Figure 2 ( A - C ) 3D rendering of Z - stack confocal image showing Isolectin B4 - stained laser - burn with FITC - dextran - labeled CNV and IBA - 1 - stained MPs . ( D ) Representative FACS plots of retinas and sclera - choroid - RPE - cell complexes from Regular - chow Diet ( RD ) - fed mice without and 3 , 7 and 14 days after laser - burn . ( E ) Quantification of MPs ( Ly6G - , F4 / 80 + , CD11b + ) at p3 , p7 and p14 ; fold naïve ( no burn ) ; n = 5 ( no burn ) , 5 ( p3 ) , 6 ( p7 ) , 5 ( p14 ) ; CI 95 % . ( F ) Representative FACS plots and ( G ) quantification of microglia ( Ly6G - , F4 / 80 + , CD11b + , CX3CR1hiCD45lo ) fold naïve ; n = 5 ( no burn ) , 4 ( p3 ) , 6 ( p7 ) , 5 ( p14 ) ; CI 95 % . ( H ) Representative FACS plots of retinas and sclera - choroid - RPE - cell complexes from RD - and HFD - fed mice , with vehicle or antibiotic treatment at p7 . ( I ) Quantification of MPs at p7 fold RD ; n = 6 ( RD ) , 4 ( HFD ) , 3 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % . ( J ) Representative FACS plots and ( K ) quantification of microglia ( Ly6G - , F4 / 80 + , CD11b + , CX3CR1hiCD45lo ) fold RD ; n = 5 ( RD ) , 5 ( HFD ) , 3 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % . ( L ) Representative confocal images of IBA - 1 - stained MPs on choroidal flat mounts from RD - and HFD - fed mice , treated with vehicle or with neomycin . Examples of labeled macrophages ( white dots ) are presented in side - panels . ( M ) Total number of MPs around laser impact area ; n = 7 ( RD ) , 9 ( HFD ) , 4 ( HFD + AB ) , 7 ( RD + AB ) , with 23 ( RD ) , 29 ( HFD ) , 13 ( HFD + AB ) , 23 ( RD + AB ) burns total ; CI 99 % .

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 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 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 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

Figure 2(A-C) 3D rendering of Z-stack confocal image showing Isolectin B4-stained laser-burn with FITC-dextran-labeled CNV and IBA-1-stained MPs.(D) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from Regular-chow Diet (RD)-fed mice without and 3, 7 and 14 days after laser-burn. (E) Quantification of MPs (Ly6G -, F4/80+, CD11b+) at p3, p7 and p14; fold naïve (no burn); n= 5(no burn), 5(p3), 6(p7), 5(p14); CI 95%. (F) Representative FACS plots and (G) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold naïve; n= 5(no burn), 4(p3), 6(p7), 5(p14); CI 95%.(H) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from RD- and HFD-fed mice, with vehicle or antibiotic treatment at p7. (I) Quantification of MPs at p7 fold RD; n= 6(RD), 4(HFD), 3(HFD+AB), 4(RD+AB); CI 95%. (J) Representative FACS plots and (K) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold RD; n= 5(RD), 5(HFD), 3(HFD+AB), 4(RD+AB); CI 95%.(L) Representative confocal images of IBA-1-stained MPs on choroidal flat mounts from RD- and HFD-fed mice, treated with vehicle or with neomycin. Examples of labeled macrophages (white dots) are presented in side-panels. (M) Total number of MPs around laser impact area; n= 7(RD), 9(HFD), 4(HFD+AB), 7(RD+AB), with 23(RD), 29(HFD), 13(HFD+AB), 23(RD+AB) burns total; CI 99%.


Figure 3 ( A ) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular - chow Diet ( RD ) - and high - fat diet ( HFD ) - fed mice , treated with vehicle or with antibiotic treatment ; n = 6 ( RD ) , 5 ( HFD ) , 5 ( HFD + AB ) , 5 ( RD + AB ) ; CI 99 % . ( B ) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks ; fold RD ; n = 3 for all groups ; CI 99 % . ( C ) Serum cytokine profiles determined by Bio - Plex assay ( Bio - Rad ) of IL - 1β , n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) ; CI 95 % ; IL - 6 , n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) , CI 99 . 9 % ; TNF - α n = 11 for all groups , CI 99 % ; IL - 17A n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) ; CI 95 % ; and IFN - γ ; n = 12 for all groups ; CI 95 % . ( D ) mRNA expression of Il - 1β ; n = 5 ( RD ) , 7 ( HFD ) , 7 ( HFD + AB ) , 3 ( RD + AB ) ; CI 95 % , Il - 6 ; n = 5 ( RD ) , 5 ( HFD ) , 4 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % , Tnf - α ; n = 6 ( RD ) , 9 ( HFD ) , 4 ( HFD + AB ) , 5 ( RD + AB ) ; CI 95 % and Vegf - a ; n = 6 ( RD ) , 5 ( HFD ) , 5 ( HFD + AB ) , 6 ( RD + AB ) ; CI 95 % in choroids .

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 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 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

Figure 3(A) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular-chow Diet (RD)- and high-fat diet (HFD)-fed mice, treated with vehicle or with antibiotic treatment; n= 6(RD), 5(HFD), 5(HFD+AB), 5(RD+AB); CI 99%.(B) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks; fold RD; n=3 for all groups; CI 99%.(C) Serum cytokine profiles determined by Bio-Plex assay (Bio-Rad) of IL-1β, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; IL-6, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB), CI 99.9%; TNF-α n=11 for all groups, CI 99%; IL-17A n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; and IFN-γ; n= 12 for all groups; CI 95%.(D) mRNA expression of Il-1β; n= 5(RD), 7(HFD), 7(HFD+AB), 3(RD+AB); CI 95%, Il-6; n= 5(RD), 5(HFD), 4(HFD+AB), 4(RD+AB); CI 95%, Tnf-α; n= 6(RD), 9(HFD), 4(HFD+AB), 5(RD+AB); CI 95% and Vegf-a; n= 6(RD), 5(HFD), 5(HFD+AB), 6(RD+AB); CI 95% in choroids.


Figure 4 ( C ) Weight gain and ( D ) area under the curve of percentage weight gain of RD x RDT , HFD x RDT and HFD x HFDT mice ; n = 13 ( RDxRDT ) , 12 ( HFDxHFDT ) , 14 ( HFDxRDT ) ; CI 99 % . ( E ) Representative circle charts of relative abundance of bacterial phyla in gut microbiota of RD - fed mice receiving a RD transfer and HFD - fed mice receiving a HFD or RD transfer ( F ) relative proportion per experimental group of different phyla ; n = 5 ( RDxRDT ) , 5 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; Bacteroidetes ; CI 99 % , Firmicutes ; CI 95 % , Proteobacteria ; CI 95 % , Actinobacteria and Spirochaetes . ( G ) Compilation of compressed Z - stack confocal images of FITC - dextran - labeled CNV and single - plane confocal images of isolectin B4 - stained choroidal flat mounts from RD x RDT , HFD x RDT and HFD x HFDT mice . ( H ) Quantification of area of FITC - dextran - labeled CNV over area of isolectin B4 - stained laser impact area fold RD x RDT ; n = 6 ( RDxRDT ) , 9 ( HFDxHFDT ) , 9 ( HFDxRDT ) , with 15 ( RDxRDT ) , 21 ( HFDxHFDT ) , 16 ( HFDxRDT ) burns total ; CI 95 % . ( I ) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in RD - and HFD - fed mice , after RD or HFD microbiotal transplant ; n = 3 ( RDxRDT ) , 3 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 95 % . ( J ) mRNA expression of Il - 1β ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 5 ( HFDxRDT ) , Il - 6 ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 99 % , Tnf - α ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 95 % and Vegf - a ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 5 ( HFDxRDT ) ; CI 95 % in choroids .

