Figure 4 B , C , Immunoblots ( B ) and viability assay ( C ) of H358 and A549 cells preincubated overnight with SP600125 ( 2 µM ) and SB203580 ( 1 µM ) and subsequently treated with AZD 4547 ( 5 µM ) and BI2536 ( 5 nM ) for 24 hours . Data were shown as mean ± SD ( n = 3 ) ; * * * P < 0 . 001 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . D , E , Immunoblots ( D ) and viability assay ( E ) of H358 and A549 cells transfected with E2F1 - specific or control siRNAs followed by treatment ( 48 h post transfection ) with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) for additional 24 hours . Data were shown as mean ± SD ( n = 3 ) . * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . F , H358 cells transfected with E2F1 - specific or control siRNAs for 24h were subsequently treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination for 48h before apoptotic assay . Data were shown as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


Figure 5 B , A549 cells stably expressing mCHerry - GFP - LC3B were exposed to vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 24h . Cell were then fixed and processed for immunofluorescence , scale bar = 10 µm . E , Flow cytometry - based ROS measure in H358 , A549 and MIA PaCa - 2 cells treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or the combination , in the presence or absence of HCQ ( 10 µM ) for 24h . Quantification of relative ROS levels was shown to the right . Data are presented as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . F , Immunoblots of H358 cells transfected with ATG5 - specific or control siRNAs for 48h and before treated for 24h with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm.  E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).


Figure 6 A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar : SD D , E , H & E and IHC analysis ( Ki67 and Caspase - 3 ) of PDX ( BE 564T ) tumors after the treatment ( D ) . Quantification of the IHC data for the positivity of Ki - 67 and Caspase - 3 are shown in ( E ) . * P < 0 . 05 and * * P < 0 . 01 by two - way ANOVA with Tukey ' s multiple comparisons test . Data are shown as mean ± SD ( error bar ) of three independent experiments ( n = 3 ) . F - K , Growth curves ( F , I ) of KRAS - mutant lung cancer PDXs ( PF139 , PF563 ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 4 mice / group ) ; error bar : SD Relative tumor volume ( G , J ) and weights ( H , K ) of the PDX tumors after the treatment . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by unpaired two - tailed t - test . Data are the mean ± SD ( error bar ) of tumor weights of each group ( 4 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD  D, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).  F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).


Figure 7 F , Box plots showing the percentage of Ki67 - positive cells in different treatment groups . Data are based on IHC staining of lung tissue section of LSL - KrasG12D mice . * P < 0 . 05 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . The boxplots span from the first to third quartile , the whiskers extend to 1 . 5 × the interquartile range ( IQR ) . The horzontal line within the boxes represents the median of each group of 10 replicates ( n = 10 ) .

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

Figure 7 F, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10).


Figure 1A . Immunoblot of cell extracts following depletion of WDR26 , RanBP9 , ARMC8 , and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells . Endogenous levels of the indicated proteins were monitored by western blotting ( n = 3 ) . Western blotting of samples after ectopic overexpression of HBP1 ( B ) alone , or together with WDR26 or GID4 in HEK - 293T cells . HBP1 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) . Western blotting of samples after ectopic overexpression of ZMYND19 ( C ) alone , or together with WDR26 or GID4 in HEK - 293T cells . ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

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 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

Figure 1A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3).Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


Figure 2C . , D . Western blot analysis of in vitro ubiquitinated HBP1 , which was performed by mixing purified HBP1 with ubiquitin E1 , UBCH5a , and ubiquitin in the presence of the indicated hGID sub - complexes ( n = 3 ) . E . Immunoblots of in vitro ubiquitinated ZMYND19 , which was performed by mixing purified ZMYND19 with ubiquitin E1 , UBE2H , ubiquitin and the 6 - subunit hGID complex ( ARCM8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) in the presence or absence of GID4 and a 10 - fold excess of the PGLV GID4 - specific peptide ( n = 2 )

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

Figure 2C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)


Figure 3D . Western blotting of samples following ectopic overexpression of HBP1 either alone , or with full - length ( FL ) or WD40 - truncated WDR26 ( ΔWD40 ) in HEK - 293T cells . HBP1 levels were monitored in cells treated with MG132 or DMSO for 12 - 14 h ( n = 3 ) . E . Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 ( WT ) or the WDR26 ( ΔWD40 ) mutant ( n = 2 ) .

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

Figure 3D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).


Figure 4C . Transiently expressed FLAG - GID4 and HSS - tagged ARMC8 isoforms ( α or β ) in HEK293T cells . The presence of GID4 in isoform - specific ARMC8 immunoprecipitates was visualized by immunoblotting ( left panels ) . The right panel shows a western blot of transiently expressed and immunoprecipitated HSS - WDR26 from HEK - 293T cells and the presence of endogenous ARMC8 isoforms ( α or β ) was probed by immunoblotting ( n = 2 ) . D . Baculoviral co - expression in Sf9 cells of the 5 - subunit hGID complex ( 5mer ; Strep - RanBP9 , His - WDR26 , FLAG - MAEA , His - RMND5a and His - TWA1 ) along with His - GID4 in the presence of ARMC8α or ARMC8β . Strep - or His - pulldowns revealed the presence of GID4 in ARMC8α , but not in ARMC8β , complexes ( n = 3 ) . E . Immunoblot analysis of in vitro ubiquitinated GID4 - hGID complexes containing either ARMC8α or ARMC8β ( 6mer ; ARMC8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) . Where indicated , the reaction was carried out in the presence of 20 - fold molar excess of the PGLV GID4 - binding peptide ( n = 3 ) . F . Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5 - subunit complex in the presence of the different ARMC8 isoforms ( α or β ) ( n = 2 ) .

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 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 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

Figure 4C. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2).D. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3).E. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3).F. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2).


Figure 1Subcellular localization of dLap1mGFP and dTormGFP - E177Q in the wild - type ( w - ) or dLap1 + / ∆ 11 fat body of 3rd instar fly larvae , when expressed as UAS transgenes . Neutral lipid staining of the 5DO larval fat body . White arrows ( O ) indicate nuclear - localized neutral lipid staining , blue signal shows DAPI .

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 O O O O O O O

Figure 1Subcellular localization of dLap1mGFP and dTormGFP-E177Q in the wild-type (w-) or dLap1+/∆11 fat body of 3rd instar fly larvae, when expressed as UAS transgenes. Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI.


Figure 2GFP , Phalloidin and LipidTOX detect the expression of dTormGFP chimeras ( green ) , cell boundaries ( red ) , and LD ( red ) , respectively , in dTorKO fat body cells .

