Figure 2Strong PTPδ - tdTomato ( red ) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus ( 2 months ) , revealed by quantitative analysis of PTPδ - tdTomato fluorescence across hippocampal lamina . DAPI was used for nuclear staining . CC , corpus callosum ; SO , stratum oriens ; SP , stratum pyramidale ; SR , stratum radiatum ; SLM , stratum lacunosum moleculare ; MO , molecular layer ; SG , granule cell layer ; PO , polymorph layer . Scale bar 500 μm . Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer . Micro - dissected SLM and SR lysates ( 2 months ) were immunoblotted for PTPδ ( C - terminal antibodies ) and α - tubulin ( control ) . The asterisk indicates a non - specific band . Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR . ( n = 4 mice for SR and SLM , mean ± SEM , * * * p < 0 . 001 , Student ' s t - test ; See Dataset EV2 for statistical details for these and all following results ) . Gene dose - dependent reductions in PTPδ protein levels in whole brains of Ptprd + / - ( HT ) and Ptprd - / - ( KO ) mice relative to Ptprd + / + ( WT ) mice ( P21 ) , shown by immunoblot analyses . Note that the C - terminal antibody also detects a non - specific band of similar size indicated by an asterisk , possibly either PTPσ or LAR . Suppressed basal excitatory synaptic transmission in the Ptprd - / - hippocampal SLM layer , but not SO or SR layer ( 2 months ; male ) , as shown by the I / O ratio ( n = 14 slices from 5 mice [ WT - SO ] , 14 , 5 [ KO - SO ] , 11 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 14 , 6 [ WT - SLM ] , 12 , 6 [ KO - SLM ] , mean ± SEM , * p < 0 . 05 ; * * p < 0 . 01 , two - way repeated - measures / RM ANOVA , Holm - Sidak post - hoc multiple comparisons test ) . ( F , H , and J ) Normal presynaptic release in the Ptprd - / - hippocampal SLM , SO , and SR layers ( P22 - 27 ; male ) , as shown by the PPF ratio ( n = 13 slices from 5 mice [ WT - SO ] , 13 , 5 [ KO - SO ] , 10 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 15 , 6 [ WT - SLM ] , 14 , 6 [ KO - SLM ] , mean ± SEM , ns , not significant , two - way RM ANOVA ) . Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs . Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs . ( n = 18 images from 3 mice [ WT and KO ] , * * * p < 0 . 001 , ns , not significant , Student ' s t - test for L , O - R , Mann - Whitney U test for M , N ) . Scale bar , 500 nm . Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . ( n = 3 mice [ WT and KO ] , mean ± SEM , * * * p < 0 . 001 , ns , not significant , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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

Figure 2Strong PTPδ-tdTomato (red) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus (2 months), revealed by quantitative analysis of PTPδ-tdTomato fluorescence across hippocampal lamina. DAPI was used for nuclear staining. CC, corpus callosum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer; SG, granule cell layer; PO, polymorph layer. Scale bar 500 μm.Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer. Micro-dissected SLM and SR lysates (2 months) were immunoblotted for PTPδ (C-terminal antibodies) and α-tubulin (control). The asterisk indicates a non-specific band. Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR. (n = 4 mice for SR and SLM, mean ± SEM, ***p < 0.001, Student's t-test; See Dataset EV2 for statistical details for these and all following results).Gene dose-dependent reductions in PTPδ protein levels in whole brains of Ptprd+/- (HT) and Ptprd-/- (KO) mice relative to Ptprd+/+ (WT) mice (P21), shown by immunoblot analyses. Note that the C-terminal antibody also detects a non-specific band of similar size indicated by an asterisk, possibly either PTPσ or LAR.Suppressed basal excitatory synaptic transmission in the Ptprd-/- hippocampal SLM layer, but not SO or SR layer (2 months; male), as shown by the I/O ratio (n = 14 slices from 5 mice [WT-SO], 14, 5 [KO-SO], 11, 4 [WT-SR], 12, 5 [KO-SR], 14, 6 [WT-SLM], 12, 6 [KO-SLM], mean ± SEM, *p < 0.05; **p < 0.01, two-way repeated-measures/RM ANOVA, Holm-Sidak post-hoc multiple comparisons test). (F, H, and J) Normal presynaptic release in the Ptprd-/- hippocampal SLM, SO, and SR layers (P22-27; male), as shown by the PPF ratio (n = 13 slices from 5 mice [WT-SO], 13, 5 [KO-SO], 10,4 [WT-SR], 12, 5 [KO-SR], 15, 6 [WT-SLM], 14, 6 [KO-SLM], mean ± SEM, ns, not significant, two-way RM ANOVA).Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs.Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs. (n = 18 images from 3 mice [WT and KO], ***p < 0.001, ns, not significant, Student's t-test for L, O-R, Mann-Whitney U test for M, N). Scale bar, 500 nm.Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd-/- SLM (P21; male), revealed by EM. (n = 3 mice [WT and KO], mean ± SEM, ***p < 0.001, ns, not significant, Student's t-test).


Figure 3A volcano plot of proteins that showed significant up - or downregulation of pTyr levels ( absolute fold change > 1 . 5 , p < 0 . 05 ) in the whole brain of Ptprd - / - mice ( P21 ) . Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels . ( n = 3 for WT and 3 for KO ) . See Dataset EV1 , ' Phosphoscan Results ' tab for a full list of significantly changed proteins . A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) . A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) .