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 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 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 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 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

Figure 4(C) Weight gain and (D) area under the curve of percentage weight gain of RD x RDT, HFD x RDT and HFD x HFDT mice; n= 13(RDxRDT), 12(HFDxHFDT), 14(HFDxRDT); CI 99%.(E) Representative circle charts of relative abundance of bacterial phyla in gut microbiota of RD-fed mice receiving a RD transfer and HFD-fed mice receiving a HFD or RD transfer(F) relative proportion per experimental group of different phyla; n= 5(RDxRDT), 5(HFDxHFDT), 4(HFDxRDT); Bacteroidetes; CI 99%, Firmicutes; CI 95%, Proteobacteria; CI 95%, Actinobacteria and Spirochaetes.(G) Compilation of compressed Z-stack confocal images of FITC-dextran-labeled CNV and single-plane confocal images of isolectin B4-stained choroidal flat mounts from RD x RDT, HFD x RDT and HFD x HFDT mice. (H) Quantification of area of FITC-dextran-labeled CNV over area of isolectin B4-stained laser impact area fold RD x RDT; n= 6(RDxRDT), 9(HFDxHFDT), 9(HFDxRDT), with 15(RDxRDT), 21(HFDxHFDT), 16(HFDxRDT) burns total; CI 95%.(I) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in RD- and HFD-fed mice, after RD or HFD microbiotal transplant; n= 3(RDxRDT), 3(HFDxHFDT), 4(HFDxRDT); CI 95%.(J) mRNA expression of Il-1β; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT), Il-6; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 99%, Tnf-α; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 95% and Vegf-a; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT); CI 95% in choroids.


Figure 1B Social interaction ratio in control , susceptible and resilient mice following the CSDS protocol ( n = 44 , 55 , 44 mice ) . C Sucrose preference in control , susceptible and resilient mice ( n = 20 , 20 , 12 mice . Treatment F2 , 49 = 16 . 05 , P < 0 . 0001 , one - way ANOVA with Fisher ' s LSD test ) . D The immobility time in TST in susceptible mice , compared to control mice ( n = 16 , 32 mice . Susceptible vs control , P = 0 . 002 , Student ' s t test ) . E The immobility time in FST in susceptible mice , compared to control mice ( n = 17 , 35 mice . Susceptible vs control , P = 0 . 0003 , Student ' s t test ) . F , G The time in the open arms ( F ) and closed arms ( G ) in the EPM in susceptible mice , compared to control mice ( n = 19 , 33 mice . Susceptible vs control , for open arms , P < 0 . 0001 ; For closed time , P < 0 . 0001 , Student ' s t test ) . H The central distance in the OFT in susceptible mice , compared to control mice ( n = 13 , 28 mice . Susceptible vs control , P = 0 . 0089 , Student ' s t test ) . I The mRNA level of CD39 , ENTPD2 and ENTPD3 in the hippocampus of susceptible mice after CSDS ( n = 7 , 8 mice . CSDS vs control , CD39 P = 0 . 0002 , ENTPD2 P = 0 . 0025 , Student ' s t test ) . J The CD39 protein expression in the hippocampus of susceptible mice induced by CSDS ( n = 6 , 7 mice . CSDS vs control , P = 0 . 0003 , Student ' s t test ) . K The ATPase activity in the hippocampus of stressed animals ( n = 6 mice / group . CSDS vs control , P = 0 . 0041 , Student ' s t test ) .

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 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 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 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 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 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 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

Figure 1B Social interaction ratio in control, susceptible and resilient mice following the CSDS protocol (n = 44, 55, 44 mice).C Sucrose preference in control, susceptible and resilient mice (n = 20, 20, 12 mice. Treatment F2, 49 = 16.05, P < 0.0001, one-way ANOVA with Fisher's LSD test).D The immobility time in TST in susceptible mice, compared to control mice (n = 16, 32 mice. Susceptible vs control, P = 0.002, Student's t test).E The immobility time in FST in susceptible mice, compared to control mice (n = 17, 35 mice. Susceptible vs control, P = 0.0003, Student's t test).F, G The time in the open arms (F) and closed arms (G) in the EPM in susceptible mice, compared to control mice (n = 19, 33 mice. Susceptible vs control, for open arms, P < 0.0001; For closed time, P < 0.0001, Student's t test).H The central distance in the OFT in susceptible mice, compared to control mice (n = 13, 28 mice. Susceptible vs control, P = 0.0089, Student's t test).I The mRNA level of CD39, ENTPD2 and ENTPD3 in the hippocampus of susceptible mice after CSDS (n = 7, 8 mice. CSDS vs control, CD39 P = 0.0002, ENTPD2 P = 0.0025, Student's t test).J The CD39 protein expression in the hippocampus of susceptible mice induced by CSDS (n = 6, 7 mice. CSDS vs control, P = 0.0003, Student's t test).K The ATPase activity in the hippocampus of stressed animals (n = 6 mice/group. CSDS vs control, P =0.0041, Student's t test).


Figure 2B The effects of apyrase ( 40 , 80 and 160 U / ml ) infusion into cerebral ventricle on social interaction time ( n = 8 , 10 , 10 , 10 mice . Interaction F3 , 34 = 3 . 917 , P = 0 . 0167 ; Target F1 , 34 = 0 . 0004 , P = 0 . 9834 ; Drug F3 , 34 = 2 . 295 , P = 0 . 0954 ; For target , Apyrase 80 U / ml vs control , P = 0 . 0008 , Two - way ANOVA with Tukey ' s post - test ) . C The effects of apyrase ( 40 , 80 and 160 U / ml ) infusion into cerebral ventricle on sucrose preference ( n = 8 , 10 , 9 , 11 mice . Treatment F3 , 34 = 2 . 87 , P = 0 . 0505 ; Apyrase 80 U / ml vs control , P = 0 . 007 , one - way ANOVA with Fisher ' s LSD test ) . E The effect of apyrase ( 40 U / ml ) infusion into hippocampal on the social interaction time ( n = 14 , 20 mice . Interaction F1 , 32 = 7 . 0754 , P = 0 . 0121 ; Target F1 , 32 = 0 . 68447 , P = 0 . 4142 ; Drug F1 , 32 = 18 . 211 , P = 0 . 0002 ; Apyrase vs control , P < 0 . 0001 , Student ' s t test ) . F The effect of apyrase ( 40 U / ml ) infusion into hippocampal on the sucrose preference ( n = 10 , 16 mice . Apyrase vs control , P = 0 . 0003 , Student ' s t test ) . G The social avoidance behavior in mice with ATP intra - hippocampal infusion after apyrase exposure ( n = 14 , 10 , 10 mice . Interaction F2 , 31 = 2 . 051 , P = 0 . 1457 ; Target F1 , 31 = 2 . 666 , P = 0 . 1127 ; Drug F2 , 31 = 3 . 711 , P = 0 . 0359 ; For Target , Apyrase - ACSF vs Vehicle , P = 0 . 0387 ; Apyrase - ATP vs Apyrase - ACSF , P = 0 . 0099 , Two - way ANOVA with Tukey ' s post - test ) . H The sucrose preference in mice with ATP intra - hippocampal infusion after apyrase exposure ( n = 10 , 8 , 8 mice . Treatment F2 , 23 = 3 . 32 , P = 0 . 0540 ; Apyrase - ACSF vs Vehicle , P = 0 . 0466 , one - way ANOVA with Fisher ' s LSD test ) .