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

Figure 2GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.


Figure 3Bars show the fraction ( mean ± SEM ) of membrane GL and GPL lipids with each fatty acyl chain characteristic . n = 3 MS analyses of 4DO fat body samples for each genotype . MUFA , mono - unsaturated ; PUFA , poly - unsaturated . ‡ indicate a significant difference between w - and dTorKO expressing Luciferase RNAi , * indicate a significant difference compared to dTorKO expressing Luciferase RNAi . Two - way ANOVA , Dunnett ' s multiple comparison test . ‡ p < 0 . 05 and * 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 O O O O O O O O O O O O

Figure 3Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.


Figure 4Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg - Gal4 driver , and labeled with anti - Calnexin ( red ) and a neutral lipid dye ( LipidTOX , magenta ) . Note that dTorKO cells are smaller . White arrowhead indicates NE enrichment of LipinmGFP . Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co - expresses CTDNEP1myc - WT or - CD ( red ) and NEP1R1V5 ( magenta ) . ( G ' ) Upper : enlargement of panel G , white arrow highlights NE enrichment . Lower : plot showing the intensity of Lipin1mGFP signal ( mean ± SD , n = 5 cells ) along a 3 µm profile that transects the NE at 0 µm . Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP . Chi - square test . * * * * p < 0 . 0001 .

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 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 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

Figure 4Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP.Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co-expresses CTDNEP1myc-WT or -CD (red) and NEP1R1V5 (magenta). (G') Upper: enlargement of panel G, white arrow highlights NE enrichment. Lower: plot showing the intensity of Lipin1mGFP signal (mean ± SD, n = 5 cells) along a 3 µm profile that transects the NE at 0 µm.Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.


Figure 5Control ( w - ) and dTorKO fat body nuclei labeled with anti - LaminDm0 , Megator / Tpr , mAb414 , or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg - Gal4 driver .

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

Figure 5Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver.


Figure 6mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA , Luciferase RNAi , or RNAi against Nep1r1 , Ctdnep1 , or Lipin . Nuc , nucleus . The percentage of fat body cells with NE - localized mAb414 . * * and * * * * indicate that Lipin , Ctdnep1 , and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE - specific mAb414 labeling compared to the Luciferase RNAi ( Luc ) . Chi - square test followed by individual post hoc Chi - square tests . * * p < 0 . 01 and * * * * p < 0 . 0001 . NE ultrastructure in 5DO dTorKO cells . Red arrows , nuclear pores ; yellow arrows , abnormal INM herniations . Nuc , nucleus ; Cyto , cytosol . The percentage that Lipin , Ctdnep1 , and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body . 0 % " normal " reflects lipid abundance in dTorKO expressing Luciferase RNAi , while 100 % " normal " reflects lipid abundance in w - expressing Luciferase RNAi . Percentage normalization was separately calculated for PI , PA , DAG , PC , PE and PS ( for dTorKO expressing Lipin , Ctdnep1 , and Nep1r1 RNAi ) . Points show the mean of all lipid class normalization per sample , bars show mean ± SEM for the group .

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 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

Figure 6mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin. Nuc, nucleus. The percentage of fat body cells with NE-localized mAb414. ** and **** indicate that Lipin, Ctdnep1, and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE-specific mAb414 labeling compared to the Luciferase RNAi (Luc). Chi-square test followed by individual post hoc Chi-square tests. ** p < 0.01 and **** p < 0.0001. NE ultrastructure in 5DO dTorKO cells. Red arrows, nuclear pores; yellow arrows, abnormal INM herniations. Nuc, nucleus; Cyto, cytosol.The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.


Figure 7NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes , or from dLap1 - / - animals . Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture . Red arrows indicate nuclear pores . R1 , region 1 ; R2 , region2 ; Nuc , nucleus ; Cyto , cytosolData from FIB - SEM imaging of fat body nuclei expressing Sec61b ( control ) , or CTDNEP1mGFP . ( J - L ) 3D reconstructions , ( M ) distance between the INM and ONM at NPC in wild - type cells versus CTDNEP1mGFP - induced structures . Kolmogorov - Smirnov non - parametric T - Test . * * * * p < 0 .

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

Figure 7NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes, or from dLap1-/- animals. Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture. Red arrows indicate nuclear pores. R1, region 1; R2, region2; Nuc, nucleus; Cyto, cytosolData from FIB-SEM imaging of fat body nuclei expressing Sec61b (control), or CTDNEP1mGFP. (J - L) 3D reconstructions, (M) distance between the INM and ONM at NPC in wild-type cells versus CTDNEP1mGFP-induced structures. Kolmogorov-Smirnov non-parametric T-Test. **** p < 0.0001


Figure 8mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1 - / - animal . * * * and * * * * indicate significant difference compared to control mGFPSec61b expressing cells . Chi - square test followed by individual post hoc Chi - square tests . * * * p < 0 . 001 and * * * * p < 0 . 0001 . NE ultrastructure of 5DO fat body cells from a ( I ) control animal , ( J ) homozygous Nup35BG01311 and ( K ) homozygous Nup35MB02683 animals . Nuc , nucleus ; Cyto , cytosol .

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

Figure 8mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1-/- animal. *** and **** indicate significant difference compared to control mGFPSec61b expressing cells. Chi-square test followed by individual post hoc Chi-square tests. *** p < 0.001 and **** p < 0.0001.NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35BG01311 and (K) homozygous Nup35MB02683 animals. Nuc, nucleus; Cyto, cytosol.


Figure 1 ( H Anti - FruM signals in 3 - 6h WTCS males are stronger than those in 4 - 6d WTCS males ( H ) n = 7 for each . Data information : Scale bars , 100 μm . ( J and Anti - GFP signals are grossly similar in 3 - 6h and 4 - 6d UAS - myrGFP ; fruGAL4 males ( J ) , n = 6 for each . Data information : Scale bars , 100 μm .

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

Figure 1(H Anti-FruM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (H) n = 7 for each. Data information: Scale bars, 100 μm.(J and Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP; fruGAL4 males (J), n = 6 for each. Data information: Scale bars, 100 μm.


