( B ) Volcano plots of the enriched proteins identified by PABPN1 TurboID - MS . Significantly enriched proteins are colored in orange , and a few representative proteins are highlighted with labels .

(B) Volcano plots of the enriched proteins identified by PABPN1 TurboID-MS. Significantly enriched proteins are colored in orange, and a few representative proteins are highlighted with labels.


( C ) RT - PCR analysis of the last intron splicing of five representative PABPN1 - dependent endogenous genes upon RBM26 / 27 knockdown with quantified IR values . Data information Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

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(C) RT-PCR analysis of the last intron splicing of five representative PABPN1-dependent endogenous genes upon RBM26/27 knockdown with quantified IR values. Data information Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


( G ) Cumulative frequency distributions of up - regulated RIs upon PABPN1 ( red ) , RBM26 & 27 ( blue ) , ZC3H3 ( green ) , and ZFC3H1 ( yellow ) knockdown by their relative intronic rank in genes .

O O O O O O O O O O O O O O O O O O O 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) Cumulative frequency distributions of up-regulated RIs upon PABPN1 (red), RBM26&27 (blue), ZC3H3 (green), and ZFC3H1 (yellow) knockdown by their relative intronic rank in genes.


( H ) Western blot validation of endogenous PABPN1 interacting with the myc - tagged RBM26 and RBM27 .

(H) Western blot validation of endogenous PABPN1 interacting with the myc-tagged RBM26 and RBM27.


( I ) Representative immunofluorescence images showing the localization of endogenous PABPN1 relative to SC35 , and the mCherry - tagged RBM26 & 27 relative to SRRM2 in vivo .

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(I) Representative immunofluorescence images showing the localization of endogenous PABPN1 relative to SC35, and the mCherry-tagged RBM26&27 relative to SRRM2 in vivo.


( K - L ) Western blot of endogenous PABPN1 immunoprecipitated myc - tagged RBM27 mutated proteins .

(K-L) Western blot of endogenous PABPN1 immunoprecipitated myc-tagged RBM27 mutated proteins.


The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . Immunoblotting ( IB ) analysis of EV71 VP1 in cells , with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation .

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. Immunoblotting (IB) analysis of EV71 VP1 in cells, with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . EV71 viral RNA levels in cell lysates and supernatants were detected by RT - qPCR with GAPDH as a control . ( n = 3 , mean ± SD , ns stands for no significance , paired t - test )

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. EV71 viral RNA levels in cell lysates and supernatants were detected by RT-qPCR with GAPDH as a control. (n=3, mean±SD, ns stands for no significance, paired t-test)


The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . The mRNA levels of the host restriction factors were detected by RT - qPCR in infected or uninfected HEK293T or RD cells , and the expression levels of the target genes were normalized to GAPDH . ( n = 3 , mean ± SD , ns stands for no significance , paired t - test ) .

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. The mRNA levels of the host restriction factors were detected by RT-qPCR in infected or uninfected HEK293T or RD cells, and the expression levels of the target genes were normalized to GAPDH. (n=3, mean±SD, ns stands for no significance, paired t-test).


The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . IB analysis of SAMHD1 and BST - 2 protein levels with tubulin as a loading control . The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin .

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. IB analysis of SAMHD1 and BST-2 protein levels with tubulin as a loading control. The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin.


SAMHD1 knockdown enhances EV71 replication . The stable cell lines pLKO . 1 and sh - SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0 . 1 and 0 . 05 respectively , and cells and supernatants were harvested at the indicated time points . IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation .

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

SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


SAMHD1 knockdown enhances EV71 replication . EV71 viral RNA in cell lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , paired t - test ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).


SAMHD1 knockdown enhances EV71 replication . Viral titers in the supernatants were measured by the cytopathic effect method .

SAMHD1 knockdown enhances EV71 replication. Viral titers in the supernatants were measured by the cytopathic effect method.


SAMHD1 knockdown enhances EV71 replication . The stable cell lines pLKO . 1 and sh - SAMHD1 constructed in RD ( H ) cells were infected with EV71 at a MOI of 0 . 1 and 0 . 05 respectively , and cells and supernatants were harvested at the indicated time points . IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation .

SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


HEK293T cells transfected with VR1012 , which is a eukaryotic expression vector , or Vpx - HA were infected with EV71 at a MOI of 0 . 1 , and then cells and supernatants were harvested at the indicated time points . IB analysis of EV71 - VP1 , SAMHD1 and Vpx - HA in cell lysates was performed with tubulin as a loading control . EV71 - VP1 was detected in the supernatants after ultracentrifugation .

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

HEK293T cells transfected with VR1012, which is a eukaryotic expression vector, or Vpx-HA were infected with EV71 at a MOI of 0.1, and then cells and supernatants were harvested at the indicated time points. IB analysis of EV71-VP1, SAMHD1 and Vpx-HA in cell lysates was performed with tubulin as a loading control. EV71-VP1 was detected in the supernatants after ultracentrifugation.


EV71 viral RNA was detected in cell lysates by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).


Viral titres in the supernatants were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * * P < 0 . 01 ) .

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

Viral titres in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).


Stable SAMHD1 - HA - overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control .

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

Stable SAMHD1-HA-overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control.


RD SAMHD1 - HA cell lines were treated with dimethyl sulfoxide ( DMSO ) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.


RD SAMHD1 - HA cell lines were treated as in ( B ) , and then HA immunoprecipitation ( IP ) and IB were performed . The IP elution was analyzed by mass spectrometry .

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

RD SAMHD1-HA cell lines were treated as in (B), and then HA immunoprecipitation (IP) and IB were performed. The IP elution was analyzed by mass spectrometry.


TRIM21 induced the degradation of SAMHD1 , and MG132 rescued the TRIM21 - mediated degradation of SAMHD1 . HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest , and then subjected to IB with tubulin as a loading control .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

TRIM21 induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.


TRIM25 ( E ) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control .

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TRIM25 (E) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control.


Reverse co - IP confirmed the interaction between SAMHD1 and TRIM21 . SAMHD1 - flag was transfected with TRIM21 - HA into HEK293T cells , and then the cells were treated as in ( B ) and subjected to HA IP and IB .

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Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.


Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1 . RD - SAMHD1 - HA cells treated as in ( B ) were subjected to TRIM21 or TRIM25 IP and IB .

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Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1. RD-SAMHD1-HA cells treated as in (B) were subjected to TRIM21 or TRIM25 IP and IB.


mRNA levels of TRIM21 in RD and HEK293T cells .

mRNA levels of TRIM21 in RD and HEK293T cells.


protein levels of TRIM21 in RD and HEK293T cells .

protein levels of TRIM21 in RD and HEK293T cells.


TRIM21 ( J ) but not TRIM25 ( K ) colocalized with SAMHD1 . Images were taken under a Zeiss LZM710 confocal microscope , Bars , 10μm .

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TRIM21 (J) but not TRIM25 (K) colocalized with SAMHD1. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.


TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . Negative control or RD - CRISPR - Cas9 - TRIM21 RD cells were infected with EV71 at a MOI of 0 . 05 for the indicated time and harvested for SAMHD1 , TRIM21 and EV71 - VP1 detection by IB . Tubulin served as a loading control .

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

TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control.


TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . EV71 RNA levels in ( B ) were detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) .

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

TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test).


TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . Viral titres in the supernatant were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * * P < 0 . 01 ) .

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

TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Viral titres in the supernatant were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).


Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1 . HEK293T cells were transfected with VR1012 or TRIM21 - HA for 24 h and then infected with EV71 at a MOI of 0 . 05 . Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control .

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

Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control.


Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1 . HEK293T cells were transfected with VR1012 or TRIM21 - HA for 24 h and then infected with EV71 at a MOI of 0 . 05 . EV71 viral RNA in cell lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T cells for 24 h and infected with EV71 . Cells were harvested and subjected to IB analysis .

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

TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Cells were harvested and subjected to IB analysis.


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T cells for 24 h and infected with EV71 . Viral titers in the supernatants were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * P < 0 . 05 ) .

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

TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).


The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT - qPCR with GAPDH as a control , and the expression of SAMHD1 and TRIM21 in the heart was set as 100 % ( n = 3 , mean ± SD , * P < 0 . 05 , paired t - test ) .

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

The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT-qPCR with GAPDH as a control, and the expression of SAMHD1 and TRIM21 in the heart was set as 100% (n=3, mean±SD, * P < 0.05, paired t-test).


The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum ( NIR ) western blot with tubulin as a control .

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

The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum (NIR) western blot with tubulin as a control.


Immunohistochemical ( IHC ) staining results of SAMHD1 in various tissues of mice . Bars , 100μm .

Immunohistochemical (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.


The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of EV71 ( D ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of , IFNα and IFNβ ( E ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 %

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

The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of , IFNα and IFNβ (E) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of SAMHD1 and TRIM21 ( F ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 %

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

The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of SAMHD1 and TRIM21 (F) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


IB analysis of THP - 1 cells treated with scrambled shRNA , TRIM21 - specific shRNA or SAMHD1 - specific shRNA . Tubulin served as loading control . Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) .

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

IB analysis of THP-1 cells treated with scrambled shRNA, TRIM21-specific shRNA or SAMHD1-specific shRNA. Tubulin served as loading control. Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD).


sh - SAMHD1 and sh - TRIM21 THP - 1 cells were treated with PMA for 24 h and infected with the VSV - G pseudotyped reporter HIV - 1 , then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , paired t - test ) .

sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).


Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3 , respectively , after MDM differentiation .

Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3, respectively, after MDM differentiation.


MDM cells treated with TRIM21 siRNA were infected with VSV - G - pseudotyped HIV - 1 reporter and then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) .

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

MDM cells treated with TRIM21 siRNA were infected with VSV-G-pseudotyped HIV-1 reporter and then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ** P < 0.01, paired t-test).


Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB analysis of Jurkat and KG - 1 cells treated with scrambled shRNA or TRIM21 - specific shRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of Jurkat and KG-1 cells treated with scrambled shRNA or TRIM21-specific shRNA.


Jurkat and KG - 1 cells treated with TRIM21 shRNA were infected with HIV - 1 , then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , ns stands for no significance , paired t - test ) .

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

Jurkat and KG-1 cells treated with TRIM21 shRNA were infected with HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ns stands for no significance, paired t-test).


TRIM21 interacts with SAMHD1 via PRY and SPRY domains . The effect of TRIM21 on the degradation of SAMHD1 . SAMHD1 - flag was cotransfected with VR1012 , TRIM21 WT or the indicated mutant into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as loading control .

TRIM21 interacts with SAMHD1 via PRY and SPRY domains. The effect of TRIM21 on the degradation of SAMHD1. SAMHD1-flag was cotransfected with VR1012, TRIM21 WT or the indicated mutant into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as loading control.


TRIM21 interacts with SAMHD1 via PRY and SPRY domains . SAMHD1 - flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB .

TRIM21 interacts with SAMHD1 via PRY and SPRY domains. SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


The effect of TRIM21 on SAMHD1 WT or its mutants . SAMHD1 - HA or the indicated mutant were cotransfected with VR1012 or TRIM21 - flag into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as a loading control .

The effect of TRIM21 on SAMHD1 WT or its mutants. SAMHD1-HA or the indicated mutant were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - flag 1 - 547 was cotransfected with VR1012 or TRIM21 - HA for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB .

SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


SAMHD1 109 - 626 followed with a His tag or TRIM21 - PRYSPRY followed with a GST tag were expressed in Rosetta ( DE3 ) , and pull - down assay were performed with Ni - Sepharose ( up ) and GST Sepharose ( down ) , respectively .

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

SAMHD1 109-626 followed with a His tag or TRIM21-PRYSPRY followed with a GST tag were expressed in Rosetta (DE3), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.