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 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

Figure 3A volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins.A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


Figure 4Decreased pTyr levels , but normal total levels of IL1RAPL1 protein , in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoprecipitation of IL1RAPL1 from whole - brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels ( 4G10 antibody ) . Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoblotting of crude synaptosomal ( P2 ) , synaptic plasma membrane ( SPM ) , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - / - brain samples . ( n = 3 mice [ WT and KO ] for each fraction [ P2 , SPM , and PSD ] , mean ± SEM , * * p < 0 . 01 , Student ' s t - test ) . Normal levels of the PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , which contrasts with the Ptprd - / - brain , where PTPδ protein is undetectable . Strongly decreased levels of tyrosine - phosphorylated PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , as shown by the immunoprecipitation of IL1RAPL1 protein from whole - brain lysates followed by immunoblotting for total and tyrosine - phosphorylated PTPδ protein . Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - meA - / - brain ( P21 - 27 ) , as revealed by immunoblotting P2 , SPM , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - meA - / - brain samples . ( n = 3 mice [ Ptprd - meA + / + and Ptprd - meA - / - ] and [ P2 , SPM , and PSD ] , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ) . Note that , while unchanged in the P2 and SPM fractions , PTPδ is significantly decreased in the PSD of Ptprd - meA - / - samples . Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the I / O ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] , 12 , 4 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 ; * * * p < 0 . 001 , two - way RM ANOVA with Holm - Sidak test ) . Normal presynaptic release in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the PPF ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] and 13 , 4 [ Ptprd - meA - / - ] , mean ± SEM , ns , not significant , two - way RM 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 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 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 O O O O O O O O O O O O O O O O O O O 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

Figure 4Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody).Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-/- brain (P21-27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-/- brain samples. (n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, **p<0.01, Student's t-test).Normal levels of the PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), which contrasts with the Ptprd-/- brain, where PTPδ protein is undetectable.Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein.Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-meA-/- brain (P21-27), as revealed by immunoblotting P2, SPM, and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-meA-/- brain samples. (n = 3 mice [Ptprd-meA+/+ and Ptprd-meA-/-] and [P2, SPM, and PSD], mean ± SEM, *p<0.05, ***p < 0.001, Student's t-test). Note that, while unchanged in the P2 and SPM fractions, PTPδ is significantly decreased in the PSD of Ptprd-meA-/- samples.Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the I/O ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+], 12, 4 [Ptprd-meA-/-], mean ± SEM, *p < 0.05; ***p<0.001, two-way RM ANOVA with Holm-Sidak test).Normal presynaptic release in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the PPF ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+] and 13, 4 [Ptprd-meA-/-], mean ± SEM, ns, not significant, two-way RM ANOVA).


Figure 5Hyperactivity of Ptprd - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off and light - on periods were strongly mimicked by Emx1 - cKO mice , but more weakly by Viaat - cKO mice and not at all by Camk2a - cKO mice . Note also that immobility , a measure that better reflects sleep behavior , showed similar changes among different mouse lines . ( n = 14 mice [ WT ] , 14 [ global KO ] , 16 [ Emx1 - cWT ] , 16 [ Emx1 - cKO ] , 11 [ Viaat - cWT ] , 13 [ Viaat - cKO ] , 18 [ Camk2a - cWT ] and 13 [ Camk2a - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; ns , not significant , two - way RM ANOVA with Holm - Sidak test ) . Hyperactive behavior and decreased immobility of Ptprd - meA - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off periods and light - on periods , similar to Ptprd - / - mice . ( n = 7 mice for Ptprd - meA + / + and 7 mice for Ptprd - meA - / - , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5Hyperactivity of Ptprd-/- mice (2.5-4 months) in Laboras cages observed during light-off and light-on periods were strongly mimicked by Emx1-cKO mice, but more weakly by Viaat-cKO mice and not at all by Camk2a-cKO mice. Note also that immobility, a measure that better reflects sleep behavior, showed similar changes among different mouse lines. (n = 14 mice [WT], 14 [global KO], 16 [Emx1-cWT], 16 [Emx1-cKO], 11 [Viaat-cWT], 13 [Viaat-cKO], 18 [Camk2a-cWT] and 13 [Camk2a-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant, two-way RM ANOVA with Holm-Sidak test).Hyperactive behavior and decreased immobility of Ptprd-meA-/- mice (2.5-4 months) in Laboras cages observed during light-off periods and light-on periods, similar to Ptprd-/- mice. (n = 7 mice for Ptprd-meA+/+ and 7 mice for Ptprd-meA-/-, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Figure 6Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . ( n = 9 mice [ Emx1 - cWT ] and 9 mice [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) . Decreased delta power ( normalized to total power ) in NREM , but not WAKE , oscillations in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . Note also the normal theta power ( normalized to total power ) in REM oscillations in Emx1 - Cre ; Ptprdfl / fl mice . ( n = 9 [ Emx1 - cWT ] and 9 [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) . ( D ) Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period and decreased REM duration in the first half of the light - on period in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . Note also that Ptprd - meA - / - mice also display increased WAKE duration and decreased NREM duration during the light - off period . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) . Decreased delta power ( normalized to total power ) in NREM ( both light - off and light - on periods ) and WAKE ( light - on period ) oscillations , and decreased theta power in REM ( mainly light - on period ) oscillations in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 6Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). (n = 9 mice [Emx1-cWT] and 9 mice [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).Decreased delta power (normalized to total power) in NREM, but not WAKE, oscillations in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). Note also the normal theta power (normalized to total power) in REM oscillations in Emx1-Cre;Ptprdfl/fl mice. (n = 9 [Emx1-cWT] and 9 [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).(D) Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period and decreased REM duration in the first half of the light-on period in Ptprd-meA-/- mice (2.5-4 months). Note also that Ptprd-meA-/- mice also display increased WAKE duration and decreased NREM duration during the light-off period. (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).Decreased delta power (normalized to total power) in NREM (both light-off and light-on periods) and WAKE (light-on period) oscillations, and decreased theta power in REM (mainly light-on period) oscillations in Ptprd-meA-/- mice (2.5-4 months). (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Figure 1The percentage of myofibroblast - like cells in total endometrial cells between controls and patients with IUA ( n = 3 samples for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Masson trichrome staining and immunostaining of α - SMA and Collagen 1 in endometrium of controls and patients with IUA ( n = 5 samples for each group ) . Scale bars : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Expression of marker genes for each cell subtype of myofibroblast - like cells ( n = 6 samples ) .