Figure 2 ( A ) Wing extension indices ( zero ) of isolated 3 - 6h juvenile w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males at 22 and 27 ℃ . n = 20 for each . ( B and C ) Wing extension indices ( B ) and latencies ( C ) of isolated 4 - 6d adult w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males at 22 and 27 ℃ . n = 20 for each . n . s . , not significant , * p < 0 . 05 , * * * p < 0 . 001 , Mann - Whitney U test for ( B ) and unpaired t test for ( C ) . NA , not applicable for latency analysis . Data information : Error bars indicate SEM . ( E and F ) Tracking indices of 3 - 6h and 4 - 6d w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males toward moving dots at 22 and 27 ℃ . n = 24 for each in ( E ) . n = 22 , 20 , 24 , 24 , 24 and 24 from left to right in ( F ) . n . s . , not significant , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Error bars indicate SEM .

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 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 2(A) Wing extension indices (zero) of isolated 3-6h juvenile w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. (B and C) Wing extension indices (B) and latencies (C) of isolated 4-6d adult w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. n.s., not significant, *p < 0.05, ***p < 0.001, Mann-Whitney U test for (B) and unpaired t test for (C). NA, not applicable for latency analysis. Data information: Error bars indicate SEM.(E and F) Tracking indices of 3-6h and 4-6d w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males toward moving dots at 22 and 27℃. n = 24 for each in (E). n = 22, 20, 24, 24, 24 and 24 from left to right in (F). n.s., not significant, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.


Figure 3 ( A and B ) qPCR experiments show that Met ( A ) and Gce ( B ) mRNA levels are significantly knocked down in actin - GAL4 > UAS - Met : : Gce - IR adult flies . n = 9 for each . * * * p < 0 . 001 , unpaired t test . Data information : Error bars indicate SEM . Courtship behaviors of 3 - 6h juvenile and 4 - 6d adult males are not significantly affected by knocking down JH receptors Met and Gce ( UAS - Met : : Gce - IR ) pan - neuronally or specifically in neurons driven by 57C10 - GAL4 ( C ) , fruGAL4 ( D ) N = 22 for each in ( C ) , n = 24 for each in ( D ) n . s . , not significant , Mann - Whitney U test . Data information : Error bars indicate SEM . Courtship behaviors of 3 - 6h juvenile and 4 - 6d adult males are not significantly affected by knocking down JH receptors Met and Gce ( UAS - Met : : Gce - IR ) pan - neuronally or specifically in R41A01 - GAL4 ( E ) , NPF - GAL4 ( F ) , TH - GAL4 ( G ) and R71G01 - GAL4 ( H ) respectively . n = 24 , 24 , 21 , and 22 from left to right in ( E ) , n = 24 , 24 , 24 , and 23 from left to right in ( F ) , n = 24 , 24 , 20 and 24 from left to right in ( G ) and n = 24 , 24 , 23 and 24 from left to right in ( H ) . n . s . , not significant , Mann - Whitney U test . Data information : Error bars indicate SEM .

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

Figure 3(A and B) qPCR experiments show that Met (A) and Gce (B) mRNA levels are significantly knocked down in actin-GAL4>UAS-Met::Gce-IR adult flies. n = 9 for each. ***p<0.001, unpaired t test. Data information: Error bars indicate SEM.Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in neurons driven by 57C10-GAL4 (C), fruGAL4 (D) N = 22 for each in (C), n = 24 for each in (D) n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in R41A01-GAL4 (E), NPF-GAL4 (F), TH-GAL4 (G) and R71G01-GAL4 (H) respectively. n = 24, 24, 21, and 22 from left to right in (E), n = 24, 24, 24, and 23 from left to right in (F), n = 24, 24, 20 and 24 from left to right in (G) and n = 24, 24, 23 and 24 from left to right in (H). n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.


Figure 4 ( A - D ) Expression pattern of R41A01 - GAL4 ( pCd , A ) , NPF - GAL4 ( B ) , TH - GAL4 ( C ) , and R71G01 - GAL4 ( P1 , D ) in adult male brains . Representative of three samples for each . Data information : Scale bars , 100 μm . ( I - L ) Courtship behaviors of 4 - 6d adult males toward virgin females are similarly intensive and not significantly influenced by activating pCd ( I ) , NPF ( J ) , TH ( K ) or P1 ( L ) neurons at 29 ℃ . n = 24 for each in ( I ) , n = 24 , 24 , 23 , and 23 from left to right in ( J ) , n = 24 , 24 , 24 , and 23 from left to right in ( K ) , and n = 24 for each in ( L ) . n . s . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Error bars indicate SEM .

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 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

Figure 4(A-D) Expression pattern of R41A01-GAL4 (pCd, A), NPF-GAL4 (B), TH-GAL4 (C), and R71G01-GAL4 (P1, D) in adult male brains. Representative of three samples for each. Data information: Scale bars, 100 μm.(I-L) Courtship behaviors of 4-6d adult males toward virgin females are similarly intensive and not significantly influenced by activating pCd (I), NPF (J), TH (K) or P1 (L) neurons at 29℃. n = 24 for each in (I), n = 24, 24, 23, and 23 from left to right in (J), n = 24, 24, 24, and 23 from left to right in (K), and n = 24 for each in (L). n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.


