C . Quantification of 5 ' - ETS RNA levels in HeLa cells infected with an anti - DCP2 or a non - targeting shRNA and expressing GFP , PNRC1WT or PNRC1W300A transgenes . The average of 2 independent experiments ± SD is reported .

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C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported.


Levels of U3 ( D ) snoRNAs immunoprecipitated by the anti - m3G cap antibody measured by Real Time PCR . Values represents the average of two biological replicates with three technical replicates and

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Levels of U3 (D) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are


Levels of U8 ( E ) snoRNAs immunoprecipitated by the anti - m3G cap antibody measured by Real Time PCR .

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Levels of U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR.


F . Levels of U3 snoRNA immunoprecipitated by the anti - m3G cap antibody in HeLa cells infected with an anti - DCP2 or a non - targeting shRNA and expressing GFP , PNRC1WT or PNRC1W300A transgenes . The average ± SD of 2 independent experiments with three technical replicates is reported .

O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported.


G . Western blot analysis of input and Flag - IP fractions of RIP experiment . H . RNA levels of U3 , U8 and U1 co - immunoprecipitating with GFP , PNRC1WT or PNRC1W300A . Results are shown as the average ± SD of two biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

G. Western blot analysis of input and Flag-IP fractions of RIP experiment. H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.


( A ) Yeast two - hybrid assays to test for interaction between Munc18 - 1 and ERK1 / 2 . pGAL4 - Munc18 - 1 ( M18 ) and pGAL4 - Empty ( control ) were used as baits and ERK1 and ERK2 as prey . Both ERK1 and ERK2 bind Munc18 - 1 and allow growth on selection plates ( minus Histidine ( - HIS ) and minus Histidine and Adenosine ( - ADE ) ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O

(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).


( B ) In vitro kinase assays showing Munc18 - 1 phosphorylation by ERK . Radioactive phosphoblot ( 32P ) and immunoblot ( IB ) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT , Munc184A ( M18T78A , S158A , S241A , T574A ) , Munc183A ( M18S158A , S241A , T574A ) , and Munc18AA ( M18S241A , T574A ) from HEK293T cells incubated with recombinant ERK2 . The phosphoblot shows phosphorylated Munc18 - 1 ( pMunc18 - 1 ) and auto - phosphorylated ERK2 ( pERK2 ) . ( C ) Quantification of phospho - Munc18 - 1 intensity in wild - type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18 - 1 normalized to total Munc18 - 1 levels . Phosphorylation of Munc18WT was set to 100 % . ( Munc18WT : 100 ± 3 % ; Munc18AA : 24 ± 2 % , n = 3 , * p = 0 . 01 ) .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).


( D ) ERK1 in vitro kinase assay showing ERK - dependent 32P - incorporation in Munc18WT immuno - precipitated from HEK293T cells . Western Blot stained with Munc18 - 1 antibody shows total precipitated Munc18 - 1 levels were identical in all conditions .

O O O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.


( A ) Experimental setup of fear conditioning paradigm . Mice placed in a fear - conditioning box received no shock ( NS ) , an immediate shock ( IS ) or delayed shock ( DS ) and were decapitated 30 min after returning to the home cage . Dorsal and ventral hippocampi were subjected to cellular fractionation ( P2 ) . DS and IS significantly increased the levels of phosphorylated ERK1 / 2 , a hallmark of ERK activation . ( B ) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group .

O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O

(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group.


( D ) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241 , total Munc18 - 1 , pSynapsin , total synapsin , pERK and GAPDH as loading control . ( E ) Quantification Munc18 - 1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18 - 1 levels . No shock was set to 1 ( NS : 1 . 0 ± 0 . 19 , n = 5 ; DS : 4 . 01 ± 0 . 39 , n = 5 ; IS : 2 . 88 ± 0 . 37 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( F ) Quantification of Munc18 - 1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18 - 1 levels . ( NS : 1 . 0 ± 0 . 32 , n = 5 ; DS : 4 . 39 ± 0 . 55 , n = 5 ; IS : 3 . 62 ± 0 . 50 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( G ) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels . ( NS : 1 . 0 ± 0 . 31 , n = 5 ; DS : 1 . 88 ± 0 . 43 , n = 5 ; IS : 1 . 18 ± 0 . 34 , n = 5 ; n . s . ) . Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels . ( NS : 1 . 0 ± 0 . 11 , n = 5 ; DS : 3 . 51 ± 0 . 41 , n = 5 ; IS : 3 . 98 ± 0 . 24 , n = 5 ; * * p < 0 . 01 ) . ( H ) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels . ( NS : 1 . 0 ± 0 . 20 , n = 5 ; DS : 3 . 32 ± 0 . 46 , n = 5 ; IS : 3 . 30 ± 0 . 60 , n = 5 ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).