FRET analysis indicates interaction between YFP - SAMHD1 and CFP - TRIM21 . A representative image of SAMHD1 - YFP ( yellow ) and ECFP - TRIM21 ( cyan ) expressing cells before and after photobleaching the acceptor fluorophore , YFP . The region chosen for photobleaching is marked ( white open box ) , Bars , 10μm . The quantization of fluorescence brightness was analyzed by Image J ( n = 3 , mean ± SD , ns stands for no significance , * P < 0 . 05 , paired t - test )

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

FRET analysis indicates interaction between YFP-SAMHD1 and CFP-TRIM21. A representative image of SAMHD1-YFP (yellow) and ECFP-TRIM21 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box), Bars, 10μm. The quantization of fluorescence brightness was analyzed by Image J (n=3, mean±SD, ns stands for no significance, * P < 0.05, paired t-test)


The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21 . Alexa Fluor ® 647 labelled SAMHD1 at a concentration of 20 nM were incubated with two - fold dilution series of unlabeled TRIM21 ( 3 μM to 9 . 16E - 05 μM ) . The curve represents the signal recorded from three measurements . The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1 : 1 binding model ( NanoTemper ® analysis software , F Norm = F Hot / F Cold ) . The Kd of this interaction was determined to be 270 ± 92nM . The data was a representive of three independent experiments using different dilution .

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

The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.


The effect of TRIM21 on SAMHD1 proteins from various species . HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector , then subjected to IB analysis . Tubulin served as a loading control .

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

The effect of TRIM21 on SAMHD1 proteins from various species. HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector, then subjected to IB analysis. Tubulin served as a loading control.


The effect of TRIM21 on hSAMHD1 mutants . HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis . Tubulin served as a loading control .

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

The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis. Tubulin served as a loading control.


SAMHD1 - flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21 - HA for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB . Tubulin served as a loading control .

SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB. Tubulin served as a loading control.


SAMHD1 - HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21 - flag into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as a loading control .

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

SAMHD1-HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - HA WT and VR1012 or TRIM21 were cotransfected with K48 - only and K63 - only ubiquitin - flag into HEK293T cells for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control .

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

SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


K48 - only ubiquitin - flag and SAMHD1 - HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated . The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control .

K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


VR1012 , SAMHD1 - HA WT or the K622R mutant was transfected into HEK293T - shSAMHD1 cells for 24 h . The cells were then infected with EV71 at a MOI of 0 . 1 and harvested at the indicated time points . SAMHD1 - HA , TRIM21 and EV71 - VP1 were detected by IB with tubulin as a loading control .

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

VR1012, SAMHD1-HA WT or the K622R mutant was transfected into HEK293T-shSAMHD1 cells for 24 h. The cells were then infected with EV71 at a MOI of 0.1 and harvested at the indicated time points. SAMHD1-HA, TRIM21 and EV71-VP1 were detected by IB with tubulin as a loading control.


( D ) Absolute numbers of lymphocytes subpopulations in the bone marrow , thymus , and spleen . B cell development in the bone marrow was assessed by FACS analysis for surface expression of : B220 + CD43 + CD19 − IgM − IgD − ( pre - pro B ) ; B220 + CD43 + CD19 + IgM − IgD − ( pro B ) ; B220 + CD43 − CD19 + IgM − IgD − ( pre B ) ; CD19 + IgM + IgD − ( immature ) ; CD19 + IgM + IgD + ( mature ) . T cell development in the thymus was assessed by FACS analysis for surface expression of : CD4 − CD8 − ( double negative , DN ) ; CD4 + CD8 + ( double positive , DP ) ; CD4 + CD8 − ( CD4 single positive , SP ) ; CD4 − CD8 + ( CD8 SP ) . Splenic B cell populations were assessed by FACS analysis for surface expression of : B220 + CD21 + CD23 + ( follicular B cells , FOB ) ; B220 + CD21 + CD23low ( marginal zone B cells , MZB ) . Each symbol represents an individual mouse . Horizontal bars indicate mean values . Two - tailed Mann - Whitney test , ns = not significant , * P < 0 . 05 , and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Absolute numbers of lymphocytes subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220+CD43+CD19−IgM−IgD− (pre-pro B); B220+CD43+CD19+IgM−IgD− (pro B); B220+CD43−CD19+IgM−IgD− (pre B); CD19+IgM+IgD− (immature); CD19+IgM+IgD+ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4−CD8− (double negative, DN); CD4+CD8+ (double positive, DP); CD4+CD8− (CD4 single positive, SP); CD4−CD8+ (CD8 SP). Splenic B cell populations were assessed by FACS analysis for surface expression of: B220+CD21+CD23+ (follicular B cells, FOB); B220+CD21+CD23low (marginal zone B cells, MZB). Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two-tailed Mann-Whitney test, ns = not significant, *P < 0.05, and **P < 0.01.


( B ) Maximum Ca2 + flux ( peak height ) at each anti - IgM concentration for FOB and MZB cells . Paired t test , ns = not significant , * P < 0 . 05 , and * * * P < 0 . 001 .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Maximum Ca2+ flux (peak height) at each anti-IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001.


( B ) Co - immunoprecipitation of HA - tagged Pacs1 by FLAG - Wdr37 and HA - Wdr37 by FLAG - Pacs1 in co - transfected 293T cells .

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

(B) Co-immunoprecipitation of HA-tagged Pacs1 by FLAG-Wdr37 and HA-Wdr37 by FLAG-Pacs1 in co-transfected 293T cells.


( E ) Wdr37 + / + and Wdr37 − / − splenocytes were labeled with Indo - 1 , stained for cell surface markers to identify FOB cells , and stimulated with the indicated amounts of anti - IgM ( black arrowhead ) . Normalized traces from three ( 2 . 5 mcg / ml anti - IgM ) or four independent experiments ( 10 mcg / ml and 5 mcg / ml anti - IgM ) are shown ( Wdr37 + / + gray , Wdr37 − / − pink ) . Mean Ca2 + flux for each genotype is overlaid in bold ( Wdr37 + / + black , Wdr37 − / − red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(E) Wdr37+/+ and Wdr37−/− splenocytes were labeled with Indo-1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti-IgM (black arrowhead). Normalized traces from three (2.5 mcg/ml anti-IgM) or four independent experiments (10 mcg/ml and 5 mcg/ml anti-IgM) are shown (Wdr37+/+ gray, Wdr37−/− pink). Mean Ca2+ flux for each genotype is overlaid in bold (Wdr37+/+ black, Wdr37−/− red).


( A ) Immunoblot of ER mass , ER stress , and autophagy markers in Pacs1 + / + and Pacs1 - / - splenic B cells that were left unstimulated or stimulated overnight with 5 mcg / ml IgM .