Figure 5 ( A and B ) Anti - GFP signals are grossly similar in 3 - 6h and 4 - 6d UAS - myrGFP / + ; dsxGAL4 / + males ( A ) , which are quantified in ( B ) . n = 6 and 8 , respectively . n . s . , not significant , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM . ( C and D ) Anti - DsxM signals in 3 - 6h WTCS males are stronger than those in 4 - 6d WTCS males ( C ) , which are quantified in ( D ) . n = 6 for each . * p < 0 . 05 , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM . ( E - L ) Synaptic transmission levels are visualized by syb - GRASP signals ( GFP ) . ( E and F ) syb - GRASP signals from NPF neurons to dsx neurons are only observed in 4 - 6d adult males but not in 3 - 6h or 1d males ( E ) , which are quantified in ( F ) . The genotype is LexAop - nSyb - spGFP1 - 10 , UAS - CD4 - spGFP11 / + ; NPF - lexA / dsxGal4 . n = 12 , 7 and 5 from left to right . ( G and H ) syb - GRASP signals from dsx neurons to NPF neurons in 4 - 6d adult males are much stronger than those in 3 - 6h and 1d males . syb - GRASP signals in ( G ) are quantified in ( H ) . The genotype is UAS - nSyb - spGFP1 - 10 , LexAop - CD4 - spGFP11 / + ; NPF - lexA / dsxGal4 . n = 13 , 7 and 6 from left to right . ( I and J ) syb - GRASP signals from TH neurons to dsx neurons increase with age from 3 - 6h juvenile males to 4 - 6d adult males ( I ) , which are quantified in ( J ) . The genotype is LexAop - nSyb - spGFP1 - 10 , UAS - CD4 - spGFP11 / TH - LexA ; dsxGal4 / + . n = 12 , 5 and 6 , respectively . ( K and L ) syb - GRASP signals from dsx neurons to TH neurons gradually increase from juvenile males to adult males . syb - GRASP signals in ( K ) are quantified in ( L ) . The genotype is UAS - nSyb - spGFP1 - 10 , LexAop - CD4 - spGFP11 / TH - LexA ; dsxGal4 / + . n = 14 , 6 and 6 , respectively . White arrowheads indicate syb - GRASP signals in the same position in brains of juvenile males and adult males . n . s . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A and B) Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP/+; dsxGAL4/+ males (A), which are quantified in (B). n = 6 and 8, respectively. n.s., not significant, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.(C and D) Anti-DsxM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (C), which are quantified in (D). n = 6 for each. *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.(E-L) Synaptic transmission levels are visualized by syb-GRASP signals (GFP). (E and F) syb-GRASP signals from NPF neurons to dsx neurons are only observed in 4-6d adult males but not in 3-6h or 1d males (E), which are quantified in (F). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 12, 7 and 5 from left to right. (G and H) syb-GRASP signals from dsx neurons to NPF neurons in 4-6d adult males are much stronger than those in 3-6h and 1d males. syb-GRASP signals in (G) are quantified in (H). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 13, 7 and 6 from left to right. (I and J) syb-GRASP signals from TH neurons to dsx neurons increase with age from 3-6h juvenile males to 4-6d adult males (I), which are quantified in (J). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 12, 5 and 6, respectively. (K and L) syb-GRASP signals from dsx neurons to TH neurons gradually increase from juvenile males to adult males. syb-GRASP signals in (K) are quantified in (L). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 14, 6 and 6, respectively. White arrowheads indicate syb-GRASP signals in the same position in brains of juvenile males and adult males. n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.


Figure 6 ( A - D ) Activation of NPF neurons with dTrpA1 induced Ca2 + rise in pC1 dsx neurons in 4 - 6d males but not in 3 - 6h males . ( A and B ) Ca2 + imaging of pC1 neurons from 22 ˚ C to 27 ˚ C and 30 ˚ C in 3 - 6h ( A ) and 4 - 6d ( B ) males . ( C ) Time - course of fluorescent changes in pC1 neurons upon activation of NPF neurons in 3 - 6h and 4 - 6d males ( UAS - GCaMP6m / LexAop - dTrpA1 ; NPF - lexA / dsxGAL4 ) , and in 4 - 6d control males ( UAS - GCaMP6m / + ; dsxGAL4 / + ) without NPF neuronal activation , which are quantified in ( D ) . n = 8 for 4 - 6d control males , 15 for 3 - 6h and 8 for 4 - 6d males with dTrpA1 . n . s . , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t test . Data information : Scale bars , 10 μm . Error bars indicate SEM . ( E - H ) Activation of TH neurons with dTrpA1 triggered Ca2 + increase in pC1 dsx neurons in both 3 - 6h and 4 - 6d males . ( E and F ) Ca2 + imaging of pC1 neurons from 22 ˚ C to 27 ˚ C and 30 ˚ C in 3 - 6h ( E ) and 4 - 6d ( F ) males . ( G ) Time - course of fluorescent changes in pC1 neurons upon activation of TH neurons in 3 - 6h and 4 - 6d males ( UAS - dTrpA1 / LexAop - GCaMP6s ; dsxLexA / TH - GAL4 ) , and in 4 - 6d control males ( LexAop - GCaMP6s / + ; dsxLexA / + ) without activating TH neurons , which are quantified in ( H ) . n = 8 for each . n . s . , not significant , * p < 0 . 05 , Mann - Whitney U test . Data information : Scale bars , 10 μm . Error bars indicate SEM .

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 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

Figure 6(A-D) Activation of NPF neurons with dTrpA1 induced Ca2+ rise in pC1 dsx neurons in 4-6d males but not in 3-6h males. (A and B) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (A) and 4-6d (B) males. (C) Time-course of fluorescent changes in pC1 neurons upon activation of NPF neurons in 3-6h and 4-6d males (UAS-GCaMP6m/LexAop-dTrpA1; NPF-lexA/dsxGAL4), and in 4-6d control males (UAS-GCaMP6m/+; dsxGAL4/+) without NPF neuronal activation, which are quantified in (D). n = 8 for 4-6d control males, 15 for 3-6h and 8 for 4-6d males with dTrpA1. n.s., not significant, *p < 0.05, **p < 0.01, ***p<0.001, unpaired t test. Data information: Scale bars, 10 μm. Error bars indicate SEM.(E-H) Activation of TH neurons with dTrpA1 triggered Ca2+ increase in pC1 dsx neurons in both 3-6h and 4-6d males. (E and F) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (E) and 4-6d (F) males. (G) Time-course of fluorescent changes in pC1 neurons upon activation of TH neurons in 3-6h and 4-6d males (UAS-dTrpA1/LexAop-GCaMP6s; dsxLexA/TH-GAL4), and in 4-6d control males (LexAop-GCaMP6s/+; dsxLexA/+) without activating TH neurons, which are quantified in (H). n = 8 for each. n.s., not significant, *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 10 μm. Error bars indicate SEM.


Figure 2A Z score for AUC and IC50 of 37 different drugs ( shown along the horizontal axis ) identified as hits in 13 of 17 samples screened . A drug hit is defined as z score of less than - 2 for both AUC and IC50 . Each dot in a column represents a sample screened for that drug . The size of the dot corresponds to the IC50 z score for that sample ( the larger the dot , the smaller the IC50 ) . Dots below the black horizontal line represent sample with AUC z score of less than - 2 . Dots are color coded for drug hit types . All color dots below the black line represent a hit for the corresponding drug .

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Figure 2A Z score for AUC and IC50 of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than -2 for both AUC and IC50. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC50 z score for that sample (the larger the dot, the smaller the IC50). Dots below the black horizontal line represent sample with AUC z score of less than -2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug.


Figure 3Event - free survival ( EFS ) and percentage of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ALL ) orthotopic models . Event - free survival ( EFS ) and percentage of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ALL ) orthotopic models . EFS and tumor volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments . EFS and tumor volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments . L EFS in a CNS orthotopic model in which drug sensitivity was observed . EFS is time of inoculation of tumor cells to event ( defined by neurologic symptoms or weight loss ) .

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Figure 3Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.L EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).


Figure 4D Treatment response by therapy tier . 14 of 29 patients with molecular - based therapeutic options received the treatment .