( A ) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 ( 10 M ) and DMSO , the GABAA receptor blocker GABAzine ( GBZ , 10 M ) or the voltage - gated sodium channel blocker TTX ( 2 M ) for 4 h . Blots were stained for pMunc18S241 , Munc18 - 1 and tubulin as loading control . ( B ) Quantification of ( A ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18 - 1 levels . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 4 % ; GBZ : 1 . 73 ± 0 . 12 ; TTX : 0 . 052 ± 0 . 002 , n = 4 , * p < 0 . 05 , * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).


( C ) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) and MEK inhibitor PD98059 ( 10 M ) for 4 h . Proteasome block increases the levels of phosphorylated Munc18 - 1 and ERK1 / 2 , which can be prevented by MEK inhibition . Blots were stained for pMunc18S241 , Munc18 , pERK1 / 2 and ERK1 / 2 . ( D ) Quantification of ( C ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18 - 1 levels . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 8 % ; MG132 : 375 ± 14 . 2 % ; MG132 + PD98059 : 24 ± 3 % , n = 3 , * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).


( E ) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 ( 1 M ) for 5 min or BDNF ( 100ng / ml ) for 48 h in the presence of 2 M TTX and stained for pMunc18S241 , Munc18 , pERK and tubulin as loading control .

O O O O B-ORGANISM B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O

(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( A ) Amplitude of excitatory synaptic response ( EPSC ) ( M18WT : 1 . 37 ± 0 . 21 nA , n = 35 ; M18S241A : 2 . 53 ± 0 . 31 nA , n = 45 ; M18S241D : 0 . 24 ± 0 . 05 nA , n = 17 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( B ) Percentage of cells that do not respond to electrical stimulation ( M18WT : 15 . 5 % ; M18S241A : 0 % ; M18S241D : 56 . 9 % ) . These cells are not included in ( A ) . Insert : representative recording of single EPSCs . Scale bar represents 10 ms and 0 . 5 nA .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( C ) Typical examples of spontaneous vesicle release . Scale bar represents 0 . 1 s and 25 pA . ( D ) Spontaneous release frequency ( M18WT : 7 . 05 ± 1 . 28 Hz , n = 32 ; M18S241A : 14 . 26 ± 2 . 04 Hz , n = 37 ; M18S241D : 0 . 64 ± 0 . 29 Hz , n = 11 ; * * * p < 0 . 001 , * * p < 0 . 01 ) . ( E ) Amplitude of spontaneous release events ( M18WT : 15 . 40 ± 1 . 06 pA , n = 17 ; M18S241A : 15 . 03 ± 0 . 61 pA , n = 20 ; M18S241D : 10 . 98 ± 0 . 72 pA , n = 10 ; * * p < 0 . 01 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( F ) Size of the readily releasable pool ( RRP ) assessed with single hyperosmotic sucrose application ( 500 mM , 3 . 5 s , M18WT : 0 . 74 ± 0 . 15 nC , n = 7 ; M18S241A : 1 . 6 ± 0 . 33 nC , n = 10 ; M18S241D : could not be determined ; * p < 0 . 05 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( G ) Neurons were stimulated with two consecutive pulses with varying inter - stimulus interval to assess release probability . Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter - stimulus interval ( M18WT , n = 25 ; M18S241A , n = 19 ; M18S241D , n = 17 ) ; * p < 0 . 05 , * * p < 0 . 01 ) . All data are expressed as mean ± SEM . See also Figure EV2 and EV3 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.


( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1null mutant neurons expressing M18WT , M18S241A or M18S241D . ( A ) Typical examples showing Munc18 - 1 protein localization and intensity in neurites of M18WT , M18S241A or M18S241Dneurons . MAP2 in blue , VAMP2 in red and Munc18 - 1 in green . Scale bar represents 2 m . ( B ) Mean somatic Munc18 - 1 intensity ( M18WT : 1682 ± 206 a . u . , n = 46 ; M18S241A : 1719 ± 238 a . u . , n = 36 ; M18S241D : 582 ± 98 a . u . , n = 33 ; * * * p < 0 . 001 ) . ( C ) Mean synapticMunc18 - 1 intensity ( M18WT : 1281 ± 161 a . u . ; M18S241A : 1390 ± 180 a . u . ; M18S241D : 210 ± 30 a . u . ; * * * p < 0 . 001 ) . ( D ) Mean synapticMunc18 - 1 intensity as a function of radial distance from the soma .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR B-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1null mutant neurons expressing M18WT, M18S241A or M18S241D.(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241Dneurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).(C) Mean synapticMunc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).(D) Mean synapticMunc18-1 intensity as a function of radial distance from the soma.


( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( E ) Mean synaptic Munc18 - 1 intensity in cultures with 3 times the standard number of neurons to increase network activity ( M18WT : 1072 ± 123 a . u . , n = 36 ; M18S241A : 1480 ± 170 a . u . , n = 32 ; M18S241D : 251 ± 70 a . u . , n = 34 ; * p < 0 . 05 , * * * p < 0 . 001 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001).


( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( F ) Mean synaptic Munc18 - 1 intensity in cultures treated with GABAA receptor antagonist Bicuculline ( 40 μM , 48 h ) . ( M18WT : 761 ± 121 a . u . , n = 26 ; M18S241A : 1482 ± 150 a . u . , n = 31 , * * p < 0 . 01 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).


O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001).


( H ) Western Blot of lysates from high - density neuronal cultures from munc18 - 1 null mutant mice rescued with M18WT , M18S241A or M18S241D . Prior to lysis , cells were treated for 6 h with MG132 ( 10 M ) or DMSO as control . Western Blots were stained for Munc18 - 1 and tubulin as loading control . ( I ) Quantification of ( H ) . Munc18 - 1 levels were normalized to tubulin levels . Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant ( M18WT : 1 . 62 ± 0 . 41 ; M18S241A : 1 . 26 ± 0 . 16 ; M18S241D : 7 . 53 ± 1 . 17 , n = 3 ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).


( J ) Ubiquitination of different Munc18 - 1 mutants in HEK293T cells . Western Blot of denatured Munc18 - 1 immuno - precipitations from cells expressing Munc18 - 1 mutants and HA - tagged ubiquitin . Prior to lysis , cells were treated for 8 h with MG132 ( 10 M ) . Ubiquitin covalently attached to Munc18 - 1 can be observed as a smear above the characteristic 68 kD Munc18 - 1 band on Western blots stained for HA - tag . ( K ) Quantification of immuno - precipitation in ( J ) . Level of Munc18 - 1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18 - 1 levels . M18WT was set to 1 ( M18WT : 1 ± 0 . 06 ; M18S241A : 0 . 84 ± 0 . 57 ; M18S241D : 2 . 70 ± 0 . 07 , n = 3 ; * * p < 0 . 01 ) . All data are expressed as mean ± SEM .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.


( A ) Western blot from whole cell lysate of munc18 - 1 null mutant neurons rescued with M18WT and incubated with WIN55 , 212 - 2 ( 5 M ) for 30 s . Cells were harvested after the indicated time . CB1R activation leads to a rapid and transient increase in pERK activity .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.