O O O O O O O O O O O O O O O O O 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) Immunoblot of ER mass, ER stress, and autophagy markers in Pacs1+/+ and Pacs1-/- splenic B cells that were left unstimulated or stimulated overnight with 5 mcg/ml IgM.


( B ) Real - time quantitative PCR of IP3R and SERCA2 transcripts in B cells . Data are presented as mean ± SD from three independent Pacs1 + / + and Pacs1 - / - pairs of mice .

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

(B) Real-time quantitative PCR of IP3R and SERCA2 transcripts in B cells. Data are presented as mean ± SD from three independent Pacs1+/+ and Pacs1-/- pairs of mice.


( D ) Peak cytosolic Ca2 + concentration based on aequorin measurements as performed in ( C ) . Symbols represent individual wells containing ~ 5 x 105 transfected 3T3 or Pacs1 - / - cells from one experiment . Horizontal bars indicate mean . Two - tailed unpaired t test , * * P < 0 . 01 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Peak cytosolic Ca2+ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~5 x 105 transfected 3T3 or Pacs1-/- cells from one experiment. Horizontal bars indicate mean. Two-tailed unpaired t test, **P < 0.01.


( E ) Pacs1 + / + and Pacs1 - / - NIH - 3T3 cells ( C1 and C2 from ( A ) ) were transfected with ER - GCamP6 - 210 . ER Ca2 + was measured before and after treatment with 10 µM ATP . Kinetic traces show the mean 488 / 405 nm excitation ratio of each cell line with error bars indicating SEM . N = 30 cells ( 3T3 ) , 16 cells ( C1 ) , 25 cells ( C2 ) . Data are representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pacs1+/+ and Pacs1-/- NIH-3T3 cells (C1 and C2 from (A)) were transfected with ER-GCamP6-210. ER Ca2+ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N=30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments.


( B , C ) Pacs1 + / + and Pacs1 − / − mice were immunized with alum - ova and one week later with NP - Ficoll . Anti - ova IgG and anti - NP IgM titers were measured at 14 days and 7 days after immunization , respectively . Each symbol represents an individual mouse .

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

(B, C) Pacs1+/+ and Pacs1−/− mice were immunized with alum-ova and one week later with NP-Ficoll. Anti-ova IgG and anti-NP IgM titers were measured at 14 days and 7 days after immunization, respectively. Each symbol represents an individual mouse.


( H , I ) Pacs1 + / + and Pacs1 - / - mice were injected with EdU and the fraction of EdU + FOB and MZB cells were measured in the spleen at 1 , 4 , and 7 days post - injection . Data are from one experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pacs1+/+ and Pacs1-/- mice were injected with EdU and the fraction of EdU+ FOB and MZB cells were measured in the spleen at 1, 4, and 7 days post-injection. Data are from one experiment.


( D , E ) The fraction of proliferating ( D ) and recovered ( E ) donor cells from the experiment in ( C ) . Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments . Two - tailed unpaired t test , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) The fraction of proliferating (D) and recovered (E) donor cells from the experiment in (C). Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments. Two-tailed unpaired t test, **P < 0.01, ***P < 0.001.


( I ) Enumeration of CD3 + B220 + cells in the peripheral blood and lymph nodes of Faslpr / lpr dependent on Pacs1 expression . Horizontal bars indicate median value . Two - tailed Mann - Whitney test , * * P < 0 . 01 .

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

(I) Enumeration of CD3+B220+ cells in the peripheral blood and lymph nodes of Faslpr/lpr dependent on Pacs1 expression. Horizontal bars indicate median value. Two-tailed Mann-Whitney test, **P < 0.01.


A - C , Immunofluorescence images ( inverted intensity ) of P14 - P15 IHCs from indicated genotypes , revealing differences in otoferlin fluorescence intensity and distribution . Maximum projection of confocal stacks , scale bar 10µm .

O O O O O O O O O O O O O O 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-C, Immunofluorescence images (inverted intensity) of P14-P15 IHCs from indicated genotypes, revealing differences in otoferlin fluorescence intensity and distribution. Maximum projection of confocal stacks, scale bar 10µm.


D - F , Upper panels , examples for IHCs , co - labelled for otoferlin ( magenta ) and Vglut3 ( green ) and position of the line for line scans ; maximum projection of few optical sections , scale bars 5µm . Lower panels , for quantification of membrane staining , the fluorescence was normalized to the cellular fluorescence for each fluorophore , then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values ( black line ) was used to determine the position of the basal membrane . At the most basal cellular point along this line which exceeds the threshold value of 2 ( yellow diamond ) , the otoferlin - Vglut3 fluorescence difference ( blue line ) gave the value for relative otoferlinplasma membrane levels ( orange diamond ) . Insets : enlargements of basal regions . G , Otoferlin protein levels were reduced in OtofI515T / I515T IHCs ( indicates numbers represent numbers of cells ) and OtofPga / Pga IHCs compared to wild - type ( Otof + / + ) controls ( mean ± SEM ) . H , ratio of apical / basal protein levels ( above / below nuclear midline depicted as green line in A ) indicates an apical shift of otoferlin in OtofPga / Pga IHCs . I , Relative levels of membrane - bound otoferlin at the basal pole of IHCs . K , Absolute amount of otoferlin at the basal IHCplasma membrane , gained by multiplication of relative plasma membrane levels ( I ) x total cellular otoferlin protein levels ( G ) . Kruskal - Wallis test ; * * * indicates p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Upper panels, examples for IHCs, co-labelled for otoferlin (magenta) and Vglut3 (green) and position of the line for line scans; maximum projection of few optical sections, scale bars 5µm. Lower panels, for quantification of membrane staining, the fluorescence was normalized to the cellular fluorescence for each fluorophore, then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values (black line) was used to determine the position of the basal membrane. At the most basal cellular point along this line which exceeds the threshold value of 2 (yellow diamond), the otoferlin-Vglut3 fluorescence difference (blue line) gave the value for relative otoferlinplasma membrane levels (orange diamond). Insets: enlargements of basal regions.G, Otoferlin protein levels were reduced in OtofI515T/I515T IHCs (indicates numbers represent numbers of cells) and OtofPga/Pga IHCs compared to wild-type (Otof+/+) controls (mean±SEM).H, ratio of apical/basal protein levels (above/below nuclear midline depicted as green line in A) indicates an apical shift of otoferlin in OtofPga/Pga IHCs.I, Relative levels of membrane-bound otoferlin at the basal pole of IHCs.K, Absolute amount of otoferlin at the basal IHCplasma membrane, gained by multiplication of relative plasma membrane levels (I) x total cellular otoferlin protein levels (G). Kruskal-Wallis test; *** indicates p<0.001; **, p<0.01; *, p<0.05.