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Figure 4D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment.


figf1 ( a ) Immunoblot analysis of caspase - 1 and IL - 1β in lysates ( Lys ) and supernatants ( Sup ) of peritoneal macrophages obtained from Map1lc3b - / - mice and Becn1 + / - mice and the corresponding wild - type littermate mice , then left unstimulated ( - ) or stimulated with LPS alone ( LPS ) or LPS followed by 30 min of ATP treatment ( L + A ) . p10 , mature form of caspase - 1 . Right margin , molecular size in kilodaltons ( kDa ) . ( b ) Enzyme - linked immunosorbent assay ( ELISA ) of cytokine secretion by peritoneal macrophages stimulated with LPS , followed by treatment for 1 h with ATP . ( c ) Immunoblot analysis of caspase - 1 and IL - 1β in lysates ( left ) and ELISA of cytokines in supernatants ( right ) of Map1lc3b + / + and Map1lc3b - / - BMDMs incubated with LPS , followed by 30 min ( immunoblot ) or 1 h ( ELISA ) of ATP treatment . ( d ) ELISA of IL - 1β secretion by LPS - primed Map1lc3b - / - mice and Becn1 + / - BMDMs incubated for 1 h with z - YVAD - fmk ( z - YVAD ; 10 μM ) or vehicle ( dimethylsulfoxide ( DMSO ) ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three or four experiments ( mean and s . d . in b - d ) .

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

figf1(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa).(b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.(c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b-/- BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment.(d) ELISA of IL-1β secretion by LPS-primed Map1lc3b-/- mice and Becn1+/- BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b-d).


figf2 ( a , b ) Transmission electron microscopy of morphological changes in mitochondria ( arrows ) in Map1lc3b − / − BMDMs ( a ) or Becn1 + / − BMDMs ( b ) left untreated or incubated for 6 h with LPS ( 10 ng / ml ) and then stimulated for 30 min with ATP ( 1 mM ) . Outlined areas ( middle ) enlarged at right . Scale bars , 500 nm . ( c ) Flow cytometry of Map1lc3b − / − and Becn1 + / − macrophages left unstained or labeled with MitoSOX . ( d ) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP ; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment . Data are representative of three experiments .

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figf2(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm.(c) Flow cytometry of Map1lc3b−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX.(d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.


figf3 ( a ) ELISA of cytokines in supernatants ( left ) and immunoblot analysis of caspase - 1 and IL - 1β in lysates ( right ) of J774A . 1 macrophages exposed to EtBr ( 0 , 10 , 100 or 500 ng / ml ; wedges ) and left unstimulated or stimulated with LPS alone or with LPS and ATP . ( b ) Immunoblot analysis of caspase - 1 and ASC in lysates of J774A . 1 or ρ0 J774A . 1 macrophages ( treated with 100 ng / ml of EtBr ) incubated with LPS , followed by stimulation for 0 , 15 or 30 min with ATP . ( c ) Cytokine secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with various combinations of LPS and ATP ( below graph ) . ( d ) Cytokine secretion ( right ) and immunoblot analysis of caspase - 1 and IL - 1β in lysates ( left ) of LPS - primed macrophages incubated for 1 h with rotenone ( + ; 5 μM ) or DMSO ( − ) before ATP treatment . ( e ) ELISA of IL - 1β secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with LPS and rotenone ( or DMSO ) , followed by ATP . ( f ) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito - TEMPO and then stimulated with various combinations of LPS and ATP . ( g , h ) ELISA of IL - 1β secretion ( left ) and immunoblot analysis for caspase - 1 in lysates ( right ) of Map1lc3b + / + and Map1lc3b − / − ( g ) or Becn1 + / + and Becn1 + / − ( h ) peritoneal macrophages incubated for 1 h with Mito - TEMPO ( 500 μM ) , followed by LPS and ATP . * P 0 . 05 ( Student ' s t - test ) . Data represent three experiments ( mean and s . d . in a , c , d - h ) .

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figf3(a) ELISA of cytokines in supernatants (left) and immunoblot analysis of caspase-1 and IL-1β in lysates (right) of J774A.1 macrophages exposed to EtBr (0, 10, 100 or 500 ng/ml; wedges) and left unstimulated or stimulated with LPS alone or with LPS and ATP.(b) Immunoblot analysis of caspase-1 and ASC in lysates of J774A.1 or ρ0 J774A.1 macrophages (treated with 100 ng/ml of EtBr) incubated with LPS, followed by stimulation for 0, 15 or 30 min with ATP.(c) Cytokine secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with various combinations of LPS and ATP (below graph).(d) Cytokine secretion (right) and immunoblot analysis of caspase-1 and IL-1β in lysates (left) of LPS-primed macrophages incubated for 1 h with rotenone (+; 5 μM) or DMSO (−) before ATP treatment.(e) ELISA of IL-1β secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with LPS and rotenone (or DMSO), followed by ATP.(f) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito-TEMPO and then stimulated with various combinations of LPS and ATP.(g,h) ELISA of IL-1β secretion (left) and immunoblot analysis for caspase-1 in lysates (right) of Map1lc3b+/+ and Map1lc3b−/− (g) or Becn1+/+ and Becn1+/− (h) peritoneal macrophages incubated for 1 h with Mito-TEMPO (500 μM), followed by LPS and ATP. *P 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d-h).


figf4 ( a ) ELISA of cytokine secretion by LPS - primed Map1lc3b + / + and Map1lc3b − / − BMDMs incubated with cyclosporine A or DMSO , followed by stimulation for 1 h with ATP . ( b ) ELISA of cytokine secretion by LPS - primed Becn1 + / + or Becn1 + / − BMDMs incubated with cyclosporine A ( 10 μM ) or DMSO , followed by ATP stimulation . ( c ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed BMDMs incubated for 1 h with rotenone ( 5 μM ) or vehicle ( DMSO ) before ATP stimulation . ( d ) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito - TEMPO ( 500 μM ) and then incubated with LPS , followed by ATP stimulation . ( e ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed BMDM incubated with cyclosporine A ( 10 μM ) , plus either rotenone ( 5 μM ) or DMSO , followed by ATP stimulation . ( f ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed Map1lc3b − / − and Becn1 + / − BMDMs stimulated for 30 min with ATP . The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells , set as 1 . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three experiments ( mean and s . d . ) .