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

Figure 1The percentage of myofibroblast-like cells in total endometrial cells between controls and patients with IUA (n=3 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Masson trichrome staining and immunostaining of α-SMA and Collagen 1 in endometrium of controls and patients with IUA (n=5 samples for each group). Scale bars: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Expression of marker genes for each cell subtype of myofibroblast-like cells (n=6 samples).


Figure 2Flow cytometric analysis of the proportion of CD14 + cells , CD301 + macrophages , CD80 + macrophages and CD163 + macrophages in the endometrium of normal controls and patients with IUA . Macro , macrophage ( n = 20 samples for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . Representative immunostaining images of CD301 and CD68 from samples of normal controls and patients with IUA ( n = 5 samples for each group ) . Representative immunostaining images of CD301 and α - SMA from samples of normal controls and patients with IUA ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . The protein levels of CD14 and CD301 in CD301 - macrophages and CD301 + macrophages examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . The protein levels of Fibronectin , Collagen 1 , α - SMA and CTGF expression in primary human endometrial stromal cells ( hESCs ) treated by the supernatants of CD301 - and CD301 + macrophages for 48h , respectively examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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 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 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 2Flow cytometric analysis of the proportion of CD14+ cells, CD301+ macrophages, CD80+ macrophages and CD163+ macrophages in the endometrium of normal controls and patients with IUA. Macro, macrophage (n=20 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Representative immunostaining images of CD301 and CD68 from samples of normal controls and patients with IUA (n=5 samples for each group). Representative immunostaining images of CD301 and α-SMA from samples of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.The protein levels of CD14 and CD301 in CD301- macrophages and CD301+ macrophages examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.The protein levels of Fibronectin, Collagen 1, α-SMA and CTGF expression in primary human endometrial stromal cells (hESCs) treated by the supernatants of CD301- and CD301+ macrophages for 48h, respectively examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Figure 3The protein levels of GAS6 and CCL8 expression in sorted human endometrial CD301 - and CD301 + macrophages ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . Localization of GAS6 and CD301 in endometrium of normal controls and patients with IUA examined by immunofluorescence ( n = 5 samples for each group ) . Localization of AXL and α - SMA in endometrium of normal controls and patients with IUA examined by immunofluorescence ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . Western blot analysis of phospho - AXL ( p - AXL ) and AXL in hESCs treated with GAS6 at dose of 10 , 20 , 50 , 100 and 200 ng / mL for 72h ( n = 3 technical replicates for each group ) . The protein levels of Collagen 1 and α - SMA expression in hESCs after treatment with GAS6 at dose of 10 , 20 , 50 , 100 and 200 ng / mL for 72h examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant . Western blot analysis of α - SMA and Collagen 1 in hESCs treated with Bemcentinib ( 1μM ) for 1 hour , followed by GAS6 ( 50ng / mL ) for 72h ( n = 3 technical replicates for each group ) . Western blot analysis of α - SMA and Collagen 1 in hESCs treated with the supernatants of CD301 - and CD301 + macrophages and GAS6 neutralizing antibody ( 2 . 5μg / mL ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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 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 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 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

Figure 3The protein levels of GAS6 and CCL8 expression in sorted human endometrial CD301- and CD301+ macrophages (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Localization of GAS6 and CD301 in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Localization of AXL and α-SMA in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Western blot analysis of phospho-AXL (p-AXL) and AXL in hESCs treated with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h (n=3 technical replicates for each group). The protein levels of Collagen 1 and α-SMA expression in hESCs after treatment with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Western blot analysis of α-SMA and Collagen 1 in hESCs treated with Bemcentinib (1μM) for 1 hour, followed by GAS6 (50ng/mL) for 72h (n=3 technical replicates for each group). Western blot analysis of α-SMA and Collagen 1 in hESCs treated with the supernatants of CD301- and CD301+ macrophages and GAS6 neutralizing antibody (2.5μg/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Figure 4Western blot analysis of phospho - p65 ( p - p65 ) and p65 expression in hESCs treated with GAS6 ( 50ng / mL ) at time of 3 , 6 , 12 , 24 and 48h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Western blot analysis of p - p65 , p65 , Collagen 1 and α - SMA expression in hESCs treated with or without GAS6 neutralizing antibody ( 2 . 5μg / mL ) or Bemcentinib ( 1μM ) for 1 hour , followed by GAS6 protein ( 50ng / mL ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Representative immunostaining images of p - p65 in endometrium of normal controls and patients with IUA ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . ( D ) Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . qRT - PCR analysis of ACTA2 and COL1A1 expression in hESCs treated with GAS6 ( 50ng / mL ) or JSH23 ( 10μM ) for 72h ( n = 3 biological replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Western blot analysis of Collagen 1 and α - SMA expression in hESCs treated with GAS6 ( 50ng / mL ) or JSH23 ( 10μM ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , 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 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 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 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 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