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 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 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 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 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

Figure 2B The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on social interaction time (n = 8, 10, 10, 10 mice. Interaction F3, 34 = 3.917, P = 0.0167; Target F1, 34 = 0.0004, P = 0.9834; Drug F3, 34 = 2.295, P = 0.0954; For target, Apyrase 80 U/ml vs control, P = 0.0008, Two-way ANOVA with Tukey's post-test).C The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on sucrose preference (n = 8, 10, 9, 11 mice. Treatment F3, 34 = 2.87, P = 0.0505; Apyrase 80 U/ml vs control, P = 0.007, one-way ANOVA with Fisher's LSD test).E The effect of apyrase (40 U/ml) infusion into hippocampal on the social interaction time (n = 14, 20 mice. Interaction F1, 32 = 7.0754, P = 0.0121; Target F1, 32 = 0.68447, P = 0.4142; Drug F1, 32 = 18.211, P = 0.0002; Apyrase vs control, P < 0.0001, Student's t test).F The effect of apyrase (40 U/ml) infusion into hippocampal on the sucrose preference (n = 10, 16 mice. Apyrase vs control, P = 0.0003, Student's t test).G The social avoidance behavior in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 14, 10, 10 mice. Interaction F2, 31 = 2.051, P = 0.1457; Target F1, 31 = 2.666, P = 0.1127; Drug F2, 31 = 3.711, P = 0.0359; For Target, Apyrase-ACSF vs Vehicle, P = 0.0387; Apyrase-ATP vs Apyrase-ACSF, P = 0.0099, Two-way ANOVA with Tukey's post-test).H The sucrose preference in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 10, 8, 8 mice. Treatment F2, 23 = 3.32, P = 0.0540; Apyrase-ACSF vs Vehicle, P =0.0466, one-way ANOVA with Fisher's LSD test).


Figure 3B The ATPase activity of mice exposure to ARL67156 ( n = 7 , 7 , 5 , 5 mice . Treatment F3 , 20 = 16 . 81 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P < 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0405 . One - way ANOVA with Fisher ' s LSD test ) . C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 ( n = 12 , 10 , 9 , 9 mice . Interaction F3 , 36 = 5 . 119 , P = 0 . 0047 ; Target F1 , 36 = 0 . 005 , P = 0 . 9437 ; Drug F3 , 36 = 3 . 626 , P = 0 . 022 . For Target , CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0054 . Two - way ANOVA with Tukey ' s post - test ) . D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 ( n = 12 , 10 , 10 , 10 mice . Interaction F1 , 38 = 5 . 352 , P = 0 . 0262 ; Group F1 , 38 = 5 . 711 , P = 0 . 0219 ; Drug F1 , 38 = 9 . 703 , P = 0 . 0035 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0083 ; Ctrl - ARL67156 vs CSDS - ACSF , P = 0 . 0027 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0031 . Two - way ANOVA with Tukey ' s post - test ) . The social avoidance behavior ( E ) in mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 9 , 9 , 9 , 8 mice . For social behavior , Interaction F3 , 31 = 5 . 579 , P = 0 . 0035 ; Target F1 , 31 = 0 . 5121 , P = 0 . 4796 ; Drug F3 , 31 = 8 . 367 , P = 0 . 0003 ; CSDS - ACSF vs Ctrl - ACSF , P < 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0131 . sucrose preference ( F ) in mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 9 , 9 , 9 , 8 mice . Sucrose , Interaction F1 , 30 = 0 . 3342 , P = 0 . 5675 ; Group F1 , 30 = 17 . 22 , P = 0 . 0003 ; Drug F1 , 30 = 0 . 05903 , P = 0 . 8097 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0761 . Two - way ANOVA with Tukey ' s post - test ) . G The immobility time in the TST of mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 16 , 12 , 21 , 19 mice . Treatment F 3 , 64 = 13 . 61 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0059 ; Ctrl - ARL67156 vs Ctrl - ACSF , P = 0 . 0011 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0002 . One - way ANOVA with Fisher ' s LSD test ) . H The immobility time in the FST with intra - hippocampal infusion of ARL67156 after CSDS ( n = 21 , 12 , 23 , 19 mice . Treatment F 3 , 71 = 12 . 94 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0036 ; Ctrl - ARL67156 vs Ctrl - ACSF , P = 0 . 0428 ; CSDS - ARL67156 vs CSDS - ACSF , P < 0 . 0001 . One - way ANOVA with Fisher ' s LSD test ) . Data information : data are expressed as the means ± SEM . NS P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Ctrl , control .

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 O O O O O O O O O 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 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 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 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 3B The ATPase activity of mice exposure to ARL67156 (n = 7, 7, 5, 5 mice. Treatment F3, 20 = 16.81, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0405. One-way ANOVA with Fisher's LSD test).C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 9, 9 mice. Interaction F3, 36 = 5.119, P = 0.0047; Target F1, 36 = 0.005, P = 0.9437; Drug F3, 36 = 3.626, P = 0.022. For Target, CSDS - ACSF vs Ctrl -ACSF, P = 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0054. Two-way ANOVA with Tukey's post-test).D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 10, 10 mice. Interaction F1, 38 = 5.352, P = 0.0262; Group F1, 38 = 5.711, P = 0.0219; Drug F1, 38 = 9.703, P = 0.0035; CSDS - ACSF vs Ctrl - ACSF, P = 0.0083; Ctrl - ARL67156 vs CSDS - ACSF, P = 0.0027; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0031. Two-way ANOVA with Tukey's post-test).The social avoidance behavior (E) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. For social behavior, Interaction F3, 31 = 5.579, P = 0.0035; Target F1, 31 = 0.5121, P = 0.4796; Drug F3, 31 = 8.367, P = 0.0003; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0131.sucrose preference (F) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. Sucrose, Interaction F1, 30 = 0.3342, P = 0.5675; Group F1, 30 = 17.22, P = 0.0003; Drug F1, 30 = 0.05903, P = 0.8097; CSDS- ACSF vs Ctrl - ACSF, P = 0.0761. Two-way ANOVA with Tukey's post-test).G The immobility time in the TST of mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 16, 12, 21, 19 mice. Treatment F 3, 64= 13.61, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0059; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0011; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0002. One-way ANOVA with Fisher's LSD test).H The immobility time in the FST with intra-hippocampal infusion of ARL67156 after CSDS (n = 21, 12, 23, 19 mice. Treatment F 3, 71 = 12.94, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0036; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0428; CSDS - ARL67156 vs CSDS - ACSF, P < 0.0001. One-way ANOVA with Fisher's LSD test).Data information: data are expressed as the means ± SEM. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.


Figure 4B Imaging of GFP expression in the hippocampus 3 weeks after virus vector injection . Scale bar : 250 μm . C Expression of CD39 in the hippocampus of mice injected with LV - siCD39 ( n = 6 mice / group . LV - siCD39 vs Control , P = 0 . 0006 , Student ' s t test ) . D The social avoidance behavior for the stressed mice after knocking down CD39 ( n = 15 , 15 , 14 mice . Interaction F2 , 41 = 17 . 96 , P < 0 . 0001 ; Target F1 , 41 = 3 . 75e - 005 , P = 0 . 9951 ; Drug F2 , 41 = 6 . 099 , P = 0 . 0048 ; For Target CSDS - LV - GFP vs Ctrl - LV - GFP , P < 0 . 0001 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P < 0 . 0001 . Two - way ANOVA with Tukey ' s post - test ) . E The immobility time in the TST of mice injected with LV - siCD39 ( n = 11 , 19 , 15 mice . Treatment F 2 , 42 = 5 . 807 , P = 0 . 0059 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0069 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0065 . One - way ANOVA with Fisher ' s LSD test ) . F The immobility time spent in the FST of mice treatment with LV - siCD39 ( n = 12 , 19 , 15 mice . Treatment F2 , 43 = 5 . 237 , P = 0 . 0092 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0095 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0093 . One - way ANOVA with Fisher ' s LSD test ) . G The ATPase hyperactivity of mice after knocking down CD39 in the hippocampal ( n = 5 , 6 , 5 mice . Treatment F2 , 13 = 8 . 45 , P = 0 . 0044 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0016 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P = 0 . 0166 . One - way ANOVA with Fisher ' s LSD test ) . H The ATP level of mice after knocking down CD39 in the hippocampal ( n = 5 mice / group . Treatment F2 , 12 = 6 . 04 , P = 0 . 0153 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0444 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P = 0 . 0051 . One - way ANOVA with Fisher ' s LSD test ) .

O B-PANEL_START 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 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 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 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 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

Figure 4B Imaging of GFP expression in the hippocampus 3 weeks after virus vector injection. Scale bar: 250 μm.C Expression of CD39 in the hippocampus of mice injected with LV-siCD39 (n = 6 mice/group. LV-siCD39 vs Control, P = 0.0006, Student's t test).D The social avoidance behavior for the stressed mice after knocking down CD39 (n = 15, 15, 14 mice. Interaction F2, 41 = 17.96, P < 0.0001; Target F1, 41 = 3.75e-005, P = 0.9951; Drug F2, 41 = 6.099, P = 0.0048; For Target CSDS - LV-GFP vs Ctrl - LV-GFP, P < 0.0001; CSDS - LV-siCD39 vs CSDS - LV-GFP, P < 0.0001. Two-way ANOVA with Tukey's post-test).E The immobility time in the TST of mice injected with LV- siCD39 (n = 11, 19, 15 mice. Treatment F 2, 42 = 5.807, P = 0.0059; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0069; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0065. One-way ANOVA with Fisher's LSD test).F The immobility time spent in the FST of mice treatment with LV-siCD39 (n = 12, 19, 15 mice. Treatment F2, 43 = 5.237, P = 0.0092; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0095; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0093. One-way ANOVA with Fisher's LSD test).G The ATPase hyperactivity of mice after knocking down CD39 in the hippocampal (n = 5, 6, 5 mice. Treatment F2, 13 = 8.45, P = 0.0044; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0016; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0166. One-way ANOVA with Fisher's LSD test).H The ATP level of mice after knocking down CD39 in the hippocampal (n = 5 mice/group. Treatment F2, 12 = 6.04, P = 0.0153; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0444; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0051. One-way ANOVA with Fisher's LSD test).


Figure 5B Immunostaining for BrdU ( blue ) and DCX ( green ) in the DG region of control , susceptible and ARL67156 treatment mice . Arrows indicate the BrdU + DCX + cells . Scale bar : 50 μm . C , D Quantification of BrdU + cells ( C ) and BrdU + DCX + cells ( D ) in the DG of susceptible mice with ARL67156 compared with vehicle treatment ( n = 4 mice / group . For BrdU + Treatment F2 , 9 = 10 . 07 , P = 0 . 0051 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0147 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0018 ; BrdU + DCX + Treatment F2 , 9 = 9 . 005 , P = 0 . 0071 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0211 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0024 , one - way ANOVA with Fisher ' s LSD test ) . F Confocal images of BrdU ( blue ) and DCX ( red ) in mice treatment with LV - siCD39 in the DG . Arrows indicate the BrdU + DCX + cells . Scale bar : 50 μm . G , H Quantitative of BrdU + ( G ) and BrdU + DCX + ( H ) cells in the DG of susceptible mice with LV - siCD39 compared with LV - GFP treatment ( n = 3 , 6 , 6 mice . For BrdU + Treatment F2 , 12 = 15 . 79 , P = 0 . 0004 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0012 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 ; BrdU + DCX + Treatment F2 , 12 = 12 . 79 , P = 0 . 0011 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 05 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 , one - way ANOVA with Fisher ' s LSD test ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B Immunostaining for BrdU (blue) and DCX (green) in the DG region of control, susceptible and ARL67156 treatment mice. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.C, D Quantification of BrdU+ cells (C) and BrdU+ DCX+ cells (D) in the DG of susceptible mice with ARL67156 compared with vehicle treatment (n = 4 mice/group. For BrdU+ Treatment F2, 9 = 10.07, P =0.0051; CSDS - ACSF vs Ctrl - ACSF, P = 0.0147; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0018; BrdU+ DCX+ Treatment F2, 9 = 9.005, P = 0.0071; CSDS - ACSF vs Ctrl - ACSF, P = 0.0211; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0024, one-way ANOVA with Fisher's LSD test).F Confocal images of BrdU (blue) and DCX (red) in mice treatment with LV-siCD39 in the DG. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).


Figure 6Representative confocal z - stack images of LV - infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment . White arrows indicate stubby spines , yellow arrows indicate mushroom spines , and blue arrows indicate thin spines . B - E Quantification of total ( B ) , stubby ( C ) , mushroom ( D ) and thin ( E ) spine density in DG from control and susceptible mice infected with LV - GFP or LV - siCD39 ( n = 15 segments from 4 mice / group . For total density : Treatment F2 , 42 = 3 . 12 , P = 0 . 0544 ; Ctrl - LV - GFP vs CSDS - LV - siCD39 , P = 0 . 0312 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0425 ; stubby density : Treatment F2 , 42 = 10 . 93 , P = 0 . 0002 ; P = 0 . 0026 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P < 0 . 0001 ; mushroom density : Treatment F2 , 42 = 0 . 34 , P = 0 . 7161 ; thin density : F2 , 42 = 1 . 00 , P = 0 . 3758 . One - way ANOVA with Fisher ' s LSD test ) . F Representative confocal z - stack images of LV - infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment . White arrows indicate stubby spines , yellow arrows indicate mushroom spines , and blue arrows indicate thin spines . G - J Quantification of total ( G ) , stubby ( H ) , mushroom ( I ) and thin ( J ) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 ( n = 11 , 13 , 10 segments from 2 , 4 , 2 mice , respectively . For total density : Treatment F2 , 31 = 4 . 474 , P = 0 . 0196 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 0366 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0086 ; stubby density : Treatment F2 , 31 = 3 . 327 , P = 0 . 0491 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 1026 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0179 ; mushroom density : Treatment F2 , 31 = 1 . 986 , P = 0 . 1543 ; thin density : F2 , 31 = 1 . 475 , P = 0 . 2445 . One - way ANOVA with Fisher ' s LSD test ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 6Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.B-E Quantification of total (B), stubby (C), mushroom (D) and thin (E) spine density in DG from control and susceptible mice infected with LV-GFP or LV-siCD39 (n = 15 segments from 4 mice/group. For total density: Treatment F2, 42 = 3.12, P = 0.0544; Ctrl - LV-GFP vs CSDS - LV-siCD39, P = 0.0312; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0425; stubby density: Treatment F2, 42 = 10.93, P=0.0002; P = 0.0026; CSDS - LV-CD39 vs CSDS - LV-GFP, P < 0.0001; mushroom density: Treatment F2, 42 = 0.34, P = 0.7161; thin density: F2, 42 = 1.00, P = 0.3758. One-way ANOVA with Fisher's LSD test).F Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.G-J Quantification of total (G), stubby (H), mushroom (I) and thin (J) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 (n = 11, 13, 10 segments from 2, 4, 2 mice, respectively. For total density: Treatment F2, 31 = 4.474, P = 0.0196; CSDS - ASCF vs Ctrl - ACSF, P = 0.0366; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0086; stubby density: Treatment F2, 31 = 3.327, P=0.0491; CSDS - ASCF vs Ctrl - ACSF, P = 0.1026; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0179; mushroom density: Treatment F2, 31 = 1.986, P = 0.1543; thin density: F2, 31 = 1.475, P = 0.2445. One-way ANOVA with Fisher's LSD test).


Figure 1RT - PCR analysis of SPPL2c expression in different murine tissues . Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification ( + RT ) or , as negative control , used directly as template ( - RT ) . A fragment of the actin ORF was amplified as control . Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c - / - using antibodies against C - ( D ) or N - terminal ( E ) epitopes of SPPL2c . Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel . SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C - terminally Myc - tagged murine SPPL2c isoform A . For detection of SPPL2c , either anti - Myc or the SPPL2c antiserum ( C - terminal epitope ) were employed as indicated together with anti - KDEL , anti - GM130 or anti - ERGIC53 as indicated in order to label the ER , the cis Golgi apparatus or the ER - Golgi intermediate compartment ( ERGIC ) , respectively . Lysates from HEK293 cells transiently transfected with murine SPPL2c - myc ( isoform A ) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti - SPPL2c ( C - term ) . Deglycosylation of endogenously expressed LIMP2 was analysed as control . N - glycosylation of endogenous SPPL2c is similar to the overexpressed protein . Total lysates from testis of wild type or SPPL2c - / - mice were treatedSubcellular fractionation of postnuclear supernatant from murine testis using a self - forming Percoll density gradient . Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c . Protein disulfide isomerase ( PDI ) , Cathepsin D and CD44 were detected as organelle marker proteins .

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Figure 1RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c-/- using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel.SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either anti-Myc or the SPPL2c antiserum (C-terminal epitope) were employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively.Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control.N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treatedSubcellular fractionation of postnuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulfide isomerase (PDI), Cathepsin D and CD44 were detected as organelle marker proteins.


Figure 2HEK293 cells were transiently transfected with murine N - terminally HA - tagged heme oxygenase 1 ( HA - mHO - 1 ) alone or in combination with active or inactive ( D / A ) murine SPP or SPPL2c . Substrate levels were determined by Western blot analysis with anti - HA . For detection of SPP , an antiserum recognizing both endogenous human ( h ) and overexpressed murine ( m ) SPP was employed . Murine SPPL2c was visualised using the antiserum generated against the C - terminus of the protein . Actin was detected to control for equal protein loading . Immunofluorescence analysis of HeLa cells transiently expressing murine HO - 1 ( HA - mHO - 1 ) alone or together with active or inactive ( D / A ) SPPL2c - myc . Substrate and proteases were visualised using the HA and Myc epitopes , respectively . The TA protein Ube2J1 is not proteolysed by co - expressed SPP or SPPL2c in HEK293 cells . Western blot analysis was performedDifferential cleavage of CYB5a , RAMP4 - 2 and RAMP4 by SPPL2c and SPP . Substrates and proteases were co - expressed in HEK293 cells followed by Western blot analysis as described above . Densitometric quantification from at least n = 3 independent experiments was performed . Substrate levels were normalised and compared to cells without protease - overexpression . HEK293 cells expressing HA - RAMP4 - 2 alone or in combination with SPPL2c or SPP were treated with 100 µM ( Z - LL ) 2 - ketone ( ZLL ) , 5 µM inhibitor X ( InX ) , 5 µM DAPT or DMSO as control . RAMP4 - 2 band intensity was quantified from blots of three independent experiments and normalised to cells just over - expressing the substrate . Microsomes isolated from testis of wild type and SPPL2c - / - mice were solubilised in 1 % digitonin and proteins were separated by blue native PAGE . After transfer to PVDF membrane , SPPL2c and SPP were detected using the polyclonal antisera introduced above .

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 B-PANEL_START 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 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 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

Figure 2HEK293 cells were transiently transfected with murine N-terminally HA-tagged heme oxygenase 1 (HA-mHO-1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Substrate levels were determined by Western blot analysis with anti-HA. For detection of SPP, an antiserum recognizing both endogenous human (h) and overexpressed murine (m) SPP was employed. Murine SPPL2c was visualised using the antiserum generated against the C-terminus of the protein. Actin was detected to control for equal protein loading.Immunofluorescence analysis of HeLa cells transiently expressing murine HO-1 (HA-mHO-1) alone or together with active or inactive (D/A) SPPL2c-myc. Substrate and proteases were visualised using the HA and Myc epitopes, respectively.The TA protein Ube2J1 is not proteolysed by co-expressed SPP or SPPL2c in HEK293 cells. Western blot analysis was performedDifferential cleavage of CYB5a, RAMP4-2 and RAMP4 by SPPL2c and SPP. Substrates and proteases were co-expressed in HEK293 cells followed by Western blot analysis as described above. Densitometric quantification from at least n=3 independent experiments was performed. Substrate levels were normalised and compared to cells without protease-overexpression.HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate.Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.


Figure 3Histochemical visualization of β - galactosidase ( β - gal ) reporter activity revealed SPPL2c expression within seminiferous tubules . Cryo - sections from PFA - fixed wild type and SPPL2c - / - testis were stained with X - Gal as β - gal substrate . SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D . While for 4C , 2C and 1C fractions 10 µg of protein were loaded , for the testis control samples ( = total lysates ) 20 µg of protein were subjected to electrophoresis . * , non - specific band . Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin - fixed wild type and SPPL2c - / - testis . The SPPL2c antiserum directed against a C - terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed . Nuclei were stained with hematoxylin . Scale bar , 100 µm ( upper panel ) or 50 µm ( lower panel ) . SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4 , 6 , 8 or 12 weeks by Western blotting . A testis lysate from an adult SPPL2c - / - mouse was used as negative control . The weight of both testicles of wild type and SPPL2c - / - mice ( n = 12 per genotype ) was determined and normalised to the body weight . Paraffin sections from Bouin - fixed wild type and SPPL2c - / - testis were stained with hematoxylin & eosin ( H & E ) . Scale bars , 100 µm . In H & E stained paraffin - sections from Bouin - fixed wild type and SPPL2c - / - testis , the mean number of elongated spermatids per seminiferous tubule was determined . Cells were counted in 20 tubuli in sections from n = 6 mice per genotype . SPPL2c is not present in mature spermatozoa . Western blot analysis of total lysates of wild type ( + / + ) and SPPL2c - / - testis , epididymis and spermatozoa recovered from the cauda epididymidis / vas deferens for SPPL2c . Mature sperm were isolated from the epididymis of wild type or SPPL2c - deficient mice and analysed by phase - contrast microscopy . Representative pictures of morphologically normal spermatozoa are depicted . Scale bars , 10 µm . Reduced motility of SPPL2c - / - sperm cells . Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c - / - mice ( n = 3 ) and subjected to computer - assisted sperm analysis . Average path velocity ( VAP ) , straight line velocity ( VSL ) and curvilinear velocity ( VCL ) were determined in micrometer per second . Mean litter sizes for different breeding schemes . The data depicted are based on n = 12 ( + / + X + / + ) , n = 57 ( + / - X + / - ) , n = 11 ( - / - X - / - ) , n = 10 ( - / - X + / + ) and n = 11 ( + / + X - / - ) different breedings , respectively .

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 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 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 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 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 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 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 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 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

Figure 3Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate.SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control.The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight.Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype.SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c.Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm.Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second.Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively.


Figure 4Carbonate - washed total membrane fractions from wild type and SPPL2c - / - testis ( n = 5 ) were subjected to a label - free quantitative proteome analysis . For each quantified protein , a log2 intensity ratio between SPPL2c - deficient and wild type samples was calculated . Negative values indicate a depletion and positive values an enrichment in SPPL2c - / - samples . In the depicted volcano plot the negative log10 of the p value ( y axis ) is plotted versus the log2 intensity ratio of SPPL2c - / - versus wild type samples ( x axis ) . All proteins above the significance level of p < 0 . 01 ( unpaired Student ' s t test ) are coloured in red . The hyperbolic curve indicates the threshold for a permutation - based FDR correction for multiple hypotheses with p = 0 . 05 and s0 = 0 . 1 . HeLa cells were transiently transfected with N - terminally HA - tagged PLN ( HA - PLN ) and inactive ( D / A ) SPPL2c - myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes . HEK293 cells were transiently transfected with HA - PLN alone or in combination with active or inactive ( D / A ) murine SPPL2c Isoform A ( D ) or Isoform B ( E ) . Substrate processing was analysed by Western blotting with anti - HA . SPPL2c expression was confirmed with the antiserum against the C - terminal epitope . Actin was detected to confirm equal protein loading . Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti - HA in combination with anti - protein disulphide isomerase A6 ( PDIA6 ) to stain the ER . The 37 - 41A and 47 - 51 PLN mutants were not expressed in relevant amounts . HEK293 cells were transiently transfected with wild type or mutated HA - PLN alone or together with SPPL2c isoform A . Quantification of n = 5 experiments as shown in ( H ) . For each PLN variant , the residual PLN in SPPL2c co - expressing cells is depicted as % of PLN in absence of the protease .

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Figure 4Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes.HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading.Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts.HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A.Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease.


Figure 5Accumulation of endogenous PLN in the testis of SPPL2c - / - mice . PLN was immuno - precipitated ( IP ) from equal protein amounts of total lysates of wild type or SPPL2c - / - testis with the mouse monoclonal antibody . Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody . SPPL2c and actin were detected from total lysates directly . Quantification of normalised PLN / Actin ratios from n = 3 biological replicates per genotype . Expression of PLN in murine testis from wild type ( wt ) SPPL2c - / - ( 2c - / - ) was quantified by qPCR . Immunohistochemical detection of PLN in paraformaldehyde ( PFA ) - fixed testis from wild type and SPPL2c - / - mice . Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate . Testis suspensions were stained with 2 . 5 µM DRAQ5 to determine cellular DNA amount . 1C - co = elongated spermatids , 1C = other haploid cells including round spermatids , 2C = spermatogonia , secondary spermatocytes , Sertoli cells , other somatic cells , 4C = primary spermatocytes , G2 spermatogonia . Intracellular Ca2 + concentrations were analysed in testis suspensions from either wild type or SPPL2c - deficient mice using the Ca2 + - sensitive probe Fluo4 - AM . Individual germ cell populations were identified Bars indicate Median Fluo4 - AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples . Median forward scatter ( FSC ) was controlled in the 1C - co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype . Expression of SPPL2c in human testis . ( H ) Western blot analysis of lysates from HEK293 cells expressing human ( h ) SPPL2c , human and murine testis using antisera against a C - terminal epitope of murine SPPL2c or human SPPL2c as indicated . Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin - sections from Bouin ' s - fixed human testis . In addition , sections were incubated with control immunoglobulins . Arrows indicate elongated spermatids .

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Figure 5Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly.Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype.Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR.Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate.Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples.Median forward scatter (FSC) was controlled in the 1C-co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype.Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated.Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids.


Figure 1 ( A ) Co - localization of c - terminally GFP - or mCherry - tagged Apl5 , Sec7 and Vps41 in a vam3 temperature sensitive ( ts ) background strain . Cells were grown over night at 23 ° C and where indicated shifted to 37 ° C for 3 h prior to imaging . White arrowheads indicate co - localization events . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( B ) Quantification of co - localization events Single events were counted and the percentage of co - localization normalized to the number of Apl5 - punctae was plotted ( n ≥ 187 cells ) . Bars show the mean percentage of co - localization ± standard deviation . ( C ) Re - localization of Apl5 upon Vps41 re - localization . Vps41 was tagged with a C - terminal FRBGFP tag in a strain , where Apl5 carries a C - terminal mCherry and Pma1 a FKBP - tag Cells were grown in either the absence ( control ) or presence of 10 μM rapamycin , and localization of Vps41 and Apl5 was analysed by fluorescence microscopy . Scale bar , 5 μm . ( D ) Effect of clathrin deletion on the AP - 3 pathway . GFP - tagged Nyv1 with a C - terminal Snc1 transmembrane domain ( GNS ) was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Labeling of the cell surface by GFP indicates a defect in the AP - 3 pathway ( E ) Quantification of AP - 3 defect from ( D ) . Linear intensity plots were laid over the cells , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 30 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation .

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Figure 1(A) Co-localization of c-terminally GFP- or mCherry-tagged Apl5, Sec7 and Vps41 in a vam3 temperature sensitive (ts) background strain. Cells were grown over night at 23°C and where indicated shifted to 37°C for 3 h prior to imaging. White arrowheads indicate co-localization events. Scale bar, 5 µm. Scale bar in inset, 2 µm.(B) Quantification of co-localization events Single events were counted and the percentage of co-localization normalized to the number of Apl5-punctae was plotted (n ≥ 187 cells). Bars show the mean percentage of co-localization ± standard deviation.(C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm.(D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


Figure 2 ( C ) Tethering of AP - 3 vesicles to the mitochondrial surface . Cells were grown at 30 ° C in YPG and kept in logarithmic phase . Just prior to imaging , the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC ( difference interference contrast ) image of cells . Scale bar , 5 µm . The inset shows the indicated magnification . Scale bar in inset , 2 µm . Apl5 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - dots . Bars show the mean percentage of co - localization ± standard deviation ( n ≥ 60 cells ) . ( E , F ) Purification of mitochondria from selected strains . Mitochondria were purified from the indicated strains as described in the methods . The amount corresponding to 1 % of the total volume was loaded of the ' load ' ' sample , 5 % were used for ' crude ' and ' pure ' mitochondria fractions . Samples were loaded on a 10 % SDS PAGE gel and analysed by Western Blot . White asterisks indicate unspecific bands . ( G ) Quantification of relative Nyv1 signal intensity from ( F ) . Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted . Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments .

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Figure 2(C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm.Apl5-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5-dots. Bars show the mean percentage of co-localization ± standard deviation (n ≥ 60 cells).(E, F) Purification of mitochondria from selected strains. Mitochondria were purified from the indicated strains as described in the methods. The amount corresponding to 1 % of the total volume was loaded of the 'load'' sample, 5% were used for 'crude' and 'pure' mitochondria fractions. Samples were loaded on a 10% SDS PAGE gel and analysed by Western Blot. White asterisks indicate unspecific bands. (G) Quantification of relative Nyv1 signal intensity from (F). Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted. Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments.


Figure 3 ( A - H ) Indicated strains were grown in YPG for expression of Vps41 - GFP - Fis constructs . Two representative images are shown for each strain . Mitochondria ( M ) are highlighted in red and vesicular structures in teal ( unlabeled for VPS41 - GFP - Fis ) or green ( unlabeled for VPS41 - GFP - Fis ) . Cell wall ( CW ) , plasma membrane ( PM ) , and vacuoles ( V ) are indicated . Scale bars , 250 nm .

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Figure 3(A-H) Indicated strains were grown in YPG for expression of Vps41-GFP-Fis constructs. Two representative images are shown for each strain. Mitochondria (M) are highlighted in red and vesicular structures in teal (unlabeled for VPS41-GFP-Fis) or green (unlabeled for VPS41-GFP-Fis). Cell wall (CW), plasma membrane (PM), and vacuoles (V) are indicated. Scale bars, 250 nm.


Figure 4 ( A ) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects . First column shows DIC image of cells . Scale bar , 5 µm . ( B ) Quantification of the AP - 3 defect from ( A ) . Linear intensity plots were laid over cells grown in medium containing glucose ( D ) or galactose ( G ) , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 15 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation . ( C ) Effect of different truncations and deletions on AP - 3 targeting to mitochondria . Indicated strains were grown at 30 ° C in YPG and kept in logarithmic phase . Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC image of cells . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( D ) Quantification of ( C ) . ( - ) Control corresponds to Vps41 ( ΔPEST ) - GFP - Fis1 Apl6 - mCherry ( CUY12558 ) and ( + ) corresponds to Vps41 ( S - D ) - GFP - Fis1 Apl6 - mCherry ( CUY12560 ) . Apl5 - / Apl6 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - / Apl6 - dots ( n ≥ 63 ) . Bars show the mean percentage of co - localization ± standard deviation .

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Figure 4(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm. (B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.(C) Effect of different truncations and deletions on AP-3 targeting to mitochondria. Indicated strains were grown at 30°C in YPG and kept in logarithmic phase. Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC image of cells. Scale bar, 5 µm. Scale bar in inset, 2 µm. (D) Quantification of (C). (-) Control corresponds to Vps41(ΔPEST)-GFP-Fis1 Apl6-mCherry (CUY12558) and (+) corresponds to Vps41(S-D)-GFP-Fis1 Apl6-mCherry (CUY12560). Apl5- /Apl6-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5- /Apl6-dots (n ≥ 63). Bars show the mean percentage of co-localization ± standard deviation.


Figure 5 ( C ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells , from a label - free proteomics analysis of streptavidin - biotin pulldowns . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 05 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black dots , ( E ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells in a GAL1pr - Vps41 - GFP - Fis1 background , from a label - free proteomics analysis of streptavidin - biotin pulldowns with cells grown in YPG . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 001 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black

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Figure 5(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,(E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots


Figure 6 ( B ) AP - 3 sorting defects in GAP deletions . The AP - 3 reporter was localized relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows the DIC image of cells . Scale bar , 5 µm . ( D ) Localization of AP - 3 in ArfGAP deletion strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows DIC image of cells . Scale bar , 5 µm . ( E ) Quantification of Apl5 - dots in ( D ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean . ( F ) Co - localization of C - terminally GFP - tagged Apl5 and C - terminally mCherry - tagged Sec7 in wt and age2Δ background . Cells were grown at 30 ° C and kept in logarithmic phase . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( G ) Quantification of co - localization from ( F ) normalized per cell . Number of co - localizing dots was determined and normalized to the number of co - localization events per cell . Bars show the mean number of co - localizing dots per cell ± standard deviation ( n ≥ 60 ) .

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 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 O O O O

Figure 6(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm. (E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.(F) Co-localization of C-terminally GFP-tagged Apl5 and C-terminally mCherry-tagged Sec7 in wt and age2Δ background. Cells were grown at 30°C and kept in logarithmic phase. Scale bar, 5 µm. Scale bar in inset, 2 µm. (G) Quantification of co-localization from (F) normalized per cell. Number of co-localizing dots was determined and normalized to the number of co-localization events per cell. Bars show the mean number of co-localizing dots per cell ± standard deviation (n ≥ 60).


Figure 7 ( A , B ) AP - 3 sorting defects in strains overexpressing Gcs1 ( A ) or Age2 ( B ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( C ) Localization of AP - 3 in ArfGAP overexpression strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( D ) Quantification of Apl5 dots in ( C ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean . ( E , F ) AP - 3 sorting defects in GAP - dead mutants of Gcs1 ( E ) and Age2 ( F ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( G ) Localization of AP - 3 in GAP - dead mutants of Gcs1 and Age2 . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( H ) Quantification of Apl5 dots in ( G ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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Figure 7(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. (E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


Figure 1B : Log2 gene expression fold changes of significantly induced genes ( FDR = 1 % ) across different treatment scenarios . Gene induction of immediate , delayed and late responding genes is sustained upon constant activation ( ON scenario ) and transient upon two - hour pulse activation ( ON / OFF scenario ) . Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes . Genes were ranked by their model - derived response time .

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Figure 1B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.


Figure 3B : pERK2 log2 fold change upon sustained activation ( left ) and deduced input function ( right ) used for model fitting . Average pERK2 log2 fold change upon 4OHT treatment equals 100 % signalling amplitude . C : Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes ( FDR = 1 % ) . Gene expression is shown as percentage of response amplitude . Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure .

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Figure 3B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.C: Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes (FDR = 1%). Gene expression is shown as percentage of response amplitude. Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure.


Figure 4B : Signalling input conditions ( left side shows deduced input function , right side shows pERK2 measurements ) : Sustained ERK signalling ( 4OHT ) , two - hour pulse ERK signalling ( 4OHT + U0126 ) , growth factor signalling ( EGF : Epidermal growth factor , FGF : Fibroblast growth factor ) . Deduced input functions : 100 % signalling amplitude corresponds to mean induction in training condition ( 4OHT ) . Growth factor - induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition . C : Predictions are verified with actual gene expression data . Heat maps show log2 fold changes of induced mRNAs . P : Model prediction . D : Gene expression data . E : Mean error = mean of absolute residuals .

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 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

Figure 4B: Signalling input conditions (left side shows deduced input function, right side shows pERK2 measurements): Sustained ERK signalling (4OHT), two-hour pulse ERK signalling (4OHT+U0126), growth factor signalling (EGF: Epidermal growth factor, FGF: Fibroblast growth factor). Deduced input functions: 100% signalling amplitude corresponds to mean induction in training condition (4OHT). Growth factor-induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition.C: Predictions are verified with actual gene expression data. Heat maps show log2 fold changes of induced mRNAs. P: Model prediction. D: Gene expression data. E: Mean error = mean of absolute residuals.


Figure 5 Figure 5 - Immediate late genes ( ILGs ) have long mRNA half - lives , are transcribed immediately and have GC - rich promoters . A : Boxplot comparison of mRNA half - life estimates based on modelling of gene induction ( model - derived ) , transcriptional shutdown ( ActD - derived ) and metabolic labelling ( 4SU - derived ) . Estimates from 4OHT - pretreated HEK293 ∆ RAF1 : ER cells ( ON panel ) are more appropriate to characterise induced genes than estimates from unstimulated cells ( OFF panel ) . Genes not assigned to any cluster are shown in grey . C : Log2 fold changes of transcription rate in 4OHT - treated HEK293 ∆ RAF1 : ER cells derived from metabolic labelling ( 4SU ) RNA - sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs .

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 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

Figure 5 Figure 5 - Immediate late genes (ILGs) have long mRNA half-lives, are transcribed immediately and have GC-rich promoters. A: Boxplot comparison of mRNA half-life estimates based on modelling of gene induction (model-derived), transcriptional shutdown (ActD-derived) and metabolic labelling (4SU-derived). Estimates from 4OHT-pretreated HEK293∆RAF1:ER cells (ON panel) are more appropriate to characterise induced genes than estimates from unstimulated cells (OFF panel). Genes not assigned to any cluster are shown in grey. C: Log2 fold changes of transcription rate in 4OHT-treated HEK293∆RAF1:ER cells derived from metabolic labelling (4SU) RNA-sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs.


Figure 6 Figure 6 - Immediate late genes ( ILGs ) translate signal duration into response amplitude . A : Upper panel : pERK2 log2 fold changes upon different input scenarios ( sustained : 4OHT ; two - hour pulse : 4OHT + U0126 ) . Lower panel : Response amplitude across temporal clusters and signal durations . Bold lines show median cluster amplitude at each time point . C : qPCR validation to test different ERK signal durations . HEK293 ∆ RAF1 : ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0 . 5 to 8 hours ( cf . Fig EV1 C ) . mRNAs of IEGs EGR1 and FOS relay signal duration to response duration , whereas ILGs CLU and FOSL1 decode signal duration to response amplitude ( qPCR data for all 17 validated mRNAs is shown in Fig EV6 A ) . E : Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling ( 4OHT - induced ) and transient ERK signalling ( EGF - induced ) in HEK293 ∆ RAF1 : ER cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 6 Figure 6 - Immediate late genes (ILGs) translate signal duration into response amplitude. A: Upper panel: pERK2 log2 fold changes upon different input scenarios (sustained: 4OHT; two-hour pulse: 4OHT+U0126). Lower panel: Response amplitude across temporal clusters and signal durations. Bold lines show median cluster amplitude at each time point. C: qPCR validation to test different ERK signal durations. HEK293∆RAF1:ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0.5 to 8 hours (cf. Fig EV1 C). mRNAs of IEGs EGR1 and FOS relay signal duration to response duration, whereas ILGs CLU and FOSL1 decode signal duration to response amplitude (qPCR data for all 17 validated mRNAs is shown in Fig EV6 A).E: Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling (4OHT-induced) and transient ERK signalling (EGF-induced) in HEK293∆RAF1:ER cells.


Figure 1A ) In our chemoattraction - assay setup , nematode chemoattraction behavior is quantified using a measurable chemoattraction index ( C . I . ) ; the higher the C . I . measured in the assay , the stronger the attraction of the males to the sex pheromone . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . B ) After vaporization ( VAC ) at 4 ° C for 24 h , the C . remanei female extract failed to attract wild - type C . elegans males . Conversely , a drop of the sex pheromone extract placed on the lid of a 60 - mm Petri dish evoked chemoattractive behavior in wild - type C . elegans males ( volatility test ) . Thus , the functional components in C . remanei female sex pheromone are volatile substances . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown . C ) Mutant males with established defects in amphid neurons responded to C . remanei sex pheromone to a lesser degree relative to wild - type , demonstrating that some or all of the amphid neurons are required for sex pheromone perception . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1A) In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value.B) After vaporization (VAC) at 4°C for 24 h, the C. remanei female extract failed to attract wild-type C. elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.C) Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.