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figf4(a) ELISA of cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with cyclosporine A or DMSO, followed by stimulation for 1 h with ATP.(b) ELISA of cytokine secretion by LPS-primed Becn1+/+ or Becn1+/− BMDMs incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation.(c) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDMs incubated for 1 h with rotenone (5 μM) or vehicle (DMSO) before ATP stimulation.(d) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito-TEMPO (500 μM) and then incubated with LPS, followed by ATP stimulation.(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDM incubated with cyclosporine A (10 μM), plus either rotenone (5 μM) or DMSO, followed by ATP stimulation.(f) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed Map1lc3b−/− and Becn1+/− BMDMs stimulated for 30 min with ATP. The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells, set as 1. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


figf5 ( a ) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I , lactate dehydrogenase LDH ( Control ) or heat - inactivated DNase I ( HI DNase I ) and then stimulated with LPS , followed by stimulation for 1 h with ATP . ( b ) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS , followed by stimulation for 15 min with ATP . ( c ) ELISA of IL - 1β secretion by Aim2 + / + and Aim2 − / − BMDMs primed for 4 h with LPS ( 200 ng / ml ) , then transfected for 6 h with 1 μg mtDNA ( through the use of liposomes as the vehicle ) , followed by stimulation for 1 h with ATP . * P 0 . 05 , versus Aim2 − / − cells treated with LPS and ATP ( Student ' s t - test ) . ( d ) ELISA of cytokine secretion by LPS - primed Aim2 − / − BMDMs transfected for 6 h with mtDNA , poly ( dA : dT ) or mtDNA predigested by DNase I ( + DNase ; far left ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three experiments ( mean and s . d . ) .

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figf5(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP.(b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP.(c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test).(d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


figf6 ( a ) Cytokine secretion by LPS - primed Map1lc3b + / + and Map1lc3b − / − ( a ) or Becn1 + / + and Becn1 + / − ( b ) peritoneal macrophages incubated for 15 min with glybenclamide ( Glyb ; 100 μM ( a ) or 50 - 100 μM ( b ) ) , Bay 11 - 7082 ( Bay ; 12 μM ) or DMSO ( - ) , followed by ATP . ( c ) Cytokine secretion by LPS - primed wild - type ( WT ) and NALP3 - deficient ( NALP3 - KO ) macrophages incubated with rotenone , followed by stimulation for 1 h with ATP . ( d ) Quantitative PCR analysis of cytosolic mtDNA in wild - type and NALP3 - deficient BMDMs left unstimulated ( - ) or stimulated with LPS , followed by treatment for 15 or 30 min with ATP ( left ) , and immunoblot analysis of cytosolic cytochrome c and activation of caspase - 1 in the cells at left ( right ) . ( e ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed wild - type , NALP3 - deficient or ASC - deficient ( ASC - KO ) BMDMs stimulated for 15 min with ATP . * P 0 . 05 , NALP3 - deficient or ASC - deficient versus wild - type treated with LPS and ATP ( Student ' s t - test ) . ( f ) Flow cytometry of untreated macrophages ( control ) or of LPS - primed macrophages left unstained or stained with MitoSOX , then stimulated for 15 min with ATP . ( g ) Flow cytometry of untreated macrophages ( control ) or of macrophages stimulated with LPS and ATP , stained with MitoTracker Deep Red . Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential . ( h ) Flow cytometry of LPS - primed wild - type , ASC - deficient and NALP3 - deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V . Data represent three experiments ( mean and s . d . in a - e ) .

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

figf6(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50-100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (-), followed by ATP.(c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP.(d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (-) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right).(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test).(f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP.(g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential.(h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).


figf7 ( a ) ELISA of serum IL - 1β and IL - 18 at 24 h after intraperitoneal injection of LPS ( 12 mg per kg body weight ) into male Map1lc3b + / + and Map1lc3b − / − mice . ( b ) Survival of Map1lc3b + / + mice ( n = 11 ) and Map1lc3b − / − mice ( n = 10 ) after LPS challenge ( 100 mg per kg body weight ) , assessed over a period of 2 weeks . ( c , d ) ELISA of serum IL - 1β and IL - 18 in male Map1lc3b + / + and Map1lc3b − / − mice ( c ) and Becn1 + / + and Becn1 + / − mice ( d ) at 24 h after CLP or sham laparotomy . In a , c , d , each symbol represents an individual mouse ; small horizontal lines indicate the mean . P values , unpaired two - tailed Student ' s t - test ( a , c , d ) or log - rank test ( b ) . Data are representative of two experiments .

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figf7(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice.(b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks.(c,d) ELISA of serum IL-1β and IL-18 in male Map1lc3b+/+ and Map1lc3b−/− mice (c) and Becn1+/+ and Becn1+/− mice (d) at 24 h after CLP or sham laparotomy. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or log-rank test (b). Data are representative of two experiments.


Figure 1B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2 - KO PSCs , NPCs , and neurons . Scale bar = 10 µm . C RT - qPCR array revealed differential gene expression between control and MECP2 - KO 28 - day neurons . The P values are calculated based on a Student ' s t - test of the replicate 2 - ΔΔCt values for each gene in the control group and KO groups ( WT83 / Q83X cell lines were used ; N = 3 clones from each genotype ) . D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function ( days 14 and 28 post differentiation ; normalized by control NPC expression ) . E Single - cell RT - qPCR analysis distinguished populations of control and KO 28 - day neurons . Principal Component Analysis ( PCA ) of 441 cells projected onto the first two components . Overlaid populations of WT83 / Q83X and WT82 / K82fs neurons are shown . F Violin plots of selected genes showing the comparison between control and MECP2 - KO neurons from the single - cell analyses ( log2 ( expression ) values ) that overlapped with the results obtained via RT - PCR array . G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single - cell analyses . The dotted lines represent differentially expressed genes between the groups at P < 0 . 05 ( unpaired Student ' s t - test ) . H Left : Schematic of laminar cortical markers . Right : Top : Quantitation of laminar cell markers based on the single - cell RT - qPCR analysis ( two - way analysis of variance ( ANOVA ) , N = 3 replicates per genotype , F3 , 24 = 3 . 31 , * P = 0 . 04 , with difference in the proportion of cortical layers V and VI , 95 % confidence interval ( CI ) [ 8 . 31 , 42 . 41 ] ) ; Bottom : altered proportions of neurotransmitter markers ( two - way ANOVA , N = 3 replicates per genotype , F5 , 24 = 27 . 33 , * * * P < 0 . 0001 , with differences in the proportions of glutamatergic , 95 % CI [ 22 . 33 , 33 . 60 ] , and cholinergic , 95 % CI [ 0 . 166 , 11 . 43 ] , neurons ) .