Figure 4Western blot analysis of phospho-p65 (p-p65) and p65 expression in hESCs treated with GAS6 (50ng/mL) at time of 3, 6, 12, 24 and 48h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Western blot analysis of p-p65, p65, Collagen 1 and α-SMA expression in hESCs treated with or without GAS6 neutralizing antibody (2.5μg/mL) or Bemcentinib (1μM) for 1 hour, followed by GAS6 protein (50ng/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Representative immunostaining images of p-p65 in endometrium of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (D) Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.qRT-PCR analysis of ACTA2 and COL1A1 expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 biological replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Western blot analysis of Collagen 1 and α-SMA expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 5Representative images of Masson trichrome staining and immunostaining for Collagen 1 between endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Flow cytometric analysis of the proportions of CD11b + F4 / 80 + cells ( pan - macrophages ) and CD301b + macrophages in the uterus of Sham and IUA mice ( n = 5 mice for each group ) . Macro , macrophage . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Representative images of immunostaining for GAS6 ( red ) and CD301b ( green ) in the endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Representative images of immunostaining for AXL in the endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . ( F ) Mann - Whitney U 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 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 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 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 5Representative images of Masson trichrome staining and immunostaining for Collagen 1 between endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Flow cytometric analysis of the proportions of CD11b+F4/80+ cells (pan-macrophages) and CD301b+ macrophages in the uterus of Sham and IUA mice (n=5 mice for each group). Macro, macrophage. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for GAS6 (red) and CD301b (green) in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for AXL in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (F) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 6qRT - PCR analysis of Acta2 and Col1a1 expression in the uterus of Sham , IUA and IUA + DT mice ( n = 5 mice for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Representative images of immunostaining for AXL , Collagen 1 and p - p65 and Masson trichrome staining in the endometria of Sham , IUA and IUA + DT mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , 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 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

Figure 6qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of Sham, IUA and IUA+DT mice (n=5 mice for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for AXL, Collagen 1 and p-p65 and Masson trichrome staining in the endometria of Sham, IUA and IUA+DT mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 7qRT - PCR analysis of Acta2 and Col1a1 expression in the uterus of IUA + PBS and IUA + BEM mice ( n = 5 mice for each group ) . Data are presented as mean ± SEM . ( B , Two - tailed Student ' s t - test . Representative images of immunostaining for AXL , Collagen 1 , p - p65 and Masson trichrome staining in the endometrium of IUA + PBS group and IUA + BEM group ( n = 5 mice for each group ) . Scale bar , 100μm . Data are presented as mean ± SEM . C for AXL and Collagen 1 Two - tailed Student ' s t - test . ( C for p - p65 ) Mann - Whitney U 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 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

Figure 7qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of IUA+PBS and IUA+BEM mice (n=5 mice for each group). Data are presented as mean ± SEM. (B, Two-tailed Student's t-test.Representative images of immunostaining for AXL, Collagen 1, p-p65 and Masson trichrome staining in the endometrium of IUA+PBS group and IUA+BEM group (n=5 mice for each group). Scale bar, 100μm. Data are presented as mean ± SEM. C for AXL and Collagen 1 Two-tailed Student's t-test. (C for p-p65) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 1Loss of LRRC8D causes resistance to carboplatin and cisplatin , but not to oxaliplatin . Survival of parental , vector ‐ transduced , or LRRC8D ‐ deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin , carboplatin , and oxaliplatin . The corresponding IC50 values and 95 % confidence interval ( CI ) are given in Appendix Table S2 . Data are presented as mean ± SEM .

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

Figure 1Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.


Figure 3A , BVRAC currents ( ICl , vol ) of the HAP1 ( A ) and KBM7 ( B ) haploid cell lines . Left panels , example current traces of ICl , vol fully activated by hypotonic cell swelling measured with the voltage ‐ clamp protocol shown in ( A ) . Dashed lines indicate zero current . Right panels , averaged current / voltage relationships of maximally activated ICl , vol . Consistent with VRAC currents , they needed hypotonic swelling for activation , displayed an I − > Cl − permeability sequence , and were blocked by DCPIB ( Appendix Fig S2A - H ) . The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E ( Fig EV1 ) which accelerates VRAC inactivation ( Voss et al , 2014 ) . At potentials > + 100 mV , also KBM7 currents inactivated ( Appendix Fig S2I ) . Data are presented as mean ± SEM ; n = 5 - 10 . CCDependence of regulatory volume decrease ( RVD ) of HEK cells on LRRC8 genes . Cells were exposed to hypotonic medium starting at t = 0 , and intracellular calcein fluorescence was followed over ˜ 1 h as semiquantitative measure of cell volume . Data are presented as mean values ± SEM from sixteen wells .

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 O O O O O O O O O O O

Figure 3A, BVRAC currents (ICl,vol) of the HAP1 (A) and KBM7 (B) haploid cell lines. Left panels, example current traces of ICl,vol fully activated by hypotonic cell swelling measured with the voltage‐clamp protocol shown in (A). Dashed lines indicate zero current. Right panels, averaged current/voltage relationships of maximally activated ICl,vol. Consistent with VRAC currents, they needed hypotonic swelling for activation, displayed an I− > Cl− permeability sequence, and were blocked by DCPIB (Appendix Fig S2A-H). The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E (Fig EV1) which accelerates VRAC inactivation (Voss et al, 2014). At potentials > +100 mV, also KBM7 currents inactivated (Appendix Fig S2I). Data are presented as mean ± SEM; n = 5-10.CCDependence of regulatory volume decrease (RVD) of HEK cells on LRRC8 genes. Cells were exposed to hypotonic medium starting at t = 0, and intracellular calcein fluorescence was followed over ˜1 h as semiquantitative measure of cell volume. Data are presented as mean values ± SEM from sixteen wells.


Figure 4LRRC8 subunit ‐ and osmolarity ‐ dependent caspase induction in HCT116 cellsA , BCisplatin ‐ induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions ( A ) , or after 1 h exposure to 200 μM cisplatin under iso ‐ and hypotonic conditions ( B ) , was followed over time in WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Results from HAP1 − / − and LRRC8D − / − were obtained with two different clonal cell lines each and averaged . CCaspase activation after 1 ‐ h exposure to 4 μM staurosporine under iso ‐ or hypotonic conditions of WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Data information : Data are presented as mean ± SEM , n = 3 - 6 . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . Similar results were obtained in three independent experiments . Fold change in ( A ) refers to t = 0 . Control experiments indicated that hypotonicity per se had no effect ( Appendix Fig S4 ) .

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

Figure 4LRRC8 subunit‐ and osmolarity‐dependent caspase induction in HCT116 cellsA, BCisplatin‐induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions (A), or after 1 h exposure to 200 μM cisplatin under iso‐ and hypotonic conditions (B), was followed over time in WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells. Results from HAP1−/− and LRRC8D−/− were obtained with two different clonal cell lines each and averaged.CCaspase activation after 1‐h exposure to 4 μM staurosporine under iso‐ or hypotonic conditions of WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells.Data information: Data are presented as mean ± SEM, n = 3-6. *P < 0.05; **P < 0.01; and ***P < 0.001. Similar results were obtained in three independent experiments. Fold change in (A) refers to t = 0. Control experiments indicated that hypotonicity per se had no effect (Appendix Fig S4).


Figure 5Activation of LRRC8 channels by pro ‐ apoptotic stimuliA - CCisplatin ‐ induced iodide influx into WT ( black ■ ) , but not HAP1 − / − ( green ▼ ) HEK cells indicates VRAC halide current activation during apoptosis . Cells expressing an iodide ‐ sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0 . 5 h ( A ) , 4 . 5 h ( B ) , or 8 . 5 h ( C ) before adding extracellular I − ( 50 mM final ) . The difference in slopes of YFPfluorescence quenching between control and cisplatin ‐ treated cells semiquantitatively reflects VRAC current activation . Note that increased YFP quenching with cisplatin preincubation is not due to large non ‐ specific leaks resulting from cell morbidity . Such leaks should lead to a fast component of YFP quenching in WT , but not HAP1 − / − cells after the pipetting artifact that immediately follows addition of iodide ( indicated by arrows ) . DSwelling ‐ induced iodide influx into WT ( black ■ ) and HAP1 − / − ( green ▼ ) HEK cells for comparison . Iodide ( 50 mM final ) was added in isotonic or hypotonic ( 230 mOsm final ) solution at the time indicated by arrow . E , FTime course of VRAC activation by 200 μM cisplatin ( E ) or 4 μM staurosporine ( F ) determined as in ( A - C ) . Averaged maximal slopes of YFP quenching from eight wells ( E ) or 16 wells ( F ) each were evaluated to estimate iodide influx rates . WT ( black ■ ) and HAP1 − / − ( green ▼ ) . Data are presented as mean ± SEM .

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

Figure 5Activation of LRRC8 channels by pro‐apoptotic stimuliA-CCisplatin‐induced iodide influx into WT (black ■), but not HAP1−/− (green ▼) HEK cells indicates VRAC halide current activation during apoptosis. Cells expressing an iodide‐sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0.5 h (A), 4.5 h (B), or 8.5 h (C) before adding extracellular I− (50 mM final). The difference in slopes of YFPfluorescence quenching between control and cisplatin‐treated cells semiquantitatively reflects VRAC current activation. Note that increased YFP quenching with cisplatin preincubation is not due to large non‐specific leaks resulting from cell morbidity. Such leaks should lead to a fast component of YFP quenching in WT, but not HAP1−/− cells after the pipetting artifact that immediately follows addition of iodide (indicated by arrows).DSwelling‐induced iodide influx into WT (black ■) and HAP1−/− (green ▼) HEK cells for comparison. Iodide (50 mM final) was added in isotonic or hypotonic (230 mOsm final) solution at the time indicated by arrow.E, FTime course of VRAC activation by 200 μM cisplatin (E) or 4 μM staurosporine (F) determined as in (A-C). Averaged maximal slopes of YFP quenching from eight wells (E) or 16 wells (F) each were evaluated to estimate iodide influx rates. WT (black ■) and HAP1−/− (green ▼). Data are presented as mean ± SEM.


Figure 6Carboplatin uptake into control KBM7 cells and two LRRC8D ‐ deficient clones ( n = 6 ) . Hypotonicity ‐ stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone ( CBX ) , a non ‐ specific blocker of VRAC ( n = 3 ) . Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long ‐ term isotonic ( C ) or 200 μM cisplatin in short ‐ term hypo ‐ and isotonic ( D ) conditions as function of time ( n = 3 ) . Similar results were obtained in HCT116 cells ( Fig EV4 ) . Cisplatin uptake ( 200 μM ) into HEK cells of indicated genotypes . LRRC8 ( B , C , E ) − / − and LRRC8 ( B , D , E ) − / − cells express only LRRC8A and LRRC8D , and LRRC8A and LRRC8C , respectively ( n = 3 for WT , 6 for LRRC8D − / − , 9 for LRRC8A − / − in E ; n = 3 in F ) . Mean current densities of maximally activated ICl , vol at − 80 mV . The number of cells is indicated for each column . Ratio of LRRC8 ‐ dependent swelling ‐ activated cisplatin uptake ( 60 min ) to mean ICl , vol ( as in G ) as function of genotype . Data information : Data are presented as mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 ( for ( C , E ) compared to HAP1 − / − cells ) . Dotted lines in ( D ) and ( F ) highlight that there is no significant difference in isotonic 60 ‐ min uptake between the genotypes .

O B-PANEL_START 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 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 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 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 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

Figure 6Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F).Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column.Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.


Figure 7Cisplatin - DMSO inhibition of ICl , vol depends on the LRRC8D subunitUpper panel , example current traces ( as in Fig 3A ) of fully activated ICl , vol in HEK cells exposed to hypotonic solution containing vehicle ( 0 . 3 % DMSO ) or 200 μM cisplatin in 0 . 3 % DMSO . Dashed lines indicate zero current . Lower panel , ICl , vol current densities ( at − 100 mV and 100 mV ) of WT HEK cells treated with different cisplatin concentrations . No effect of 200 μM cisplatin / DMSO on ICl , vol in LRRC8D − / − HEK cells . Data information : Data are presented as mean ± SEM ; the number of experiments is given for each bar ; * P < 0 . 05 ; * * P < 0 . 01 .