( B - D ) Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A . CB1R agonist WIN55 , 212 - 2 ( 5 M ) was applied at t = 60 s for 30 s . ( B ) Amplitude of excitatory synapse response normalized to t = 0 s . Neurons were stimulated by a single action potential every 30 s for 4 min ( M18WTn = 21 , M18S241An = 24 , * p < 0 . 05 ) . Note the broken y - axis . ( C ) Ratio of the frequency of spontaneous release events after ( t = 180 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 58 . 0 ± 2 . 3 % , n = 21 ; M18S241A : 84 . 2 ± 5 . 6 % , n = 24 , * p < 0 . 05 ) . ( D ) Ratio of amplitude of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 85 . 0 ± 2 . 7 % , n = 21 ; M18S241A : 83 . 1 ± 2 . 6 % , n = 24 ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O 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-D) Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WTn = 21, M18S241An = 24, *p<0.05). Note the broken y-axis.(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).


( E - G ) Autaptic neurons of munc18 - 1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55 , 212 - 2 ( 1 M ) , WIN55 , 212 - 2 ( 1 M ) plus AM251 ( 1 M ) or vehicle only ( DMSO ) . ( E ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) or vehicle only ( DMSO ) were applied at t = 60 s for 30 s . ( WT + WIN n = 13 ; WT + DMSO n = 16 , * p < 0 . 05 ) . Note the broke Y - axis . ( F ) Paired pulse ratio ( 20 ms interval ) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55 , 212 - 2 ( 1 M ) at t = 60 s for 30 s . WIN55 , 212 - 2 application increases PP ratios indicative of a reduction of synaptic release probability ( t = 0 s : 0 . 94 ± 0 . 1 , n = 13 ; t = 240 s : 1 . 36 ± 0 . 2 , n = 16 , * p < 0 . 05 ) . ( G ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) was applied together with the CB1R antagonist AM251 ( 1 M ) at t = 60 s for 30 s . ( WT + WIN n = 13 , same trace as 6F ; WT + WIN + AM251 n = 15 , * p < 0 . 05 ) . Note the broke Y - axis .

O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.


( H - J ) Hippocampal autaptic neurons of munc18 - 1 null mutant mice were rescued with M18WT or M18S241A . mGluR2 / 3 agonist LY379268 ( 1 M ) was applied by bath - perfusion for 4 min . ( H ) Ratio of amplitude of evoked release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 55 . 0 ± 4 . 7 % , n = 14 ; M18S241A : 81 . 1 ± 3 . 6 % , n = 16 , * p < 0 . 05 ) . ( I ) Ratio of frequency of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 63 . 0 ± 5 . 7 % , n = 14 ; M18S241A : 85 . 1 ± 3 . 4 % , n = 16 , * p < 0 . 05 ) . ( J ) Ratio of the amplitude of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 81 . 0 ± 1 . 3 % , n = 14 ; M18S241A : 82 . 1 ± 3 . 3 % , n = 16 ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).


( A - D ) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre - treatment with PD98059 ( PD ; 10 µM ) compared to 0 . 1 % DMSO ( control ) . ( A ) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 196 . 36 + / - 16 . 51 pA , n = 10 ; PD : 196 . 42 + / - 18 . 61 pA , n = 11 , p = 0 . 9 ) . ( B ) DSE in neurons of DMSO pre - treated slices , induced by a 10s depolarization to 0 mV ( arrow ) . Inset : example traces of 2 averaged EPSCs before ( black ) and after depolarization ( grey ) . ( C ) DSE in slices pre - treated with PD . Insets : example traces of 2 averaged EPSCs before ( red ) and after depolarization ( grey ) . ( D ) Comparison of DSE between control and PD . PD pre - treatment limits amplitude and duration of DSE . * * = p < 0 . 01 ; unpaired T - test with Welch ' s correction . Numbers in bars represent the number of neurons in each group .

O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O

(A-D) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.


( E - H ) ERK - pathway inhibition by pre - treatment with PD98059 ( PD ; 10 µM ) blocks the effect of WIN55 , 212 - 2 ( WIN ; 2 µM ) on the amplitude of EPSCs recorded in CA1 pyramidal neurons . ( E ) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 295 . 69 + / - 28 . 73 pA , n = 6 ; PD : 270 . 86 + / - 32 . 14 pA , n = 6 , p = 0 . 6 ) . ( F ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of 0 . 1 % DMSO ( control ) pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( black ) and the last 5 min ( grey ) . ( G ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of PD pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( red ) and the last 5 minutes ( grey ) . ( H ) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference . * = p < 0 . 05 ; unpaired T - test . Numbers in bars represent number of slices in each group .