A , ABR thresholds in OtofI515T / I515T ( red , n = 5 ) and Otof - / I515T ( blue , n = 7 ) mice were elevated compared to Otof + / + mice ( black , n = 5 ) at an age of 3 - 4 weeks . The grey dotted line indicates the maximum loudspeaker output of 90dB ; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM . At 12kHz , only the threshold increase for Otof - / I515T vs Otof + / + is significant ( Kruskal - Wallis test with Dunn ' s multiple comparisons test between all 3 groups ) . B , Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A : The small wave preceding ABR wave I probably represents the summating potential ( SP , hair cell receptor potential ) , which is intact in Otof mutants . ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T / I515T mice ( Fig EV1 ) . C , Mean ABR wave I amplitude ± SEM for different stimulus intensities ( all differences between genotypes are significant ; 2 - way ANOVA with Tukey ' s multiple comparison test ) . D , At 8 weeks ( circles ) and 25 weeks ( open squares ) , OtofI515T / I515T mice showed highly elevated ABR thresholds compared to Otof + / + mice ( n = 7 - 8 each ; p < 0 . 001 at 12kHz , Mann - Whitney - U - test ) . Grey dotted line as in A . E , Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks . OtofI515T / I515T ( n = 8 ) have drastically reduced ABR amplitudes compared to Otof + / + mice ( n = 7 ) . Otof - / - mice have no ABR ( green , n = 9 ) . F , Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T / I515T and Otof + / + mice ( p < 0 . 001 , 2 - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, ABR thresholds in OtofI515T/I515T (red, n=5) and Otof-/I515T (blue, n=7) mice were elevated compared to Otof+/+ mice (black, n=5) at an age of 3-4 weeks. The grey dotted line indicates the maximum loudspeaker output of 90dB; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM. At 12kHz, only the threshold increase for Otof-/I515T vs Otof+/+ is significant (Kruskal-Wallis test with Dunn's multiple comparisons test between all 3 groups).B, Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A: The small wave preceding ABR wave I probably represents the summating potential (SP, hair cell receptor potential), which is intact in Otof mutants. ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T/I515T mice (Fig EV1).C, Mean ABR wave I amplitude ± SEM for different stimulus intensities (all differences between genotypes are significant; 2-way ANOVA with Tukey's multiple comparison test).D, At 8 weeks (circles) and 25 weeks (open squares), OtofI515T/I515T mice showed highly elevated ABR thresholds compared to Otof+/+ mice (n=7-8 each; p<0.001 at 12kHz, Mann-Whitney-U-test). Grey dotted line as in A.E, Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks. OtofI515T/I515T (n=8) have drastically reduced ABR amplitudes compared to Otof+/+ mice (n=7). Otof-/- mice have no ABR (green, n=9).F, Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T/I515T and Otof+/+ mice (p<0.001, 2-way ANOVA).


A , B , No difference in voltage dependent Ca2 + - currents ( A ) and fractional activation of ICa - channels ( B ) between OtofI515T / I515T IHCs ( n = 16 ) and IHCs of Otof + / + littermates ( n = 13 ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O 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,B, No difference in voltage dependent Ca2+-currents (A) and fractional activation of ICa-channels (B) between OtofI515T/I515T IHCs (n=16) and IHCs of Otof+/+ littermates (n=13).


C , Exocytosis was recorded by measures of changes in membrane capacitance ( ΔCm , lower panel ) in response to depolarization ( left , 20ms ; right , 100ms ) to the voltage where maximum Ca2 + - currents were elicited ( upper panel ) , typically - 14mV .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

C, Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20ms; right, 100ms) to the voltage where maximum Ca2+-currents were elicited (upper panel), typically -14mV.


D , Upper panel , while for stimuli up to 20ms exocytosis from OtofI515T / I515T ( n = 13 ) and Otof + / + ( n = 11 ) IHCs was similar , sustained exocytosis , representing most likely the release of replenished vesicles , was significantly reduced in OtofI515T / I515T IHCs ( t - test ) . Lower panel , Ca2 + - current integrals were of similar size .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

D, Upper panel, while for stimuli up to 20ms exocytosis from OtofI515T/I515T (n=13) and Otof+/+ (n=11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in OtofI515T/I515T IHCs (t-test). Lower panel, Ca2+-current integrals were of similar size.


E , Flash photolysis of caged Ca2 + elicited a smaller exocytic response in OtofI515T / I515T IHCs .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.


F , The kinetics of the fast component from E was comparable between OtofI515T / I515T IHCs and Otof + / + littermates ( b ) . Open circles represent previously published data on IHCs of hearing wild - type mice ( a ; Beutner et al , 2001 ; Pangrsic et al , 2010 ) .

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

F, The kinetics of the fast component from E was comparable between OtofI515T/I515T IHCs and Otof+/+ littermates (b). Open circles represent previously published data on IHCs of hearing wild-type mice (a; Beutner et al, 2001; Pangrsic et al, 2010).


A , Spontaneous rates of SGNs from OtofI515T / I515T ( red , n = 35 ) and Otof + / + littermates ( black , n = 39 ) were not significantly different ( p = 0 . 83 , Kolmogorov - Smirnov test ) .

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

A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test).


B - D , Averaged poststimulus time histograms ± SEM from OtofI515T / I515T mice ( n = 25 - 32 ) and Otof + / + littermate SGNs ( n = 13 - 27 ) to stimulation with 50ms tone bursts at the characteristic frequency ( CF ) of each fiber , 30dB above threshold at indicated stimulus rates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Averaged poststimulus time histograms ±SEM from OtofI515T/I515T mice (n=25-32) and Otof+/+ littermate SGNs (n=13-27) to stimulation with 50ms tone bursts at the characteristic frequency (CF) of each fiber, 30dB above threshold at indicated stimulus rates.


