6 ) . ( C ) As described in ( B ) , bovine tubulin binds to GST - Aut2p glutathione - Sepharose 4B ( lane 12 ) , but not to GST glutathione - Sepharose 4B ( lane 9 ) . Protein staining with Coomassie is shown in lanes 1 - 6 ; in lanes 7 - 12 tubulin was detected using monoclonal antibodies ( Boehringer Mannheim ) . Further details are given in Materials and methods . ( D ) Similar to ( C ) , bovine tubulin does not bind to GST - Aut7p glutathione - Sepharose 4B ( lane 12 ) and to GST glutathione - Sepharose 4B ( lane 9 ) . Lanes 1 - 6 , protein staining with Coomassie ; lanes 7 - 12 probed with antibodies directed against tubulin .

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Figure 5(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods).(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).(C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods.(D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.


Figure 6Benomyl sensitivity of wild ‐ type cells overexpressing AUT2 and AUT7 , and of aut2Δ and aut7Δ cells is not altered . Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg / ml benomyl . Plates were incubated at 30 ° C .

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Figure 6Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.


Figure 7 ( A ) Autophagosome ‐ like vesicles ( arrowhead ) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1 % K ‐ acetate in the presence of PMSF . Bar represents 100 nm . ( B ) Electron micrographs unravels the accumulation of double membrane ‐ layered autophagosome ‐ like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen ‐ free medium in the presence of PMSF . Arrowheads indicate the clearly distinguishable two membrane layers . Bar represents 100 nm . ( C and D ) Cytosol containing autophagosome ‐ like vesicles accumulating in the cytosol of aut2Δ cells ( D ) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction . In wild ‐ type cells ( C ) , no P100 is detectable . Fatty acid synthase ( Fas ) was used as a cytosolic protein . Further details are given in the text .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.(B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.


Figure 1Human placental syncytiotrophoblast cells express markers of cellular senescence , p16 Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - staining for senescence regulators ( A ) p16 Green label : the syncytiotrophoblast marker βHCG , red label : p16 Blue label : DAPI nuclear stain . Arrowheads indicate positively stained syncytiotrophoblast cells βHCG + / p16 + ( A ) Scale bar , 50 µm . Human placental syncytiotrophoblast cells express markers of cellular senescence , p21 . Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - staining for senescence regulators ( B ) p21 . Green label : the syncytiotrophoblast marker βHCG , red label : p21 . Blue label : DAPI nuclear stain . Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG + / p21 + ( B ) . Scale bar , 50 µm . ( C - F ) Sections of human normal third - trimester placentas post - partum ( n = 4 normal placentas ) and IUGR - complicated placentas of the same gestational age ( n = 4 IUGR placentas ) were stained for senescence - related markers of p15 ( C ) , p16 ( D ) , p21 ( E ) , and p53 ( F ) in the syncytiotrophoblast ( n = 3 sections , derived from each normal and IUGR placenta ) . Percentages of positively stained syncytia were quantified ( n = at least 12 fields of view from all sections ) . Scale bar , 100 µm . Arrowheads indicate syncytiotrophoblast cells . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 . ( G ) Immunoblot analysis of the protein content of p16 , p21 , p53 and DCR in normal ( n = 4 placentas ) and in IUGR - complicated placentas ( n = 4 placentas ) . Each lane represents one independent placenta . ( H ) Quantitative RT - PCR analysis of p16 , p21 and DCR2 expression in IUGR - complicated and in normal placentas . Results were obtained from four human normal and four IUGR placentas . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 Green label: the syncytiotrophoblast marker βHCG, red label: p16 Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A) Scale bar, 50 µm.Human placental syncytiotrophoblast cells express markers of cellular senescence, p21. Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (B) p21. Green label: the syncytiotrophoblast marker βHCG, red label: p21. Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG+/p21+ (B). Scale bar, 50 µm.(C-F) Sections of human normal third-trimester placentas post-partum (n=4 normal placentas) and IUGR-complicated placentas of the same gestational age (n=4 IUGR placentas) were stained for senescence-related markers of p15 (C), p16 (D), p21 (E), and p53 (F) in the syncytiotrophoblast (n=3 sections, derived from each normal and IUGR placenta). Percentages of positively stained syncytia were quantified (n= at least 12 fields of view from all sections). Scale bar, 100 µm. Arrowheads indicate syncytiotrophoblast cells. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.(G) Immunoblot analysis of the protein content of p16, p21, p53 and DCR in normal (n=4 placentas) and in IUGR-complicated placentas (n=4 placentas). Each lane represents one independent placenta.(H) Quantitative RT-PCR analysis of p16, p21 and DCR2 expression in IUGR-complicated and in normal placentas. Results were obtained from four human normal and four IUGR placentas. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


Figure 2DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( A ) Representative T1 - weighted image of a pregnant WT mouse and a selection of four different placentas ( outlined by colored lines ) . DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( B ) Representative SI dynamics of a single WT placenta ( marked in A ) , following biotin - BSA - GdDTPA administered for 60 min . SI parameters of Initial Enhancement and Recovery are illustrated . DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( C ) Representative SI dynamics of a single WT placenta ( marked in A ) over time . Scale bar , 2 . 5 mm . DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( D − G ) Representative SI plots of Cdkn1a - / - ( D ) , p53 - / - ( E ) , Cdkn2a - / - ( F ) , and Cdkn2a - / - ; p53 - / - ( G ) placentas , compared to their WT or HET littermates . ( H − I ) Quantification of the SI Initial Enhancement ( H ) and SI Recovery ( I ) in WT , Cdkn1a - / - , p53 - / - , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas . MRI experiments were repeated at least three times for each murine genotype ( n > = 3 placental SI measurements from each genotype ) . Values are means + SEM ; Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines). DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated.DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm.DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates. (H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


Figure 3Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , and Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( A − E ) The labyrinth zone of the WT ( A ) differs from those of Cdkn2a - / - ( B ) and Cdkn2a - / - ; p53 - / - ( C ) placentas . Black arrows indicate regular - sized nuclei in the WT placenta and polyploidy in the Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas . Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas . Labyrinths of p53 - / - ( D ) and Cdkn1a - / - ( E ) placentas did not differ from that of the WT placenta . L , labyrinth . Staining for each placenta was performed in triplicates . Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , and Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( F − G ) Ki67 immunostaining in the labyrinth zones of murine WT , p53 - / - , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas , on day E14 . 5 ( n = 3 placentas from each genotype ) . ( F ) Representative images of Ki67 staining in the labyrinth zone are shown . Positive Ki67 trophoblast cells are indicated with arrows . L , labyrinth . ( G ) Proliferation scores ( n = 3 scores , derived from three placentas of each genotype ) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas relative to WT . Values are means + SEM of three scores for each genotype . * P < 0 . 05 by a one - tailed unpaired Student ' s t - test . Data information : Scale bars , 50 µm . Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( H ) Representative images of SA - β - gal staining in the labyrinth zones of WT , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas on day E14 . 5 . ( I ) SA - β - gal activity , quantified by ImageJ sofware in Cdkn2a - / - and Cdkn2a - / - ; p53 - / - labyrinths , is significantly reduced relative to those in WT labyrinth ( n > = 7 fields of view from each genotype ) . Values are means + SEM . ( * * * ) p < 0 . 001 by a two - tailed unpaired Student ' s t - test . Data information : Scale bars , 50 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (A−E) The labyrinth zone of the WT (A) differs from those of Cdkn2a-/- (B) and Cdkn2a-/-;p53-/- (C) placentas. Black arrows indicate regular-sized nuclei in the WT placenta and polyploidy in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Labyrinths of p53-/- (D) and Cdkn1a-/- (E) placentas did not differ from that of the WT placenta. L, labyrinth. Staining for each placenta was performed in triplicates. Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (F−G) Ki67 immunostaining in the labyrinth zones of murine WT , p53-/- ,Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas, on day E14.5 (n=3 placentas from each genotype). (F) Representative images of Ki67 staining in the labyrinth zone are shown. Positive Ki67 trophoblast cells are indicated with arrows. L, labyrinth. (G) Proliferation scores (n=3 scores, derived from three placentas of each genotype) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas relative to WT. Values are means +SEM of three scores for each genotype. *P < 0.05 by a one-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5) placentas on day E14.5, compared to WT placentas (n=6). (H) Representative images of SA-β-gal staining in the labyrinth zones of WT, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas on day E14.5. (I) SA-β-gal activity, quantified by ImageJ sofware in Cdkn2a-/- and Cdkn2a-/-;p53-/- labyrinths, is significantly reduced relative to those in WT labyrinth (n>=7 fields of view from each genotype). Values are means + SEM. (***) p < 0.001 by a two-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.


Figure 4Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . On days 1 and 5 after seeding , cell fusion was monitored by staining for phalloidin ( F - actin ) ( green ) and DAPI ( blue ) . Scale bar , 50 μm . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( B ) Trophoblast cell fusion was quantified on days 1 , 3 , and 5 post - seeding . Values are means + SEM , derived independently from human placentas of three pregnancies . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( C ) Quantification of the human βhCG hormone , normally secreted by functional syncytiotrophoblast cells , in the medium of a representative trophoblast culture on post - delivery days 2 , 3 and 5 . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( D ) Gene set enrichment analysis ( GSEA ) of published datasets from human primary trophoblast cultures on days 5 and 2 ( n = 4 trophoblast culture of day 5 and day 2 ) . Gene sets related to pregnancy , aging , immune response , cytokine activity , MAPK , JAK - STAT , NF - κB and metalloproteinase activity were found to be enriched in day 5 cultures , whereas on the same day gene sets related to cell cycle , E2F targets , mitosis and DNA replication were downregulated . Statistical significance was determined by permutation testing with normalized enrichment score ( NES ) Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the cell cycle - related genes p16 , p21 and CCNE1 Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the syncytiotrophoblast markers GCM1 , CGA and CGB ( F ) . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( G ) Immunoblot analysis of the indicated proteins in human trophoblast cells , derived independently from two placentas , on days 2 and 5 . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the SASP components CCL2 , CCL5 , CCL8 , IL6 , IL1β , TGF - β and PDG - FC ( H ) in human primary trophoblast cultures on days 5 and 2 . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 . Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the metalloproteases MMP2 , MMP3 and MMP9 ( I ) , in human primary trophoblast cultures on days 5 and 2 . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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Figure 4Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (B) Trophoblast cell fusion was quantified on days 1, 3, and 5 post-seeding. Values are means + SEM, derived independently from human placentas of three pregnancies.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (C) Quantification of the human βhCG hormone, normally secreted by functional syncytiotrophoblast cells, in the medium of a representative trophoblast culture on post-delivery days 2, 3 and 5.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (D) Gene set enrichment analysis (GSEA) of published datasets from human primary trophoblast cultures on days 5 and 2 (n=4 trophoblast culture of day 5 and day 2). Gene sets related to pregnancy, aging, immune response, cytokine activity, MAPK, JAK-STAT, NF-κB and metalloproteinase activity were found to be enriched in day 5 cultures, whereas on the same day gene sets related to cell cycle, E2F targets, mitosis and DNA replication were downregulated. Statistical significance was determined by permutation testing with normalized enrichment score (NES)Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the cell cycle-related genes p16, p21 and CCNE1 Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the syncytiotrophoblast markers GCM1, CGA and CGB (F). Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (G) Immunoblot analysis of the indicated proteins in human trophoblast cells, derived independently from two placentas, on days 2 and 5.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the SASP components CCL2, CCL5, CCL8, IL6, IL1β, TGF-β and PDG-FC (H) in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the metalloproteases MMP2, MMP3 and MMP9 (I), in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.