O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H) ERK-pathway inhibition by pre-treatment with PD98059 (PD; 10 µM) blocks the effect of WIN55,212-2 (WIN; 2 µM) on the amplitude of EPSCs recorded in CA1 pyramidal neurons.(E) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 295.69 +/- 28.73 pA, n = 6; PD: 270.86 +/- 32.14 pA, n = 6, p= 0.6).(F) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of 0.1% DMSO (control) pre-treated slices. Inset: example traces of averaged EPSCs of baseline (black) and the last 5 min (grey).(G) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of PD pre-treated slices. Inset: example traces of averaged EPSCs of baseline (red) and the last 5 minutes (grey).(H) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference. * = p < 0.05; unpaired T-test. Numbers in bars represent number of slices in each group.


E ) Caco - 2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h . Western blotting of cell lysates is shown .

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E) Caco-2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h. Western blotting of cell lysates is shown.


D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h . D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with mouse ( m ) IFITM plasmids or vector control for 24 h . C ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


D ) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in ( C ) and one experiment with MOI 0 . 1 . Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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D) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in (C) and one experiment with MOI 0.1. Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu - 3 cells transfected with mCherry and SARS - CoV - 2 Spike ( S ) or vector control . Cells were co - cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye . A ) Representative example fluorescent imaging of the indicated co - cultures showing the presence of EGFP / mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs . Scale bar indicates 50 μm . B ) Quantification of nuclei per EGFP / mCherry double positive cell syncytia in co - cultures involving the indicated IFITM3 constructs as compared to vector control . Bars depict averages of three independent experiments with circles representing the individual data points . For each condition , nuclei within 50 - 75 individual syncytia were counted . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h . B ) Graph depicts normalized infection percentage measurements from 3 independent experiments , each performed in triplicate , from cells infected as in ( B ) . Bars represent averages with individual data points shown as circles . Error bars represent SD . The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection . # p < 0 . 05 by ANOVA followed by Tukey ' s multiple comparisons test . NS , not significant . Not indicated , additional comparisons of interest with p < 0 . 05 include IFITM3 ( - ) vs . IFITM3 - Y20 ( - ) , IFITM3 ∆ 59 - 68 ( - ) , and IFITM3 - S61A , N64A , T65A ( - ) .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h. B) Graph depicts normalized infection percentage measurements from 3 independent experiments, each performed in triplicate, from cells infected as in (B). Bars represent averages with individual data points shown as circles. Error bars represent SD. The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test. NS, not significant. Not indicated, additional comparisons of interest with p<0.05 include IFITM3 (-) vs. IFITM3-Y20 (-), IFITM3∆59-68 (-), and IFITM3-S61A,N64A,T65A (-).


B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2 - KO PSCs , NPCs , and neurons . Scale bar = 10 µm .

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B 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 ) .

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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 ) .

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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 .

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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 .

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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 ) .

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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 O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O

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).


D , 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 ) .

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D, 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 .

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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 ) .

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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 O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-EXP_ASSAY O O O O O

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).


B , 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 .

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B, 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 .

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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 .

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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 ) .

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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 ) .

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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 ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B 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 .

O O O O O O O B-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O

B 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 .

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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 .

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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 .

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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 .

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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 .

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 .

O B-GENEPROD O B-SUBCELLULAR O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O

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 )

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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 .

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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 ) .

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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 ) .

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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).


Quantification of fibroblast density ( number of PDGFRαH2BEGFP + cells , right ; n = 3 biological replicates per time point ) and dermis volume ( left ) with age ( n = 7 E12 . 5 ; n = 8 P0 , P6 ; n = 9 E16 . 5 , E17 . 5 , P4 ; n = 10 P10 , P50 ; n = 11 E10 . 5 ; n = 12 E18 . 5 , P2 , P21 ; n = 20 P12 biological replicates ) .

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Quantification of fibroblast density (number of PDGFRαH2BEGFP+ cells, right; n=3 biological replicates per time point) and dermis volume (left) with age (n=7 E12.5; n=8 P0, P6; n=9 E16.5, E17.5, P4; n=10 P10, P50; n=11 E10.5; n=12 E18.5, P2, P21; n=20 P12 biological replicates).