E , Quantification of onset ( largest 0 . 5ms bin , Mann - Whitney U test ) and adapted responses ( averaged 35 - 45ms from response onset , t - test ) from data in B - D .

O O O O O O O O O O O O O O O O O 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, Quantification of onset (largest 0.5ms bin, Mann-Whitney U test) and adapted responses (averaged 35-45ms from response onset, t-test) from data in B-D.


F , The jitter of the first sound - evoked spike was significantly increased ( p < 0 . 001 , Mann - Whitney U test ) but retained its inverse correlation with spike rates .

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

F, The jitter of the first sound-evoked spike was significantly increased (p<0.001, Mann-Whitney U test) but retained its inverse correlation with spike rates.


G , Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T / I515T SGNs , both for repetitive stimulation with 50ms tone bursts ( left , p = 0 . 014 , t - test ) and for continuous stimulation with amplitude modulated tones ( right , p < 0 . 001 , Mann - Whitney U test ) .

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

G, Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T/I515T SGNs, both for repetitive stimulation with 50ms tone bursts (left, p=0.014, t-test) and for continuous stimulation with amplitude modulated tones (right, p<0.001, Mann-Whitney U test).


H , Phase locking to amplitude modulated tones ( assessed as the maximal synchronization index ) was typically less precise in OtofI515T / I515T SGNs than in Otof + / + SGNs ( p = 0 . 09 , Mann - Whitney U test ) .

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

H, Phase locking to amplitude modulated tones (assessed as the maximal synchronization index) was typically less precise in OtofI515T/I515T SGNs than in Otof+/+SGNs (p=0.09, Mann-Whitney U test).


I , 100ms masker tone and 15ms probe tones ( both at CF , 30dB above threshold ) were separated by silent intervals of variable duration . Inter - masker intervals were 500ms for Otof + / + , and 1000ms for OtofI515T / I515T . The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T / I515T ( pink ; mean ± SEM red ) compared to Otof + / + ( grey ; mean ± SEM black ; for 4ms interval : p = 0 . 001 , t - test ) and a slowed time course of recovery ( x : half time of recovery , taken from normalized recovery functions ; p < 0 . 001 , Mann - Whitney U test ) .

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

I, 100ms masker tone and 15ms probe tones (both at CF, 30dB above threshold) were separated by silent intervals of variable duration. Inter-masker intervals were 500ms for Otof+/+, and 1000ms for OtofI515T/I515T. The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T/I515T (pink; mean ±SEM red) compared to Otof+/+ (grey; mean ±SEM black; for 4ms interval: p=0.001, t-test) and a slowed time course of recovery (x: half time of recovery, taken from normalized recovery functions; p<0.001, Mann-Whitney U test).


K , Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps . 5 OtofI515T / I515T mice ( pink , mean ± SEM red ) avoided drinking less efficiently than 2 Otof + / + mice ( grey , mean ± SEM black ) for shorter gap durations . See also Figs S2 / S3 .

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

K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.


A , Exemplary ABR traces ( Click 100dB ) from one OtofI515T / I515T mouse recorded at indicated bullatemperatures during local heating . Note that ABRs never disappeared completely , but wave I amplitude changed reversibly with temperature .

O O O O O O O O O O O O O O O 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, Exemplary ABR traces (Click 100dB) from one OtofI515T/I515T mouse recorded at indicated bullatemperatures during local heating. Note that ABRs never disappeared completely, but wave I amplitude changed reversibly with temperature.


B , C , ABR wave I amplitudes in OtofI515T / I515T ( A , Click 100dB ) and Otof + / + mice ( B , Click 80dB ) decreased with increasing temperature in the bulla . Each color represents a different experiment , dashed lines are line fits . Open symbols indicate the beginning of the experiments ; subsequent recordings are connected by lines . The four indicated data points in B correspond to ABRs in A .

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

B,C, ABR wave I amplitudes in OtofI515T/I515T (A, Click 100dB) and Otof+/+ mice (B, Click 80dB) decreased with increasing temperature in the bulla. Each color represents a different experiment, dashed lines are line fits. Open symbols indicate the beginning of the experiments; subsequent recordings are connected by lines. The four indicated data points in B correspond to ABRs in A.


D , Explanted organ of Corti from an Otof + / + mouse at P4 after two days in vitro ( DIV2 ) at 37 ° C immunostained against otoferlin ( magenta ) and Vglut3 ( green ) . Note the intense immunostaining of otoferlin at the plasma membrane . Scale bar 10 µm . E - J , Explanted organs of Corti from Otof - / - mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2 . E , Example cell transfected with wild - type mouseotoferlin cDNA . F , G , Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif ( see Fig EV4 ) . Cells in G were incubated at 38 . 5 ° C for 30 min prior to fixation . The RXR - otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence / eGFP fluorescence at the basal pole of the cells ( arrows ) , similar as in controls ( A , B ) . H , J , Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA , and cells were incubated at 37 ° C ( H ) or for 30min at 38 . 5 ° C ( J ) before fixation . Here , green fluorescence surrounds the otoferlin immunofluorescence ( arrows ) , suggesting loss of otoferlin from the plasma membrane . K , Quantification of the relative plasma membrane levels of otoferlin ( as in Fig 1 ) revealed normal plasma membrane abundance when the human RXR motif was present , but a strong reduction for otoferlin with RXR and Ile515Thr ( individual cells ; mean ± SEM ; t - test ) .

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

D, Explanted organ of Corti from an Otof+/+ mouse at P4 after two days in vitro (DIV2) at 37°C immunostained against otoferlin (magenta) and Vglut3 (green). Note the intense immunostaining of otoferlin at the plasma membrane. Scale bar 10 µm.E-J, Explanted organs of Corti from Otof-/- mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2.E, Example cell transfected with wild-type mouseotoferlin cDNA.F,G, Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif (see Fig EV4). Cells in G were incubated at 38.5°C for 30 min prior to fixation. The RXR-otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence/eGFP fluorescence at the basal pole of the cells (arrows), similar as in controls (A,B).H,J, Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA, and cells were incubated at 37°C (H) or for 30min at 38.5°C (J) before fixation. Here, green fluorescence surrounds the otoferlin immunofluorescence (arrows), suggesting loss of otoferlin from the plasma membrane.K, Quantification of the relative plasma membrane levels of otoferlin (as in Fig 1) revealed normal plasma membrane abundance when the human RXR motif was present, but a strong reduction for otoferlin with RXR and Ile515Thr (individual cells; mean ±SEM; t-test).