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

Figure 7Cisplatin-DMSO inhibition of ICl,vol depends on the LRRC8D subunitUpper panel, example current traces (as in Fig 3A) of fully activated ICl,vol in HEK cells exposed to hypotonic solution containing vehicle (0.3% DMSO) or 200 μM cisplatin in 0.3% DMSO. Dashed lines indicate zero current. Lower panel, ICl,vol current densities (at −100 mV and 100 mV) of WT HEK cells treated with different cisplatin concentrations.No effect of 200 μM cisplatin/DMSO on ICl,vol in LRRC8D−/− HEK cells.Data information: Data are presented as mean ± SEM; the number of experiments is given for each bar; *P < 0.05; **P < 0.01.


Figure 8Swelling ‐ induced efflux of 3 [ H ] ‐ taurine from WT and LRRC8D − / − HEK cells ( A , B ) and partial rescue by transient transfection of LRRC8D ( B ) . Rescue is incomplete due to low transfection / expression efficiency of LRRC8D ( Voss et al , 2014 ) . C - ESwelling ‐ induced efflux of 3 [ H ] ‐ taurine from LRRC8 ( B , C , E ) − / − ( C ) , LRRC8 ( B , D , E ) − / − ( D ) , or LRRC8 ( B , C , D ) − / − ( E ) HEK cells compared to WT cells . FCisplatin ‐ induced taurine efflux ( over 30 min ) from WT and HAP1 − / − HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h , or without cisplatin ( 0 h ) . Carbenoxolone ( CBX ; 100 μM ) blocks taurine efflux from WT cells treated for 12 h with cisplatin , excluding taurine flux through unspecific leaks . Data information : Data are presented as mean ± SEM . Dashed lines in ( A - E ) represent isotonic efflux from the same individual experiments . Red arrows , change to hypotonic solution . Each panel shows the average of two independent experiments ( n = 4 for each experiments , i . e . , n = 8 in total ) .

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 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 8Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D −/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).C-ESwelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E) −/− (C), LRRC8(B,D,E) −/− (D), or LRRC8(B,C,D) −/− (E) HEK cells compared to WT cells.FCisplatin‐induced taurine efflux (over 30 min) from WT and HAP1−/−HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.Data information: Data are presented as mean ± SEM. Dashed lines in (A-E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).


Figure 1 ( A ) Netrin - 1 ( NTN1 ) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas . ( B ) Reporter gene expression in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) ) of netrin - 1 + / lacZ + adult ( P60 ) mice incubated with S - Gal . ( Scale bar , 2000 µm ) . Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel . ( C ) Netrin - 1 protein localization in the SN of adult rat . ( Scale bar , 200 µm ) . Upper panel , confocal image of the SN . Higher magnification of the SNpc ( bottoms panels ) . Netrin - 1 ( red ) , Tyrosine hydroxylase ( TH ) ( green ) . ( D ) Localization of netrin - 1 protein in the adult rat striatum . ( Scale bar , 600µm ) . Left panel , confocal image of the rat striatum . Netrin - 1 ( red ) , Dopamine transporter ( DAT ) ( green ) . Right panels , higher magnification .

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 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 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

Figure 1(A) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.(C) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.


Figure 2 ( A ) Representative images of dopamine neurons ( TH - positive ) in the substantia nigra ( SN ) ( left panel ) and in the SN and striatum ( right panel top , and bottom respectively ) by immunofluorescence , six weeks after the intranigral injection of control ( CTL ) ( AAV6 - GFP ) or Cre ( AAV6 - GFP Cre ) adenovirus vectors in Netrin - 1fl / fl mice ( left panel : green - GFP , red - TH ; right panel : Cy - 5 - TH ) ( Scale bar , 1000 μm ) . Filled arrows indicate SN TH dopamine neurons . ( B ) Representative images of TH and netrin - 1 stainings by immunofluorescence ( scale bar , 2000 μm ) six weeks after the intranigral injection of CTL adenovirus vector ( left panel ) or Cre adenovirus vector ( right panel ) , in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) of Netrin - 1fl / fl mice . ( C ) Quantification of fluorescence intensity of TH - positive neurons , in the substantia nigra ( SN ) ( left ) , and fibres , in the striatum ( right ) , six weeks after adenovirus vector injection , in Netrin - 1fl / fl mice . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 . N = 3 each group . ( D ) Terminal deoxynucleotidyl transferase ( TdT ) dUTP Nick - End Labelling ( TUNEL ) reactivity in the SN after Cre and control adenovirus vector injection , in Netrin - 1fl / fl mice ( Scale bar , 50μm ) . The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * * P < 0 . 01 . N = 3 each group . ( E ) Motor behavior assays at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ( F ) Immunoblot of SN lysates from netrin - 1 wild type ( WT ) and netrin - 1 conditional KO mice ( left upper panel ) . Densitometry band quantification of cleaved receptor over full length receptor ratio ( lower right panel ) . We randomly selected 2 mice from each group for immunoblot analysis . N = 3 independent experiments . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 .

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

Figure 2(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.(C) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.(D) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.(E) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05(F) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


Figure 3 ( A ) Representative images of TH + dopamine neurons and fibers staining by immunofluorescence , in the SN and the striatum ( Str ) 6 weeks after CTL ( AAV6 - GFP ) ; Cre ( AAV6 - GFP Cre ) ; sh DCC ( AAV6 - GFP Cre + Sh - DCC ) and shUNC5B ( AAV6 GFP Cre + Sh - UNC5B ) injection into the SN of Netrin - 1fl / fl mice . ( Scale bar , 2000 µm ) . ( B ) Quantification of TH fluorescent intensity in the SN ( upper bar graph ) and striatum ( Str ) of Netrin - 1fl / fl mice ( lower bar graph ) . Data are shown as mean + SEM . Statistical significance was determined by an unpaired t - test . N = 3 each group . Tukey post hoc analysis after one - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , N . S . , not significant . ( C ) DCC depletion mitigates netrin - 1 depletion - induced apoptosis in Netrin - 1fl / fl mice . TH - positive cell loss was assessed by TUNEL assay in the SN . Upper panel , TUNEL ( red ) and TH ( Cy5 - white ) ( Scale bar , 50 µm ) . Apoptotic index ( bar graph , bottom panel ) expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . N = 3 each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * P < 0 . 05 ; P * * < 0 . 01 . ( D ) Immunoblot of SN lysates ( upper panel ) and corresponding quantification ( bottom panel ) . Quantification of band intensity ( bottom ) . Mouse N = 2 . N = 3 independent immunoblot analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 . ( E ) Motor behaviour tests at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * 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 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 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

Figure 3(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.


Figure 4 ( B ) Amphetamine - induced rotations at two and six weeks after lesion . Individual values and Mean ± SD are shown , n = 11 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 compared to WT . ( C ) Optical density of TH - positive fibres in the striatum . Individual values and mean ± SD , n = 10 animals , unpaired t - test , * P < 0 . 05 . ( D ) TH - positive cell count in the SN . Individual values and mean ± SD , n = 10 animals , unpaired t - test . ( F ) TH staining by immunohistochemistry ( Scale bar , 2000µm ) in the SN ( left ) and striatum ( right ) of WT and SNCA Tg mice , 6 days after treatment . ( G ) TH - positive cell count bar graph . All experiments were repeated 3 times . We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * * P < 0 . 01 ; N . S . , not significant . Motor behavioural assays ( Rotarod rod test ( H ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 . Motor behavioural assays Cylinder test ( I ) , Grid test ( J ) ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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 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 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 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 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

Figure 4(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.Motor behavioural assays (Rotarod rod test (H) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.Motor behavioural assays Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


Figure 5 ( B ) Cumulative amphetamine - induced ipsilateral rotations . Progression of amphetamine - induced ipsilateral rotations over time ( right panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * * * * P < 0 . 0001 compared to the initial rotation score at week 2 . Comparison of the treatment ' s effect between groups ( left panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group . ( C ) Dopamine fibres in the striatum of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the striatum showing TH IHC staining ( left panel ) , intact side on the left and 6 - OHDA lesioned side on the right . ( Scale bar , 2000 µm ) . Optical density measurement of TH - positive fibres in the lesioned side of the striatum ( bar graph , right panel ) . Individual values and mean ± SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the bottom ( D ) Dopamine cell bodies in the SN of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the SN ( lesioned side ) showing TH - positive cell bodies stained by IHC ( left panel ) ( Scale bar , 2000 µm ) . TH - positive cell count in the SN ( right panel ) . Individual values and mean + SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the bottom .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.


Figure 6 ( A ) Netrin - 1 gene expression profiling by array of substantiae nigrae from PD and non - PD ( Control ) patients using the GEO dataset GSE7621 that has a total of 25 samples ( n = 9 control and n = 16 PD cases ) . Unpaired t - test , * * * P < 0 . 0005 , Mean ± SD are shown . ( B ) Immunoblot of netrin - 1 , DCC , UNC5B , active caspase - 3 , alpha synuclein FL , alpha synuclein pS129 , and Tyrosine Hydroxylase ( TH ) levels in age - matched controls ( n = 3 ) vs Parkinson ' s disease patients brain samples ( n = 3 ) ( left panel ) . Band quantification bar graph ( right panel ) . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . P * * < 0 . 01 . N = 3

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 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

Figure 6(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates


Figure 3 ( C ) Immunohistochemistry for C . burnetii in the lung on d7 after i . t . infection . Representative images and quantitation of lung tissue positive for C . burnetii ( n = 12 - 13 mice , pooled from three experiments , each normalized to the mean of WT or Acod1 + / - infected mice ) . Each dot represents one mouse . ( E ) Immunohistochemistry for C . burnetii on day 7 after intraperitoneal infection . Each dot represents one mouse .

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

Figure 3(C) Immunohistochemistry for C. burnetii in the lung on d7 after i.t. infection. Representative images and quantitation of lung tissue positive for C. burnetii (n=12-13 mice, pooled from three experiments, each normalized to the mean of WT or Acod1+/- infected mice). Each dot represents one mouse.(E) Immunohistochemistry for C. burnetii on day 7 after intraperitoneal infection. Each dot represents one mouse.


Figure 5representative images of liver sections and quantification of immunohistochemistry for C . burnetii in liver sections ( G ) . n = 6 - 11 mice ( for NMII infections ) , pooled from two experiments . Each dot represents one mouse . ( Mann - Whitney test comparing treatment and control group of NMII infected Acod1 - / - mice in specified organs , *

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

Figure 5representative images of liver sections and quantification of immunohistochemistry for C. burnetii in liver sections (G). n=6-11 mice (for NMII infections), pooled from two experiments. Each dot represents one mouse. (Mann-Whitney test comparing treatment and control group of NMII infected Acod1-/- mice in specified organs, *


Figure 1A . qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice ; n = 4 / group ; * P < 0 . 05 , Unpaired t - test , two - tailed . Note that the transgene is not detected in the brain . C . Ejection fraction , cardiac output and index are significantly decreased in CamkIIδc TG mice ( n = 8 ) when compared to control mice ( n = 5 ; * P < 0 . 05 , , * * P < 0 . 01 , * * * P < 0 . 001 , Unpaired t - test , two - tailed ) . Heart to body ratio , left ventricle to body ratio , and lung to body weight ratio are increased in CamkIIδc TG ( n = 8 ) compared to control ( n = 5 ; * P < 0 . 05 , * * P < 0 . 01 , Unpaired t - test ; two - tailed ) . H . qPCR quantification of selected genes reflecting ER - stress or protein methylation - related processes in CamkIIδc TG mice ( n = 4 ) and control mice ( n = 5 ) . * p < 0 . 05 , ns = non - significant , Unpaired t - test ; two - tailed . Data is normalized to Hprt1 expression .

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 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 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 1A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.


Figure 2D . The cumulative score of hippocampus - dependent search strategies during Barnes maze training is impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . E . Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . Data information : Bars and error bar indicates mean ± SEM .

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

Figure 2D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.


Figure 3A . Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A , Kmt2A and Kmt2b knock out mice . Fisher ' s hypergeometric test , Benjamini - Hochberg ( BH ) correction .

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

Figure 3A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.


Figure 4E . Cumulative hippocampus - dependent strategy scores during the Barnes maze training ( One - way ANOVA ) . n = 10 per group . F . Number of visits to the escape hole during probe test ( n = 10 / group , One - Way ANOVA ) . Data information : Bars and error bars indicate mean ± SEM .

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

Figure 4E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.


Figure 5E . H3K4me3 at TSS region of genes from RNA module1 . Inset shows statistical comparisons among groups ( One - way ANOVA , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Bars and error bars indicate mean ± SEM . Number of replicates analyzed for H3K4me3 profile at TSS ( Wildtype vehicle : 3 ; CamkIIδc TG - vehicle n = 4 ; CamkIIδc TG - Vorinostat n = 4 ) .

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

Figure 5E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).


Figure 1 ( A ) Raw 264 . 7 cells were pre - treated with DMSO or the EGFR inhibitor gefitinib ( 10 µM ) before cGAMP transfection ; after 5h , IFNβ mRNA was measured by qRT - PCR ( n = 3 ) . ( B ) WT and EGFR knockdown Raw 264 . 7 cells were transfected with cGAMP ; 4 . 5h post treatment , IFNβ mRNA was measured ( n = 3 ) . ( C ) WT and EGFR - / - BMDMs were transfected with cGAMP ; 5h post transfection , IFNα mRNA induction was measured ( n = 4 ) . ( D ) WT and EGFR - / - HeLa cells were transfected with cGAMP ; after 5h IFNβ mRNA was measured ( n = 3 ) . MEF cells were pre - treated with gefitinib for 1h and then transfected with cGAMP ; 4 . 5h post treatment , total RNA was harvested for analysis . ( E ) RNA - seq analysis ( n = 2 ) . For RNA - seq analysis , poly T enriched cDNA library was prepared and 30 M single - end 50 bp reads were analyzed . The heat map for some of the genes is shown . MEF cells were pre - treated with gefitinib for 1h and then transfected with cGAMP ; 4 . 5h post treatment , total RNA was harvested for analysis . ( G ) The RNA - seq analysis results were confirmed by qRT - PCR analysis of a gefitinib - sensitive and an insensitive mRNA ( n = 3 ) . ( H ) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly ( I : C ) transfection ; 4 . 5h post treatment , total RNA was harvested and IFNα mRNA induction was measured ( n = 3 ) .

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 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 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 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 1(A) Raw 264.7 cells were pre-treated with DMSO or the EGFR inhibitor gefitinib (10 µM) before cGAMP transfection; after 5h, IFNβ mRNA was measured by qRT-PCR (n=3).(B) WT and EGFR knockdown Raw 264.7 cells were transfected with cGAMP; 4.5h post treatment, IFNβ mRNA was measured (n=3).(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4).(D) WT and EGFR-/- HeLa cells were transfected with cGAMP; after 5h IFNβ mRNA was measured (n=3).MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (E) RNA-seq analysis (n=2). For RNA-seq analysis, poly T enriched cDNA library was prepared and 30 M single-end 50 bp reads were analyzed. The heat map for some of the genes is shown.MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (G) The RNA-seq analysis results were confirmed by qRT-PCR analysis of a gefitinib-sensitive and an insensitive mRNA (n=3).(H) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly (I:C) transfection; 4.5h post treatment, total RNA was harvested and IFNα mRNA induction was measured (n=3).


Figure 2 ( A ) MEF cells were pre - treated with gefitinib or DMSO for 1 hour , and then infected with HSV1 at m . o . i . of 1 . 4 hours post infection ( h . p . i . ) , IFNβ mRNA induction was monitored by qRT - PCR ( n = 3 ) . Uninfected cells were used as negative control ( U ) . ( B ) DNA was extracted from gefitinib - treated or untreated infected MEFs 0 and 16 h . p . i . and viral DNA was measured by qPCR ( n = 3 ) . ( C ) WT and EGFR KO HeLa cells were infected with HSV1 , RNA was extracted at 4 h . p . i . and IFNα mRNA induction was measured ( n = 3 ) . ( D ) Survival of uninfected mice treated with gefitinib and HSV1 - infected mice treated or untreated with gefitinib . ( E ) Survival of WT and EGFRfl + / - LysMCre + / + mice infected with HSV1 . WT and EGFRfl + / - LysMCre + / + mice infected with HSV1 . HSV1 titers in the brains of the live mice at day 9 were determined .

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 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 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 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

Figure 2(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U).(B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3).(C) WT and EGFR KO HeLa cells were infected with HSV1, RNA was extracted at 4 h.p.i. and IFNα mRNA induction was measured (n=3).(D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib.(E) Survival of WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1.WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1. HSV1 titers in the brains of the live mice at day 9 were determined.