Changes in fibroblast proliferation and collagen deposition during dermal development in mice . Immunofluorescence staining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin at indicated developmental time points ( upper panel ) . Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points ( lower panel ) .

O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-TISSUE O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O

Changes in fibroblast proliferation and collagen deposition during dermal development in mice. Immunofluorescence staining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin at indicated developmental time points (upper panel). Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points (lower panel).


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Percentage of PDGFRαH2BEGFP+ cells in G1 cell cycle phase with age (n=1 E12.5; n=2 P2, P10; n=3 E16.5, E17, P0; n=4 E10.5; n=5 E17.5, E18.5, P50; n=7 P4 biological replicates) fitted using an exponential model.


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Quantification of fibroblast proliferation (left; n= 4 E16.5, P10; n= 3 E10.5, E18.5, P2, P21, P44, P50; n= 2 E17.5, P0 biological replicates) and collagen density (right; n= 4 E16.5, P2, P6, P10, P14, P16, P21, P50; n= 3 E10.5, E18.5, P2, P21, P44, P50 biological replicates) with age.


Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus - hGem ( green ) expressing cells are in S / G2 / M phase and mCherry - hCdt1 ( red ) positive cells are arrested in G1 phase . Percentage of labelled cells in S / G2 / M cell cycle phase in the upper and lower dermis at P0 ( n = 3 biological replicates ) and P2 ( n = 4 biological replicates ) .

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Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus-hGem (green) expressing cells are in S/G2/M phase and mCherry-hCdt1 (red) positive cells are arrested in G1 phase. Percentage of labelled cells in S/G2/M cell cycle phase in the upper and lower dermis at P0 (n=3 biological replicates) and P2 (n=4 biological replicates).


Changes in dermal cell proliferation and collagen deposition in human skin . ( G ) Immunofluorescence staining for Vimentin ( red ) and Ki67 ( green ) of back skin at indicated time points ( upper panel ) . Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points ( lower panel ) . ( H ) Quantification of proliferating cells in the upper and lower dermis ( n = 3 biological replicates per time point ) . ( I ) Quantification of dermal cell proliferation ( Ki67 + ) ( left ) and collagen density ( right ) with age ( n = 3 biological replicates per time point ) . Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent , non - proliferative state before collagen was efficiently deposited . Data shown are means ± s . d . Nuclei were labelled with DAPI ( blue in B , E , G ) . Scale bars , 100µm . wk , week .

O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Changes in dermal cell proliferation and collagen deposition in human skin. (G) Immunofluorescence staining for Vimentin (red) and Ki67 (green) of back skin at indicated time points (upper panel). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points (lower panel). (H) Quantification of proliferating cells in the upper and lower dermis (n=3 biological replicates per time point). (I) Quantification of dermal cell proliferation (Ki67+) (left) and collagen density (right) with age (n= 3 biological replicates per time point). Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent, non-proliferative state before collagen was efficiently deposited. Data shown are means ±s.d. Nuclei were labelled with DAPI (blue in B,E,G). Scale bars, 100µm. wk, week.


Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in ( triangle ) and outside ( circle ) collagen gels for up to 5 days . Cell number was measured by CellTiterGlo assay at indicated time points ( n = 3 technical replicates of one representative experiment repeated 3 times ) .

O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times).


Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days ( triangle ) . Controls were kept on TCP for 3 days before plating ( circle ) . Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown ( n = 3 technical replicates ) .

O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates).


Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding . Quantification of Ki67 positive fibroblasts at indicated time point after seeding ( n = 8 section per condition of one representative experiment repeated two times ) .

O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding. Quantification of Ki67 positive fibroblasts at indicated time point after seeding (n= 8 section per condition of one representative experiment repeated two times).


ECM degradation in DED organotypic cultures by collagenase treatment . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures at indicated time points after collagenase treatment . Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment ( n = 8 section per condition of one representative experiment repeated two times ) . Note that fibroblasts around the injection sides start to proliferate after 48 hours . Nuclei were labelled with DAPI ( blue ) ( D , F ) . Scale bars , 100µm ( D , F ) . Data shown are means ± s . d . Fb , fibroblasts ; PD , post digestion ; TCP , tissue culture plastic ; h , hours .

B-SUBCELLULAR O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O 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-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O

ECM degradation in DED organotypic cultures by collagenase treatment. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.


Resulting simulation of the system of equations


Live imaging of adult PDGFRαH2BEGFP mouse back skin . Representative time - lapse images of adult PDGFRαH2BEGFP ( green ) dermis with collagen shown as second harmonic generation ( SHG ) in purple at indicated imaging time points . Scale bar , 100 µm .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-ORGANISM B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

Live imaging of adult PDGFRαH2BEGFP mouse back skin. Representative time-lapse images of adult PDGFRαH2BEGFP (green) dermis with collagen shown as second harmonic generation (SHG) in purple at indicated imaging time points. Scale bar, 100 µm.


In silico lineage tracing of fibroblasts . ( E ) Labelling of fibroblasts close to the epidermis at indicated MCS . ( F ) Labelling of fibroblasts in the middle of the dermis at indicated MCS . ( G ) Labelling of fibroblasts close to the inside of the body at indicated MCS .

O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

In silico lineage tracing of fibroblasts. (E) Labelling of fibroblasts close to the epidermis at indicated MCS. (F) Labelling of fibroblasts in the middle of the dermis at indicated MCS. (G) Labelling of fibroblasts close to the inside of the body at indicated MCS.


Fibroblast activation during wound repair . Immunofluorescence staining for α − sma ( red ) of PDGFRαH2BEGFP ( green ) wound sites at indicated post wounding ( PW ) times Nuclei were labelled with DAPI ( blue )

B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-DISEASE O O O O B-DISEASE O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O

Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times Nuclei were labelled with DAPI (blue)


Fibroblast activation during wound repair . quantification of α − sma fluorescence intensity in pixel per area inside the upper and lower wound bed ( n = 4 PW0 , PW2 , PW7 , PW21 ; n = 3 PW10 , PW14 ; n = 2 PW4 biological replicates )

B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fibroblast activation during wound repair. quantification of α−sma fluorescence intensity in pixel per area inside the upper and lower wound bed (n= 4 PW0, PW2, PW7, PW21; n= 3 PW10, PW14; n= 2 PW4 biological replicates)


Changes in fibroblast density , proliferation and collagen deposition during wound repair . Immunostaining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin sections at indicated wound healing time points . Nuclei were labelled with

O O B-CELL_TYPE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O

Changes in fibroblast density, proliferation and collagen deposition during wound repair. Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. Nuclei were labelled with DAPI


Changes in fibroblast density , proliferation and collagen deposition during wound repair . Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

O O B-CELL_TYPE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Changes in fibroblast density, proliferation and collagen deposition during wound repair. Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Quantification of fibroblast density during wound repair inside and outside the wound bed ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of fibroblast density during wound repair inside and outside the wound bed (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding .

O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O B-DISEASE O

Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding.


Quantification of proliferating ( Ki67 positive ) keratinocytes and fibroblasts during wound repair over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of proliferating (Ki67 positive) keratinocytes and fibroblasts during wound repair over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Quantification of fibroblast proliferation ( left ; n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) and collagen density ( right ; n = 4 PW0 , PW2 ; n = 3 PW4 , PW7 , PW10 , PW14 ; n = 2 PW21 biological replicates ) inside ( A ) and outside ( B ) the wound bed over time .

O O B-CELL_TYPE B-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-DISEASE O O O O

Quantification of fibroblast proliferation (left; n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates) and collagen density (right; n= 4 PW0, PW2; n= 3 PW4, PW7, PW10, PW14; n= 2 PW21 biological replicates) inside (A) and outside (B) the wound bed over time.


Temporal evolution of proliferating fibroblasts ( PF ) and dermal ECM density over time .

O O O B-EXP_ASSAY B-CELL_TYPE O O O O B-TISSUE B-SUBCELLULAR O O O O

Temporal evolution of proliferating fibroblasts (PF) and dermal ECM density over time.