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

Figure 1B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm.C RT-qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2-ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype).D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post differentiation; normalized by control NPC expression).E Single-cell RT-qPCR analysis distinguished populations of control and KO 28-day neurons. Principal Component Analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown.F Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT-PCR array.G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test).H Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT-qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons).


Figure 2D , E Western blot quantification showed decreased presynaptic Synapsin1 ( D ) and postsynaptic PSD - 95 ( E ) in untreated 6 - week MECP2 - KO neurons that can be increased by drug treatment ( Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; full Western blot , Fig EV4A ) ( WT83 / Q83X cell lines were used ; N = 3 clones from each genotype ) . F , G 6 - week MECP2 - KO neurons ( F ) showed a pharmacologically rescuable reduction of co - localized synaptic puncta ( G ; one - way ANOVA , F16 , 118 = 9 . 148 , * P < 0 . 05 ; Dunnet ' s multiple comparisons test vs untreated KO ( WT83 / Q83X cell lines were used ; N = 7 - 8 neurons / condition ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Z scores relative to KO untreated : Control = 3 . 903 ; Nefiracetam = 3 . 560 ; Carbamoylcholine = 2 . 705 ; Pirenzepine = 0 . 0448 ; PHA543613 = 2 . 121 ; Acamprosate = 3 . 339 ; Baclofen = 0 . 5672 ; GR73632 = 2 . 237 ; Hyperforin = 3 . 016 , and IGF - 1 = 3 . 791 . Scale bar = 5 µm . H Drug treatment increased calcium transient frequency in 6 - week neurons . Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest ( one - way ANOVA , F4 , 52 = 20 . 28 , Dunnett ' s multiple comparisons test vs untreated KO : Nefiracetam , * * P < 0 . 01 and Z = 2 . 364 ; PHA 543613 , * * * P < 0 . 001 and Z = 3 . 391 ; Acamprosate , * * * P < 0 . 001 and Z = 3 . 153 ; WT83 / Q83X cell lines were used ; N = 10 - 14 neurons / condition ) and the percentage of active neurons ( one - way ANOVA , F4 , 52 = 23 . 11 ; Dunnett ' s multiple comparison test vs untreated KO : Nefiracetam , * * * P < 0 . 001 and Z = 3 . 144 ; PHA 543613 , * * P < 0 . 01 and Z = 2 . 567 ; * * Acamprosate , P < 0 . 01 and Z = 2 . 285 ; N = 10 - 14 neurons / condition ) . I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2 - KO neurons on MEA ( two - sided unpaired Student ' s t - test compared to KO untreated : Nefiracetam , * P = 0 . 016 and Z = 2 . 418 ; PHA 543613 , * P = 0 . 027 and Z = 2 . 147 ; Acamprosate , P = 0 . 39 and Z = 0 . 477 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 4 - 11 MEA wells / condition ) .

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

Figure 2D, E Western blot quantification showed decreased presynaptic Synapsin1 (D) and postsynaptic PSD-95 (E) in untreated 6-week MECP2-KO neurons that can be increased by drug treatment (Kruskal-Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001; full Western blot, Fig EV4A) (WT83/Q83X cell lines were used; N = 3 clones from each genotype).F, G 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs untreated KO (WT83/Q83X cell lines were used; N = 7-8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm.H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition).I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).


Figure 3B , C Immunofluorescent staining ( B ) and quantitation ( C ) confirmed the graded decrease in MeCP2 expression from control to 50 % MECP2 mosaic to full MECP2 - KO neurospheres . Scale bar = 50 µm . D 8 - week MECP2 - KO neurospheres ( N = 44 ) exhibited decreased size ( diameter ; * * P < 0 . 01 ) , but 50 % MECP2 - mosaic neurospheres ( CTR / KO ; N = 44 ) were unchanged from controls ( one - way ANOVA , F2 , 150 = 6 . 03 , P = 0 . 003 ; Dunnett ' s multiple comparisons test ; N = 65 ) . Left : representative brightfield images of neurospheres ; Right : neurosphere size quantification . Scale bar = 200 µm . E Calcium imaging analysis showing calcium transients normalized by control ( one - way ANOVA , F2 , 72 = 15 . 62 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs control : MECP2 - mosaic , * * * P < 0 . 001 ; MECP2 - KO , * * * P < 0 . 001 ) . WT83 / Q83X cell lines were used ; N = 44 Q83X neurospheres and 65 WT83 neurospheres . F 8 - week neurosphere size ( diameter ) remained unchanged by treatment with either drug ( one - way ANOVA , F6 , 344 = 0 . 52 , P = 0 . 79 ; N = 44 Q83X neurospheres and 65 WT83 neurospheres ) . G Cell viability was improved or unharmed in treated 8 - week neurospheres ( one - way ANOVA , F9 , 104 = 6 . 81 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi + PHA 1 µM , * * P < 0 . 01 and Z = 2 . 974 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 3 . 733 ; N = 8 - 24 neurospheres per condition ) . H , I Calcium transient frequency ( number of peaks in 10 mins recording ) normalized by CTR / KO untreated ( H ; one - way ANOVA , F9 , 239 = 6 . 18 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi 1 µM , * P < 0 . 05 and Z = 0 . 780 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 1 . 972 ; N = 15 - 61 neurospheres per condition ) and calcium transient amplitude normalized by CTR / KO untreated ( I ; one - way ANOVA , F9 , 241 = 1 . 27 , P = 0 . 255 ; N = 16 - 62 neurospheres per condition ) . J , K MEA spike frequency heatmap ( J ) and quantification ( K ) normalized by CTR / KO untreated ( one - way ANOVA , F5 , 38 = 1 . 21 , P = 0 . 321 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : PHA , P = 0 . 65 and Z = 1 . 134 ; Nefi , P = 0 . 98 and Z = 0 . 06 ; PHA + Nefi , P = 0 . 86 and Z = 0 . 94 ; N = 7 - 8 wells per condition ) . ' X ' on activity heatmap signifies absence of a neurosphere in that position .

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

Figure 3B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm. D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm.  E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres.  F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres).  G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition). H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.