A - E , Capacitance increments recorded from Otof + / + ( black ) and OtofI515T / I515T ( red ) IHCs . Individual cells recorded at indicated temperatures ( light circles ) , mean ± SEM for perforated patch clamp experiments ( t - test ) .

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

A-E, Capacitance increments recorded from Otof+/+ (black) and OtofI515T/I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ±SEM for perforated patch clamp experiments (t-test).


F , G , Summary of the capacitance changes and Ca2 + - current integrals for Otof + / + ( F ) and OtofI515T / I515T IHCs ( G ) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature . * Significant differences compared to RT measurements are indicated with colors of the respective temperature , and between PT and high temperature in violet ( t - test ) .

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

F,G, Summary of the capacitance changes and Ca2+-current integrals for Otof+/+ (F) and OtofI515T/I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. *Significant differences compared to RT measurements are indicated with colors of the respective temperature, and between PT and high temperature in violet (t-test).


H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.


H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . K , Quantification of otoferlin immunofluorescence in Otof + / + IHCs ( black bars , n = 98 - 137 cells , 4 - 5 experiments ) and OtofI515T / I515T IHCs ( red bars , n = 90 - 97 cells ; Kruskal - Wallis test as in L - N ) , indicate reduced protein levels with increasing temperature . L , Apical / basal otoferlin protein distribution , revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T / I515T IHCs ( red symbols ) at 38 . 5 ° C compared to Otof + / + ( grey / black symbols ) . M , Relative levels of membrane bound otoferlin were lowered with increasing temperature . N , Absolute otoferlin membrane immunostaining strongly decreased with temperature .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.K, Quantification of otoferlin immunofluorescence in Otof+/+ IHCs (black bars, n=98-137 cells, 4-5 experiments) and OtofI515T/I515T IHCs (red bars, n=90-97 cells; Kruskal-Wallis test as in L-N), indicate reduced protein levels with increasing temperature.L, Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T/I515T IHCs (red symbols) at 38.5°C compared to Otof+/+ (grey/black symbols).M, Relative levels of membrane bound otoferlin were lowered with increasing temperature.N, Absolute otoferlin membrane immunostaining strongly decreased with temperature.


A - C , Pre - embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof + / + ( A ) and OtofI515T / I515T ( B ) but not in Otof - / - ( C ) mice . IHCs are highlighted in beige . Scale bar 100nm .

A-C, Pre-embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof+/+ (A) and OtofI515T/I515T (B) but not in Otof-/- (C) mice. IHCs are highlighted in beige. Scale bar 100nm.


D - G , Magnified synaptic ribbons ( R ) and postsynaptic afferent boutons of SGNs ( Aff ) , displaying otoferlin immunogold labelling at active zone membranes ( pink arrowheads ) but not around the synaptic ribbon , using saponin permeabilization ( D - F ) , or Triton - X - 100 treatment ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G).


H , Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle . Scale bar 200nm .

H, Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle. Scale bar 200nm.


I , J , Some otoferlin labelled structures bear clathrin coats ( red arrowheads ) , suggesting that they originate from bulk endocytosis . Scale bar 100nm . K , Otoferlin labelled vesicles were on average larger in OtofI515T / I515T IHCs ( OtofI515T / I515T , n = 86 vesicles , 18 images ; Otof + / I + , n = 82 vesicles , 6 images ; Wilcoxon - rank - sum - test , as in L - M ) . L , Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T / I515T ( 6 images each ) . M , Distal from the plasma membrane , immunogold labels were reduced in OtofI515T / I515T IHCs .

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

I,J, Some otoferlin labelled structures bear clathrin coats (red arrowheads), suggesting that they originate from bulk endocytosis. Scale bar 100nm.K, Otoferlin labelled vesicles were on average larger in OtofI515T/I515T IHCs (OtofI515T/I515T, n=86 vesicles, 18 images; Otof+/I+, n=82 vesicles, 6 images; Wilcoxon-rank-sum-test, as in L-M).L, Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T/I515T (6 images each).M, Distal from the plasma membrane, immunogold labels were reduced in OtofI515T/I515T IHCs.


N , Representative electron micrographs of IHCribbon synapses in OtofI515T / I515Tand Otof + / ( conventional embedding without immunogold ) after pre - incubation for 10min at the indicated temperature followed by high K + stimulation or 0 Ca2 + inhibition for 1min 45sec . Scale bars 100nm .

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

N, Representative electron micrographs of IHCribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm.


P - Q , Synaptic vesicles with > 45 nm diameter appeared at 39 ° C in random sections ; fraction of large membrane - proximal ( P ; Kruskal - Wallis test ) and ribbon - associated ( Q ; one way ANOVA followed by Tukey ' s test ) synaptic vesicles ( see also Appendix Table S1 ) .

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

P-Q, Synaptic vesicles with >45 nm diameter appeared at 39°C in random sections; fraction of large membrane-proximal (P; Kruskal-Wallis test) and ribbon-associated (Q; one way ANOVA followed by Tukey's test) synaptic vesicles (see also Appendix Table S1).


A - D , Exemplary virtual sections through EM tomograms for stimulatory condition at 36 ° C and 39oC . Scale bar 100nm .

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-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm.


F - I , 3D reconstruction of the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm .

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

F-I, 3D reconstruction of the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.


F - I , 3D reconstruction of the membrane - proximal pool ( top view ) and J - M , the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm . N - Q , Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane - proximal pool ( N - O ) and ribbon - associated pool ( P - Q ) . N - P , one way ANOVA followed by Tukey ' s test ; Q , Kruskal - Wallis test . Round vesicles tested in both the pools appeared larger in OtofI515T / I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes ( see also Table EV1 ) .