Figure 4B 2 - month old cortical organoid diameter ( one - way ANOVA , F2 , 173 = 91 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * * P < 0 . 001 and Z = 5 . 414 ; PHA 543613 , * * * P < 0 . 001 and Z = 5 . 351 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 28 - 90 organoids per condition ) . Left : representative brightfield images of cortical organoids ; Right : cortical organoid size quantification . Scale bar = 200 µm . C Ki67 + proliferative area ( Student ' s t - test , t10 = 0 . 36 , P = 0 . 73 , N = 6 each ) . Left : representative immunofluorescence images of progenitor , proliferative , and neuronal markers in cortical organoids ; Right : quantification of Ki67 + proliferation in cortical organoids . Scale bar = 50 µm . RNA sequencing ( D , left : heatmap ) and GO analysis ( D , right ) of untreated MECP2 - KO organoids vs Nefiracetam ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated genetic expression in synapse - relevant pathways . E , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values . RNA sequencing ( F , left : heatmap ) and GO analysis ( F , right ) of untreated MECP2 - KO organoids vs PHA 543613 ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated expression of genes in synapse - relevant pathways . G , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values . H Synapsin1 + puncta quantitation ( one - way ANOVA , F3 , 34 = 10 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * P < 0 . 01 and Z = 2 . 998 ; PHA 543613 , P > 0 . 05 and Z = 1 . 266 ; WT83 / Q83X cell lines were used ; N = 8 - 10 each ) . Left : representative image of Synapsin1 + immunofluorescent staining ; Right : Synapsin1 + puncta quantification . Scale bar = 20 µm . MECP2 - KO and control organoids had proportions of cells positive for the neuronal marker NeuN ( I ; Student ' s t - test , t14 = 0 . 26 , P = 0 . 80 , N = 8 each ) J MECP2 - KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 ( J ; one - way ANOVA , F3 , 40 = 2 . 489 , P = 0 . 074 and Z < 1 , WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 8 - 14 organoids each ) . J , left : representative image of CTIP2 + immunofluorescent staining in cortical organoids ; J , right : quantification of CTIP2 + neurons . Scale bar = 50 µm . L ; left and center : representative population spiking traces of control and MECP2 - KO organoids ; right : quantification of organoid population spiking activity , N = 6 replicates each ) . Drug treatment increased spiking network activity of MECP2 - KO cortical organoids to a level not significantly different from that of control organoids ( M ; one - way ANOVA , F3 , 31 = 3 . 775 , Dunnett ' s multiple comparisons test vs control : untreated KO , * * P < 0 . 01 and Z = 3 . 192 ; Nefiracetam , P = 0 . 98 and Z = 2 . 117 ; PHA 543613 , P = 0 . 12 and Z = 1 . 101 , WT83 / Q83X cell lines were used ; N = 7 - 10 MEA wells per condition ) .

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

Figure 4B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm.C Ki67+ proliferative area (Student's t-test, t10 = 0.36, P = 0.73, N = 6 each). Left: representative immunofluorescence images of progenitor, proliferative, and neuronal markers in cortical organoids; Right: quantification of Ki67+ proliferation in cortical organoids. Scale bar = 50 µm.RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways.E, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways.G, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm.MECP2-KO and control organoids had proportions of cells positive for the neuronal marker NeuN (I; Student's t-test, t14 = 0.26, P = 0.80, N = 8 each)J MECP2-KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 (J; one-way ANOVA, F3,40 = 2.489, P = 0.074 and Z < 1, WT83/Q83X and WT82/K82fs cell lines were used; N = 8-14 organoids each). J, left: representative image of CTIP2+ immunofluorescent staining in cortical organoids; J, right: quantification of CTIP2+ neurons. Scale bar = 50 µm.L; left and center: representative population spiking traces of control and MECP2-KO organoids; right: quantification of organoid population spiking activity, N = 6 replicates each).Drug treatment increased spiking network activity of MECP2-KO cortical organoids to a level not significantly different from that of control organoids (M; one-way ANOVA, F3,31 = 3.775, Dunnett's multiple comparisons test vs control: untreated KO, **P < 0.01 and Z = 3.192; Nefiracetam, P = 0.98 and Z = 2.117; PHA 543613, P = 0.12 and Z = 1.101, WT83/Q83X cell lines were used; N = 7-10 MEA wells per condition).


Figure 1Pre - enriched thymocytes from WT BALB / c thymi as in ( a ) were labeled by CFSE ( CFSE + ) , mixed with pre - enriched thymocytes from SKG thymi ( CFSE - ) , loaded with Indo - 1 , and stimulated as in ( a ) . Ratio of Indo ( Violet ) and Indo ( Blue ) was analyzed by flow cytometry . Comparison of cytoplasmic Ca2 + profiles between WT and SKG NKT1 ( c ) and NKT2 ( d ) cells in the same test tube are shown . Data are representative from one of at least three independent experiments . Graphs represent the average Ca2 + levels .

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Figure 1Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.


Figure 2Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet , and activated by anti - CD3ε / CD28 Dynabeads for 2 days , in the presence of different [ EGTA ] in the medium . CellTrace Violet / CTV signal in living cells at the end of the culture is shown . Cell proliferation was quantified as division index based on CTV signals in ( a ) . Thymic lobes from new born ( day 0 ) BALB / c mice were cultured in vitro for 6 days . EGTA was added 48h after the start of culture ( day 2 ) , and the expression of CXCR3 ( g ) , PLZF ( h ) and PD1 ( i ) in total iNKT cells are shown .

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Figure 2Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a).Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.


Figure 3oxygen consumption rate ( OCR ) from sorted thymic T cell populations measured in a Seahorse Mitostress test ;

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Figure 3oxygen consumption rate (OCR) from sorted thymic T cell populations measured in a Seahorse Mitostress test;


Figure 4Confocal images of Tim 23 ( Alexa Fluor 488 , green ) and MitoTracker deep red ( Alexa Fluor 647 , red ) in freshly isolated thymic NKT1 ( c1 , 2 ) and NKT2 ( c5 , 6 ) cells using Airyscan technology . 3D images reconstructed by Imaris software in NKT1 ( c3 , 4 ) and NKT2 cells ( c7 , 8 ) . Dotted line in panel ( 5 - 8 ) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells . Representative images of electron microscopy in NKT1 and NKT2 cells ; ( f ) individual mitochondria size and ( g ) cristae perimeters normalized to mitochondria perimeter based on EM in ( e ) .

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Figure 4Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.Representative images of electron microscopy in NKT1 and NKT2 cells; (f) individual mitochondria size and (g) cristae perimeters normalized to mitochondria perimeter based on EM in (e).


Figure 5Maximal cytoplasmic [ Ca2 + ] in single , freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 ( 1uM ) ; BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of Tbet in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) . BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of CXCR3 ( d ) , PLZF ( e ) , and PD1 ( f ) in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).