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

F-I, 3D reconstruction of the membrane-proximal pool (top view) and J-M, the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.N-Q, Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane-proximal pool (N-O) and ribbon-associated pool (P-Q). N-P, one way ANOVA followed by Tukey's test; Q, Kruskal-Wallis test. Round vesicles tested in both the pools appeared larger in OtofI515T/I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes (see also Table EV1).


( a ) H3R expression in cultured ratcortical neurons at DIV7 after OGD / R injury ( 2 h OGD and 24 h reperfusion ) shown by western blot ( n = 6 per condition ; * * P0 . 01 versus control with Student ' s t - test ) . Full - size blots are shown in Supplementary Fig . 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H3R expression in cultured ratcortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student's t-test). Full-size blots are shown in Supplementary Fig. 9.


( b ) When the H3R antagonist thioperamide ( THIO , 10 − 6 mol l − 1 at reperfusion ) and the agonist immepip ( IMME , 10 − 6 mol l − 1 at reperfusion ) were administered , viability was tested in cultured neurons by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide ( MTT ) assay after OGD / R ( n = 7 per condition ; * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) 2 , 3 , 5 - triphenyltetrazolium chloride - stained brain sections showing the infarct area in WT and H3R − / − mice receiving saline , the H3R antagonists A331440 , clobenpropit ( CLOB ) and thioperamide , ( THIO ; 5 mg kg − 1 , i . p . , immediately at reperfusion and 6 h later ) , and the H3R agonist immepip ( IMME , 1 μg , i . c . v . , at reperfusion ) after I / R ( 1 h MCAO followed by 24 h reperfusion ; scale bar , 5 mm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).


The bar graphs show infarct volume ( d ) and neurological score ( e ) ( n = 6 - 8 mice per condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 versus WT saline group with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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

The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) TUNEL staining of apoptotic cells in penumbral brain slices in vivo ( scale bar , 100 μm ) when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after tMCAO . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice after tMCAO ( b ) ( n = 6 - 7 per condition ; * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc test )

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)


( c ) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after OGD / R . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice in cultured neurons subjected to OGD / R ( d ) ( n = 8 per condition ; * P0 . 05 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 10 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.


( a ) 2 , 3 , 5 - Triphenyltetrazolium chloride ( TTC ) - stained brain sections from WT and HDC − / − mice showing the infarct area in those receiving saline , the H3R antagonist thioperamide ( THIO ) , the H1R antagonist pyrilamine ( PYRI ; 10 mg kg − 1 , i . p . , at reperfusion ) , the H2R antagonist cimetidine ( CIME ; 10 mg kg − 1 , i . p . , at reperfusion ) and α - FMH ( 50 mg kg − 1 , i . p . , 2 h before ischemia ) after 24 h reperfusion ( n = 6 per condition ; scale bar , 5 mm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).


( b ) Infarct volumes and ( c ) neurological deficit scores ( * P0 . 05 , * * P0 . 01 , * * * P0 . 001 with analysis of variances followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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

(b) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Akt / GSK - 3β / mTOR / P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD / R . LiCl ( 5 mmol l − 1 immediately at reperfusion ) was used to rephosphorylate GSK - 3β / mTOR / P70S6K . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( b ) Representative western blots showing the phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signaling in H3R − / − mice after OGD / R injury . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 .

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

(b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( c ) Representative images showing LC3 - positive autophagic vacuoles ( red ) with 4 ' , 6 - diamidino - 2 - phenylindole staining ( blue ) after administration of thioperamide and LiCl with OGD / R ( scale bar , 10 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).


( d ) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD / R ( n = 7 , * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( e ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II in steady - state autophagy and autophagic flux under OGD / R injury when thioperamide ( THIO ) and immepip ( IMME ) were administered ( n = 6 ; * * P0 . 01 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.


( a ) Representative immunohistochemical staining of LC3 ( green ) along with NeuN staining ( red ) in WT and H3R − / − mice ( scale bar , 25 μm ) .

O O O O O O O O O O O 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) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm).


( b , c ) When thioperamide was administered in vivo , LC3 expression was assessed by western blot after I / R in WT mice ( b ) ( * P0 . 05 , * * * P0 . 001 ; n = 6 mice per condition , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) , and in H3R − / − mice ( c ) ( * * * P0 . 001 versus H3R − / − sham group ; n = 5 - 6 mice per condition , with ANOVAs followed by the Bonferroni / Dunn post hoc test . ) Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 12 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.


( a ) Western blots showing the inhibitory effect of 3 - MA on autophagy in I / R . Full - size blots are shown in Supplementary Fig . 13 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.


( b ) TTC - stained sections showing the infarct area after administration of 3 - MA ( 100 nmol , i . c . v . , at reperfusion ) in WT and H3R − / − mice ( scale bar , 5 mm ) , and ( c ) bar graph showing infarct volume ( n = 6 - 8 mice per condition ; * P0 . 05 , * * * P0 . 001 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( d ) TTC - stained sections ( scale bar , 5 mm ) and bar graph showing the infarct area after administration of thioperamide in Atg5 + / − mice ( n = 4 - 6 mice per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).


( e ) In cultured neurons , viability was tested by MTT assay after 3 - MA ( 2 . 5 mmol l − 1 at reperfusion ) and thioperamide was administered in OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( f ) In cultured neurons , viability was tested by MTT after siRNA for Atg7 and OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

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

(f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( g ) Viability was tested by MTT assay on Atg5 + / + and Atg5 − / − MEFs after thioperamide administration after OGD / R ( n = 7 per condition ; * * P0 . 01 , * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Representative co - immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

O O O O O O O O O O O O B-CONTROLLED_VAR 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) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( b ) Cell viability was tested by MTT assay after administration of Tat - H3RCT414 - 436 ( 10 − 6 mol l − 1 at reperfusion ) under OGD / R ( data are presented as mean ± s . e . m . ; n = 7 per condition ; * P0 . 05 , * * P0 . 01 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

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

(b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414-436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) Representative western blots showing the effect of Tat - H3RCT414 - 436 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

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

(c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( d ) Representative images of the effect of Tat - H3RCT414 - 436 on LC3 - positive autophagic puncta ( red ) in 4 ' , 6 - diamidino - 2 - phenylindole - stained ( blue ) neurons after OGD / R ( scale bar , 10 μm ) .

(d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm).