Representative dual - channel kymographs ( A ) quantitative ( B showing that overexpression of Snapin , but not Snapin - L99K , a Snapin mutant deficient in dynein motor binding , reduces mitophagic accumulation within AD axons incubated with CCCP .

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Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.


Representative quantitative showing that overexpression of Snapin , but not Snapin - L99K , a

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Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a Snapin


D Mean ratios evoked by 405 nm 488 nm excitation wavelengths at mitochondria in mutant hAPP axons expressing Snapin or Snapin - L99K were quantified normalized to those of mutant hAPP controls . Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment .

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D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.


A Representative showing gene delivery into hippocampus of AD mice injected with AAV - mCherry or AAV - mCherry - Snapin . mCherry indicated by arrows is present in soma processes of transduced neurons in hippocampal CA3 dentate gyrus ( DG ) of mutant hAPP mouse brains . NeuN , a neuronal marker , indicates neurons .

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A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.


Attenuation of mitophagic accumulation in hippocampal mossy of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c * : color is converted from blue to red for better

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-DISEASE B-ORGANISM O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrast


Attenuation of mitophagic accumulation in hippocampal mossy of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( arrows ) in mutant hAPP Tg mouse brains with elevated Snapin

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-DISEASE B-ORGANISM O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-ORGANISM B-TISSUE O O B-GENEPROD

Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression


D , E Quantitative ( D ) representative ( E ) showing increased density of presynaptic terminals in hippocampal mossy of AD mice infected with AAV - mCherry - Snapin . Blue indicates signal of DAPI . of SYP - labeled presynaptic terminals was quantified normalized to that of control AD mice injected with AAV - mCherry ( D ) . Data information : Data were quantified from a total number of 40 - 44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection .

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D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.


rat cortical neurons transfected with eGFP - GA50 were examined to determine at which time points preceding are found in neurites . Two days post - transfection , formed by eGFP - GA50 ( green ) are detectable in neurites ( SMI - 32 in red ) . These remain localized to neurites at 96 hours ( 4 days ) 288 hours ( 8 days ) . Colocalization is indicated by yellow overlay of colors ( right panels ) . Inset below each image shows enlargement of neurite regions containing , Representative fields from 60x magnification z - stack confocal , scale bar indicates 20μm . Inset scale bars indicate 5μm .

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Primary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 hours (4 days) and 288 hours (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, Representative fields from 60x magnification z-stack confocal images, scale bar indicates 20μm. Inset scale bars indicate 5μm.


rat cortical neurons underwent live - imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . A ) Example of a cortical neuron expressing eGFP - GA100 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed , to better visualize within neuronal processes . Inset scale bar indicates 5μm .

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Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.


rat motor neurons underwent live - imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . B ) Example of a motor neuron expressing eGFP - GA25 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed , to better visualize within neuronal processes . Inset scale bar indicates 5μm .

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Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.


C ) of eGFP - GAn aggregate velocity within cortical neurons .

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C) Quantification of eGFP-GAn aggregate velocity within cortical neurons.


D ) of eGFP - GAn aggregate velocity within motor neuron neurites .

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D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.


rat neurons were co - transfected with Td - tomato eGFP - GAn plasmids or eGFP - alone . same neurons were imaged at 24 - hour intervals to assess viability . A ) Representative fields ( right ) of eGFP eGFP - GA50 cortical neurons at days 1 , 7 , 13 post - transfection follow survival of over time ( arrows ) . 20x magnification , scale bar indicates 25μm .

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

Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm.


B ) Kaplan - Meier survival of cortical neurons followed for 13 days post - transfection .

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B) Kaplan-Meier survival analysis of cortical neurons followed for 13 days post-transfection.


C ) Kaplan - Meier survival ( left ) of motor neurons followed for 5 days post - transfection .

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C) Kaplan-Meier survival analysis (left) of motor neurons followed for 5 days post-transfection.


Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection . Basal was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative dye loading showing defined regions along neurites 40x magnification , scale bar indicates 10μm .

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Cortical neurons were loaded with FM4-64 dye 48 hours after transfection. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative dye loading showing defined puncta regions along neurites 40x magnification, scale bar indicates 10μm.


B ) Representative intensities of FM4 - 64 regions pre - post - stimulation demonstrate loss of synaptic following stimulation in GFP but not GA50 containing neurons . These visually reflect process performed in an automated manner through our imaging software to determine . An unbiased constant threshold is placed on each image per experiment , then is measured over time . threshold applied isolates from background , giving distinct regions . 8 - bit were thresholded pseudo - color ( red ) was applied to enlarged regions of interest , scale bar indicates 10μm .

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B) Representative intensities of FM4-64 puncta regions pre- and post- stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons. These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity. An unbiased constant threshold is placed on each image per experiment, then fluorescence intensity is measured over time. The threshold applied isolates puncta from background, giving distinct regions. 8-bit images were thresholded and pseudo-color (red) was applied to enlarged puncta regions of interest, scale bar indicates 10μm.


C ) Graphical representation is change in ( ΔF ) normalized to basal ( F ) , ΔF / F . final 10 seconds of baseline recording 10 seconds of depolarization phase are shown , arrow indicates start of depolarization . Non - linear regression was performed in GraphPad Prism7 using plateau followed by one phase decay model . decay constant ( k ) for GFP was 0 . 4971 s - 1 , while for GA50 it was 219 . 5 s - 1 . Similarly tau ( τ ) values were 2 . 012 seconds for GFP 0 . 00455 seconds for GA50 , respectively . Data presented as mean ± for each imaged timepoint . Statistical significance was determined using Sidak - Bonferroni method , * * * * p < 0 . 0001 , n = 20 regions from 4 biological replicates .

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C) Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown, arrow indicates start of depolarization. Non-linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model. The decay constant (k) for GFP was 0.4971 s-1, while for GA50 it was 219.5 s-1. Similarly the tau (τ) values were 2.012 seconds for GFP and 0.00455 seconds for GA50, respectively. Data presented as mean ± SEM for each imaged timepoint. Statistical significance was determined using the Sidak-Bonferroni method, ****p < 0.0001, n=20 puncta regions from 4 biological replicates.


cortical or motor neurons were co - transfected with mCherry or mCherry - GA50 GCaMP6f . After 48 hours , mCherry positive were determined . Green was then recorded from identified neurons . Basal was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative of a co - transfected cortical neuron expressing mCherry - GA50 ( left panel ) GCaMP6f ( right two panels ) . This image shows a representative demonstration of increase in detectable green from GCaMP6f indicator pre - post - stimulation ( center right respectively ) . 40x magnification , scale bar indicates 25μm .

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Primary cortical or motor neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f. After 48 hours, mCherry positive cells were determined. Green fluorescence intensity was then recorded from identified neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative images of a co-transfected cortical neuron expressing mCherry-GA50 (left panel) and GCaMP6f (right two panels). This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre- and post- stimulation (center and right respectively). 40x magnification, scale bar indicates 25μm.


B - C ) Graphical representation of peak change in ( ΔF ) following ACSF perfusion normalized to basal ( F ) , ΔF / F . contribution of Ca2 + influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2 + . of peak change in in cortical neurons ( B ) motor neurons ( C ) .

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B-C) Graphical representation of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The contribution of Ca2+ influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2+. Quantification of the peak change in fluorescence in cortical neurons (B) and motor neurons (C).


eGFP - GAn dipeptides were expressed in cortical or motor neurons for 48 hours , then immunostained for neurofilament ( gray ) SV2 ( red ) . A - B ) Representative z - stack confocal of neurites from eGFP , eGFP - GA50 , eGFP - GA100 expressing cortical ( A ) or motor ( B ) neurons . 60x magnification , scale bar indicates 5μm .

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eGFP-GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours, then immunostained for neurofilament (gray) and SV2 (red). A-B) Representative z-stack confocal images of neurites from eGFP, eGFP-GA50, and eGFP-GA100 expressing cortical (A) or motor (B) neurons. 60x magnification, scale bar indicates 5μm.


C - D ) of SV2 along neurites in cortical neurons ( C ) or motor neurons ( D ) by ImageJ manual counting , normalized to eGFP - only expressing . ( C ) Cortical neurons demonstrated a significant GA - dependent reduction , as well as a reduction in containing GA - repeats greater than 50 ( left panel ) . ( D ) Motor neurons demonstrated a significant GA - dependent reduction , as well as a reduction in containing GA - repeats greater than 100 ( left panel ) . Evaluation of neurite by NeuronJ software revealed no differences in total neurite per in cortical neurons ( C ) or motor neurons ( D ) expressing any GA - repeat compared with eGFP alone ( right panels ) .

O O O O O B-GENEPROD O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-TISSUE B-CELL_TYPE O O O O B-TISSUE B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

C-D) Quantification of SV2 puncta along neurites in cortical neurons (C) or motor neurons (D) by ImageJ manual counting, normalized to eGFP-only expressing cells. (C) Cortical neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 50 (left panel). (D) Motor neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 100 (left panel). Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons (C) or motor neurons (D) expressing any GA-repeat length compared with eGFP alone (right panels).


i3 cortical neurons from a C9orf72 - ALS paired isogenic control were differentiated for 21 days , then were immunostained for neurofilament ( red ) , SV2 ( green ) ( A ) Shown are representative z - stack confocal of neurites .

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i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) Shown are representative z-stack confocal images of neurites.


B ) i3 cortical neurons from a C9orf72 - ALS paired isogenic control were differentiated for 21 days , then were immunostained for synaptophysin ( green ) ( B ) . Shown are representative z - stack confocal of neurites .

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B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for synaptophysin (green) (B). Shown are representative z-stack confocal images of neurites.


of SV2 ( C ) colocalized with neurofilament normalized to neurofilament . This revealed a significant reduction in SV2 in C9orf72 compared with isogenic control , Data presented as mean ± . Unpaired t - , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated .

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Quantification of SV2 (C) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.


of Synaptophysin ( D ) colocalized with neurofilament normalized to neurofilament . This revealed a significant reduction in SV2 in C9orf72 compared with isogenic control , while synaptophysin remained unaltered . Data presented as mean ± . Unpaired t - , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated .

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Quantification of Synaptophysin (D) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.


E ) mRNA was Trizol extracted from 3 independent wells of C9 - ALS isogenic i3 cortical neurons . was converted to cDNA using Superscript First - strand kit , q - PCR was performed using PowerUp SYBR Green . Measurements were normalized to housekeeping gene GAPDH then to isogenic . was performed using ΔΔCT method . 2ΔΔCT ± SE , is presented . Unpaired t - , * * p < 0 . 01 .

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

E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01.


F ) Whole lysates were generated from 3 independent wells of C9 - ALS isogenic i3 cortical neurons differentiated for 21 days . Lysates were immunoblotted for SV2 , normalized to total loading . band intensities revealed a significant decrease in SV2 in C9orf72 lines compared with isogenic . Data presented as mean ± . Unpaired t - , * p < 0 . 05 .

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F) Whole cell lysates were generated from 3 independent wells of C9-ALS and isogenic i3 cortical neurons differentiated for 21 days. Lysates were immunoblotted for SV2, and normalized to total protein loading. Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05.


Whole lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for SV2 ( G ) Loading controls were β - actin band intensities normalized to loading controls revealed a trend towards reduction in SV2 in C9orf72 lines compared with controls Data presented as mean ± . Unpaired t - . Samples from 5 independent control 4 independent C9orf72 lines were used .

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

Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for SV2 (G) Loading controls were β-actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.


Whole lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for synaptophysin ( H ) . Loading controls were total loading . synaptophysin remained unaltered . Data presented as mean ± . Unpaired t - . Samples from 5 independent control 4 independent C9orf72 lines were used .

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

Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for synaptophysin (H). Loading controls were total protein loading. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.


A ) Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection with eGFP - GA50 transduction with rSV2a - eGFP - pRRL lentivirus . Basal was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . Graphical representation is change in ( ΔF ) normalized to basal ( F ) , ΔF / F . final 60 seconds of baseline recording 60 seconds of depolarization phase are shown , arrow indicates start of depolarization . lack of synaptic release in case of GA50 expression ( red ) was rescued back to comparable to eGFP controls ( pink ) in case of coexpression of rSV2a - eGFP - pRRL ( orange ) . Statistical significance was determined using Sidak - Bonferroni method , * * * p < 0 . 001 , n = 10 regions from each of 4 biological replicates .

O O B-TISSUE B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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) Cortical neurons were loaded with FM4-64 dye 48 hours after transfection with eGFP-GA50 and transduction with rSV2a-eGFP-pRRL lentivirus. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown, arrow indicates start of depolarization. The lack of synaptic release in the case of GA50 expression (red line) was rescued back to levels comparable to eGFP controls (pink line) in the case of coexpression of rSV2a-eGFP-pRRL (orange line). Statistical significance was determined using the Sidak-Bonferroni method, ***p < 0.001, n=10 puncta regions from each of 4 biological replicates.


B ) Cortical neurons were co - transfected with mCherry or mCherry - GA50 GCaMP6f , then transduced with rSV2a - eGFP - pRRL . Green was recorded from identified mCherry positive neurons . Basal was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . of peak change in ( ΔF ) following ACSF perfusion normalized to basal ( F ) , ΔF / F . significant increase in Ca2 + influx observed following GA50 expression ( * p < 0 . 05 ) is resolved when rSV2a - eGFP - pRRL is coexpressed . Data presented as mean ± . One - way ANOVA with Sidak ' s multiple comparisons , at least 10 per condition , pooled from n = 3 independent biological experiments . No significant differences were observed between mCherry or mCherry + SV2 conditions , groups were merged for this .

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

B) Cortical neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f, then transduced with rSV2a-eGFP-pRRL. Green fluorescence intensity was recorded from identified mCherry positive neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Quantification of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The significant increase in Ca2+ influx observed following GA50 expression (*p < 0.05) is resolved when rSV2a-eGFP-pRRL is coexpressed. Data presented as mean ± SEM. One-way ANOVA with Sidak's multiple comparisons test, at least 10 cells per condition, pooled from n= 3 independent biological experiments. No significant differences were observed between mCherry or mCherry+SV2 conditions, groups were merged for this analysis.


Neurons were co - transfected with mCherry - GAn plasmids or mCherry - alone transduced with rSV2a - eGFP - pRRL , then imaged at 24 - hour intervals . ( C ) Kaplan - Meier survival of cortical neurons expressing GA50 or GA50 + SV2 followed for 13 days . significant toxicity induced by GA50 is rescued by SV2 replenishment . While hazard of GA50 was 1 . 473 , this was decreased to 0 . 7227 for GA50 + SV2 . At least 190 per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , groups were merged for this as Control . ( D ) A Kaplan - Meier survival was performed for PR50 PR50 + SV2 containing . Under these conditions significant toxicity is observed regardless of increased SV2 , with hazard of PR50 containing ( 1 . 772 ) remaining high for those expressing PR50 + SV2 ( 1 . 784 ) . At least 150 per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , groups were merged for this as Control .

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

Neurons were co-transfected with mCherry-GAn plasmids or mCherry-alone and transduced with rSV2a-eGFP-pRRL, then imaged at 24-hour intervals. (C) Kaplan-Meier survival analysis of cortical neurons expressing GA50 or GA50+SV2 followed for 13 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.473, this was decreased to 0.7227 for GA50+SV2. At least 190 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control. (D) A Kaplan-Meier survival analysis was performed for PR50 and PR50+SV2 containing cells. Under these conditions significant toxicity is observed regardless of increased SV2 levels, with the hazard ratio of PR50 containing cells (1.772) remaining high for those expressing PR50+SV2 (1.784). At least 150 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control.


( E ) Kaplan - Meier survival of motor neurons expressing GA50 or GA50 + SV2 followed for 10 days . significant toxicity induced by GA50 is rescued by SV2 replenishment . While hazard of GA50 was 1 . 345 , this was decreased to 0 . 7769 for GA50 + SV2 . At least 200 per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox , * * * * p < 0 . 0001 .

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

(E) Kaplan-Meier survival analysis of motor neurons expressing GA50 or GA50+SV2 followed for 10 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.345, this was decreased to 0.7769 for GA50+SV2. At least 200 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001.


Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for SV2 ( A ) Total were determined by imaging Mini - PROTEAN TGX stain - free gel prior to transfer . of band intensities normalized to total loading revealed a significant reduction in SV2 in GA - expressing cohort compared with wild type , Data presented as mean ± . Unpaired t - , * p < 0 . 05 . A total of 4 per group were examined .

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

Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for SV2 (A) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA-expressing cohort compared with wild type animals, Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.


Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for synaptophysin ( B ) Total were determined by imaging Mini - PROTEAN TGX stain - free gel prior to transfer . synaptophysin remained unaltered . Data presented as mean ± . Unpaired t - , * p < 0 . 05 . A total of 4 per group were examined .

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

Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for synaptophysin (B) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.


Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for PSD - 95 ( C ) . Total were determined by imaging Mini - PROTEAN TGX stain - free gel prior to transfer . PSD - 95 remained unaltered . Data presented as mean ± . Unpaired t - , * p < 0 . 05 . A total of 4 per group were examined .

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

Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for PSD-95 (C). Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. PSD-95 levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.


Muscle sections ( 30μm ) were cut from snap frozen tissue of 20 - month old GA149 mice ( Tgx GA149 ) or wild type controls . Immunohistochemical was performed to assess synaptic in proximity to NMJs . Sections were immunostained for SV2 or Synaptophysin , counterstained with neurofilament . A ) Left Panels : Representative show SV2 ( green ) in proximity to α - bungarotoxin positive NMJs ( red ) in wild type ( top ) versus GA149 ( bottom ) . Right Panels : Representative show SV2 ( green ) overlapping with neurofilament ( blue ) at synaptic terminals approaching muscle in wild type ( top ) versus GA149 ( bottom ) .

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

Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls. Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs. Sections were immunostained for SV2 or Synaptophysin, and counterstained with neurofilament. A) Left Panels: Representative images show SV2 levels (green) in proximity to α-bungarotoxin positive NMJs (red) in wild type animals (top) versus GA149 animals (bottom). Right Panels: Representative images show SV2 levels (green) overlapping with neurofilament (blue) at synaptic terminals approaching muscle in wild type animals (top) versus GA149 animals (bottom).


B - C ) of SV2 ( B ) or Synaptophysin ( C ) colocalized with neurofilament normalized to neurofilament . This revealed a significant reduction in SV2 in GA149 compared with wild types , while synaptophysin remained unaltered . Data presented as mean ± . Unpaired t - , * p < 0 . 05 . A total of 10 non - overlapping regions of muscle from each of 5 per group were evaluated .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O

B-C) Quantification of SV2 (B) or Synaptophysin (C) colocalized with neurofilament and normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 10 non-overlapping regions of muscle from each of 5 animals per group were evaluated.


( A _ B ) In situs on adult brain sections reveal that ( A ) opn4 . 1 ( B ) opn4xb are coexpressed in many brain domains including epiphysis cerebri ( indicated with E ) or pineal . A horizontal through a representation of brain above sagittal section indicates location within brain . Scale bar : 1 . 0 mm .

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

(A_B) In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain. Scale bar: 1.0 mm.


( J ) ish with asmt probe on larvae sampled on 6th dpf at ZT3 shows that asmt mRNA is confined to pineal indicates that asmt gene is not ectopically expressed in opn4 dko larvae . higher asmt mRNA level measured by qPCR in opn4 dko therefore points at a defect in pineal . Note that eyes were removed after ish for optimal visibility of expression in brain . Scale bar : 0 . 5 mm .

O O O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-TISSUE O O O B-TISSUE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O

(J) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae. The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal. Note that the eyes were removed after the ish for optimal visibility of expression in the brain. Scale bar: 0.5 mm.


( N ) ish with ddc probe on wild - type opn4 dko larvae sampled on 6th dpf represents ddc expression in pineals . Biological replicates indicated with BR . Scale bar : 0 . 05 mm ( O ) of adjusted in pineals from larvae presented in N , implies a significant higher ddc transcript level in opn4 dko pineal . Data information : boxplot divides data in quartiles : box indicates interquartile range , with horizontal in box denoting median of data set , whiskers extend to minimum maximum , meet box at median of lower ( quartile 1 ) median of upper ( quartile 3 ) half of dataset . Black dots indicate biological replicates O : n = 8 ) , red dot indicates mean , red error bars indicate confidence interval ( 95 % ) , asterisks indicate significance ( 0 . 01 < p ( ✱ ) < 0 . 05 , 0 . 001 < p ( ✱ ✱ ) < 0 . 01 , p ( ✱ ✱ ✱ ) < 0 . 001 , ns = not significant ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ish with ddc probe on wild-type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals. Biological replicates indicated with BR. Scale bar: 0.05 mm (O) Quantification of the adjusted intensity in the pineals from larvae presented in N, implies a significant higher ddc transcript level in the opn4 dko pineal. Data information: boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates O: n=8), the red dot indicates the mean, red error bars indicate the confidence interval (95%), asterisks indicate significance (0.01<p(✱)<0.05, 0.001<p(✱✱)<0.01, p(✱✱✱)<0.001, ns = not significant).


( A qRT - PCR for different ISGs relative to Rps29 using epidermal from 8 week - old ( A ) K5 - R1 / R2 control ( CTRL ) mice .

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

(A qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week-old (A) K5-R1/R2 and control (CTRL) mice.


qRT - PCR for different ISGs relative to Rps29 using epidermal from 5 day - or 9 day - old ( B ) K5 - R1 / R2 control ( CTRL ) mice .

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

qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 5 day- or 9 day-old (B) K5-R1/R2 and control (CTRL) mice.


( C ) Confocal microscopy of epidermal sheets from back skin of K5 - R1 / R2 CTRL mice stained for IRF7 K14 , counterstained with DAPI . Arrows denote IRF7 ( red ) in basal suprabasal keratinocytes of K5 - R1 / R2 mice . strong red of stratum corneum ( sc ) is unspecific background is more pronounced in K5 - R1 / R2 mice due to increased thickness of this layer .

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

(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.


( D ) qRT - PCR for different ISGs , Ifnb , Ifnl3 relative to Rps29 using from freshly isolated , non - cultured keratinocytes of K5 - R1 / R2 control ( CTRL ) mice at P7 .

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

(D) qRT-PCR for different ISGs, Ifnb, and Ifnl3 relative to Rps29 using RNA from freshly isolated, non-cultured keratinocytes of K5-R1/R2 and control (CTRL) mice at P7.


( E ) qRT - PCR for ISGs relative to Rps29 using from keratinocytes of neonatal CTRL K5 - R1 / R2 mice .

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

(E) qRT-PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5-R1/R2 mice.


( F ) qRT - PCR for ISGs Cre relative to Rps29 using from keratinocytes of neonatal K5 - Cre wild - type ( WT ) mice .

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

(F) qRT-PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5-Cre and wild-type (WT) mice.


( G ) Western blot of total ( L ) ( N ) lysates from immortalized ( imm . ) keratinocytes of CTRL K5 - R1 / R2 mice for IRF1 , IRF7 , IRF9 , Lamin A / C ( marker , loading control ) GAPDH ( marker , loading control ) .

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

(G) Western blot analysis of total (L) and nuclear (N) lysates from immortalized (imm.) keratinocytes of CTRL and K5-R1/R2 mice for IRF1, IRF7, IRF9, Lamin A/C (nuclear marker, loading control) and GAPDH (cytosolic marker, loading control).


( H ) qRT - PCR for RSAD2 ISG15 relative to RPLP0 using from HaCaT keratinocytes treated for 48 h with FGFR inhibitor BGJ398 ( 3 . 5 μM ) or vehicle in presence of serum .

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

(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.


( A ) Growth factor - starved keratinocytes from WT mice were treated for 3 or 6 h with FGF7 ( 10 ng / ml ) or vehicle ( CTRL ) . samples were analyzed by qRT - PCR for indicated ISGs relative to Rps29 .

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

(A) Growth factor-starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 (10 ng/ml) or vehicle (CTRL). RNA samples were analyzed by qRT-PCR for the indicated ISGs relative to Rps29.


( B ) Serum - starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 ( each 10 ng / ml ) . samples were analyzed by qRT - PCR for IRF7 RSAD2 relative to RPLP0 .

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

(B) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 (each 10 ng/ml). RNA samples were analyzed by qRT-PCR for IRF7 and RSAD2 relative to RPLP0.


( C ) Serum - starved HaCaT keratinocytes were treated for 16 h with FGF7 ( 10 ng / ml ) . lysates were analyzed by Western blot for IRF1 , IRF3 , IRF9 GAPDH .

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

(C) Serum-starved HaCaT keratinocytes were treated for 16 h with FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for IRF1, IRF3, IRF9 and GAPDH.


( D ) samples from untreated , serum - starved keratinocytes from WT Ifnar1 - KO mice were analyzed by qRT - PCR for Irf7 , Rsad2 , Oasl2 Ifnar1 relative to Rps29 .

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

(D) RNA samples from untreated, serum-starved primary keratinocytes from WT and Ifnar1-KO mice were analyzed by qRT-PCR for Irf7, Rsad2, Oasl2 and Ifnar1 relative to Rps29.


( E ) Growth factor - starved keratinocytes from neonatal WT , Ifnar1 - KO or Ifnar1 / Ifnlr1 - KO mice were treated for 24 h with FGF7 ( 10 ng / ml ) . samples were analyzed by qRT - PCR for Irf7 relative to Rps29 .

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

(E) Growth factor-starved primary keratinocytes from neonatal WT, Ifnar1-KO or Ifnar1/Ifnlr1-KO mice were treated for 24 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for Irf7 relative to Rps29.


( F ) HaCaT keratinocytes were transiently transfected with an ISRE - Luciferase reporter construct starved 24 h after transfection for 9 h . They were then incubated in presence or absence of FGF7 ( 10 ng / ml ) for 20 h . Lysates were analyzed using a DualGlo Luciferase .

O O O B-CELL_LINE B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O

(F) HaCaT keratinocytes were transiently transfected with an ISRE-Luciferase reporter construct and starved 24 h after transfection for 9 h. They were then incubated in the presence or absence of FGF7 (10 ng/ml) for 20 h. Lysates were analyzed using a DualGlo Luciferase Assay.


HaCaT keratinocytes were pre - treated for 2 h with proteasome inhibitors MG132 ( 10 μm ( G ) followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . samples were analyzed by qRT - PCR for IRF7 IRF1 relative to RPLP0 .

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

HaCaT keratinocytes were pre-treated for 2 h with the proteasome inhibitors MG132 (10 μm (G) followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.


HaCaT keratinocytes were pre - treated for 2 h with epoxomicin ( 100 nM ) ( H ) , followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . samples were analyzed by qRT - PCR for IRF7 IRF1 relative to RPLP0 .

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

HaCaT keratinocytes were pre-treated for 2 h with epoxomicin (100 nM) (H), followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.


Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml ( A / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . was analyzed by qRT - PCR for ISGs relative to RPLP0 (

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

Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml (A and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0 (A


Serum - starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated ( B ) ) / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . was analyzed by qRT - PCR for ISGs relative to

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

Serum-starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated (B)) and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0


Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . samples by Western blot for total pSTAT1 ( Y701 S727 ) , IRF1 , IRF9 RSAD2 GAPDH ( C ) . vertical in RSAD2 blot indicates a place where gel was broken . Densitometric of different blots ( N = 3 ) is shown on right .

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

Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml and/or IFN-α (500-1000 U/ml) for 12-16 h. protein samples by Western blot for total and pSTAT1 (Y701 and S727), IRF1, IRF9 and RSAD2 and GAPDH (C). The vertical line in the RSAD2 blot indicates a place where the gel was broken. Densitometric quantification of the different blots (N=3) is shown on the right.


( D ) Human keratinocytes were starved treated for 12 h with FGF7 ( 10 ng / ml ) / or IFN - α ( 1000 U / ml ) . samples were analyzed by qRT - PCR for ISGs relative to RPLP0 .

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

(D) Human primary keratinocytes were starved and treated for 12 h with FGF7 (10 ng/ml) and/or IFN-α (1000 U/ml). RNA samples were analyzed by qRT-PCR for ISGs relative to RPLP0.


( A ) Serum - starved HaCaT were treated for 24 h with poly ( I : C ) ( 5 μg / ml ) in presence or absence of FGF7 ( 10 ng / ml ) . samples were analyzed by qRT - PCR for IFNL1 , IFNB , RSAD2 , IRF1 , IRF7 , CGAS , TLR3 or DDX58 , relative to RPLP0 .

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

(A) Serum-starved HaCaT cells were treated for 24 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for IFNL1, IFNB, RSAD2, IRF1, IRF7, CGAS, TLR3 or DDX58, relative to RPLP0.


( B ) Serum - starved HaCaT were treated for 18 h with poly ( I : C ) ( 5 μg / ml ) in presence or absence of FGF7 ( 10 ng / ml ) . lysates were analyzed by Western blot for total pSTAT1 ( Y701 S727 ) , total pSTAT2 ( Y690 ) , IRF1 , IRF3 , pIRF3 ( S396 ) , IRF9 , RIG - 1 , RSAD2 GAPDH .

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

(B) Serum-starved HaCaT cells were treated for 18 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for total and pSTAT1 (Y701 and S727), total and pSTAT2 (Y690), IRF1, IRF3, pIRF3 (S396), IRF9, RIG-1, RSAD2 and GAPDH.


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 h post infection ( hpi ) by qPCR for HSV - 1 Glyc - B gene relative to human β - actin gene ( ACTB ) ( A )

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by qPCR for the HSV-1 Glyc-B gene relative to the human β-actin gene (ACTB) (A)


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 h post infection ( hpi ) by HSV - 1 Glyc - D immunofluorescence ( red ) DAPI counterstaining ( blue ) ( B ) .

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by HSV-1 Glyc-D immunofluorescence (red) and DAPI counterstaining (blue) (B).


Transmission microscopy of HaCaT 48 h post infection ( hpi ) .

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

Transmission microscopy images of HaCaT cells 48 h post infection (hpi).


Serum - starved HaCaT ( D ) were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 at indicated concentrations . load was determined by qPCR for HSV - 1 Glyc - B relative to ACTB 24 hpi .

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

Serum-starved HaCaT cells (D) were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 at the indicated concentrations. Virus load was determined by qPCR for HSV-1 Glyc-B relative to ACTB 24 hpi.


human keratinocytes ( HPK , from two donors ) ( E ) were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 at indicated concentrations . load was determined by qPCR for HSV - 1 Glyc - B relative to ACTB 24 hpi .

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

human primary keratinocytes (HPK, from two donors) (E) were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 at the indicated concentrations. Virus load was determined by qPCR for HSV-1 Glyc-B relative to ACTB 24 hpi.


( F ) Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 , FGF10 or FGF2 ( 10 ng / ml ) . samples were analyzed by qPCR for Glyc - D relative to ACTB lysates were analyzed by Western blot for Glyc - D vinculin 10 hpi .

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

(F) Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7, FGF10 or FGF2 (10 ng/ml). DNA samples were analyzed by qPCR for Glyc-D DNA relative to ACTB and protein lysates were analyzed by Western blot for Glyc-D and vinculin 10 hpi.


( G ) Epidermal sheets from tail skin , infected ex vivo with HSV - 1 ( MOI = 2 ) + / - FGF7 ( 10 ng / ml ) , were stained 48 hpi for Glyc - D ( red ) using DAPI as counterstain ( blue ) . infected ( red ) was measured using Image J software . Data are expressed as A . U . = arbitrary unit . Background of hairs was subtracted manually .

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

(G) Epidermal sheets from tail skin, infected ex vivo with HSV-1 (MOI = 2) +/- FGF7 (10 ng/ml), were stained 48 hpi for Glyc-D (red) using DAPI as counterstain (blue). The infected area (red staining) was measured using Image J software. Data are expressed as A.U. = arbitrary unit. Background staining of hairs was subtracted manually.


( H ) Serum - starved HaCaT were infected with HSV - 1 in presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 hpi by qPCR for HSV - 1 Glyc - B gene relative to ACTB or by Western blot for viral Glyc - D GAPDH .

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

(H) Serum-starved HaCaT cells were infected with HSV-1 in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 hpi by qPCR for the HSV-1 Glyc-B gene relative to ACTB or by Western blot for the viral protein Glyc-D and GAPDH.


( I ) Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) . Four hours hpi , infected were washed incubated in fresh serum - free medium containing FGF7 ( 10 ng / ml ) . Viral load was measured 8 h later by qPCR for Glyc - B relative to ACTB .

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

(I) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5). Four hours hpi, infected cells were washed and incubated in fresh serum-free medium containing FGF7 (10 ng/ml). Viral load was measured 8 h later by qPCR for Glyc-B relative to ACTB.


( J ) Serum - starved HaCaT keratinocytes were treated for 6 h with FGF7 ( 10 ng / ml ) . samples were analyzed by qRT - PCR for NECT1 ( encoding nectin - 1 ) relative to RPLP0 .

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

(J) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for NECT1 (encoding nectin-1) relative to RPLP0.


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at indicated time points 12 h after infection , samples were analyzed by qRT - PCR for RSAD2 , IRF7 IFNL2 relative to RPLP0 ( B ) ,

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, RNA samples were analyzed by qRT-PCR for RSAD2, IRF7 and IFNL2 relative to RPLP0 (B),


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at indicated time points 12 h after infection , , samples were analyzed by qPCR for Glyc - B relative to ACTB ( C ) ,

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, , DNA samples were analyzed by qPCR for Glyc-B DNA relative to ACTB (C),


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at indicated time points D ) shows mean Glyc - B RSAD2 , IRF7 IFNL2 mRNA ( on results shown in Fig . 6B C ) in - infected treated for different periods with FGF7 .

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points D) shows mean Glyc-B DNA and RSAD2, IRF7 and IFNL2 mRNA levels (based on results shown in Fig. 6B and C) in virus-infected cells treated for different periods with FGF7.


Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at indicated time points 12 h after infection , lysates were analyzed by Western blot for Glyc - D , total phosphorylated STAT1 ( Y701 ) , STAT2 IRF3 , for IRF1 , IRF7 , IRF9 RSAD2 for GAPDH or vinculin ( loading controls ) ( E ) . of Glyc - D , RSAD2 , IRF1 , IRF7 , IRF9 STAT2 band relative to GAPDH is shown on right side .

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

Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, protein lysates were analyzed by Western blot for Glyc-D, total and phosphorylated STAT1 (Y701), STAT2 and IRF3, for IRF1, IRF7, IRF9 and RSAD2 and for GAPDH or vinculin (loading controls) (E). Quantification of the Glyc-D, RSAD2, IRF1, IRF7, IRF9 and STAT2 band intensity relative to GAPDH is shown on the right hand side.


( F ) HaCaT were infected with LCMV in presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) analyzed for LCMV nucleoprotein ( NP ) by flow cytometry 24 hpi . Arbitrary units are shown .

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

(F) HaCaT cells were infected with LCMV in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml) and analyzed for LCMV nucleoprotein (NP) by flow cytometry 24 hpi. Arbitrary units are shown.


HaCaT were infected with ZIKV strains 976 ( Uganda isolate ) ( MOI = 0 . 1 ) or PF13 / 251013 - 18 ( French Polynesia isolate ) ( MOI = 20 ) treated 2 hpi with FGF7 ( 20 ng / ml ) or vehicle ( CTRL ) . Immunofluorescence for ZIKV envelope of of expressing ZIKV envelope ( G ) 48 hpi is shown .

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

HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). Immunofluorescence staining for the ZIKV envelope protein and quantification of the percentage of cells expressing the ZIKV envelope protein (G) 48 hpi is shown.


HaCaT were infected with ZIKV strains 976 ( Uganda isolate ) ( MOI = 0 . 1 ) or PF13 / 251013 - 18 ( French Polynesia isolate ) ( MOI = 20 ) treated 2 hpi with FGF7 ( 20 ng / ml ) or vehicle ( CTRL ) . qRT - PCR of samples for ZIKV ( H ) 48 hpi is shown .

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

HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). qRT-PCR analysis of RNA samples for ZIKV RNA (H) 48 hpi is shown.


( I , J ) HaCaT were infected with ZIKV PF13 / 251013 - 18 ( MOI = 20 ) in presence or absence of FGF7 ( 20 ng / ml ) . samples were analyzed by qRT - PCR for OAS1 MxA relative to RPLP0 72 hpi .

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

(I, J) HaCaT cells were infected with the ZIKV PF13/251013-18 (MOI = 20) in the presence or absence of FGF7 (20 ng/ml). RNA samples were analyzed by qRT-PCR for OAS1 and MxA relative to RPLP0 72 hpi.


( A ) Serum - starved HaCaT were infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of FGF7 ( 10 ng / ml ) FGFR kinase inhibitors AZD4547 ( 1 μM ) or BGJ398 ( 3 . 5 μM ) . lysates were analyzed by qPCR for Glyc - B relative to ACTB or by Western blot for Glyc - D GAPDH , respectively , 16 hpi .

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

(A) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5) in the presence or absence of FGF7 (10 ng/ml) and the FGFR kinase inhibitors AZD4547 (1 μM) or BGJ398 (3.5 μM). DNA and protein lysates were analyzed by qPCR for Glyc-B relative to ACTB or by Western blot for Glyc-D and GAPDH, respectively, 16 hpi.


HaCaT were cultured in DMEM / 10 % FCS ( B ) infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of AZD45347 ( 1 μM ) / or BGJ398 ( 3 . 5 μM ) . Viral load was determined by qPCR for Glyc - D relative to ACTB ( B )

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

HaCaT cells were cultured in DMEM/10% FCS (B) infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by qPCR for Glyc-D relative to ACTB (B)


HaCaT were cultured in DMEM / 5 % FCS ( C ) infected with HSV - 1 ( MOI = 0 . 5 ) in presence or absence of AZD45347 ( 1 μM ) / or BGJ398 ( 3 . 5 μM ) . Viral load was determined by measurement of viral titers ( plaque forming units ( PFU ) ) 14 hpi ( C ) .

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

HaCaT cells were cultured in DMEM/5% FCS (C) and infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by measurement of viral titers (plaque forming units (PFU)) 14 hpi (C).


( D , E ) Adult C57BL / 6 wild - type ( D ) or K5 - R1 / R2 mice respective controls ( E ) were infected subcutaneously with HSV - 1 ( MOI = 10 ) . from infected skin was analyzed by qRT - PCR for Fgf7 relative to Rps29 ( D ) was analyzed by qPCR for ICP0 , normalized to host gene Tbx15 48 hpi ( E ) .

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

(D, E) Adult C57BL/6 wild-type (D) or K5-R1/R2 mice and respective controls (E) were infected subcutaneously with HSV-1 (MOI = 10). RNA from infected skin was analyzed by qRT-PCR for Fgf7 relative to Rps29 (D) and DNA was analyzed by qPCR for ICP0, normalized to the host gene Tbx15 48 hpi (E).


A GS ∆ 44 myoblasts were transfected with an empty vector ( CTRL ) or with a CELF2a overexpressing construct ( OE CELF2A ) ( Martone et al , 2016 ) . were collected after 10 days in differentiation medium ( DM ) was analysed by RT - PCR for CELF2a expression DMD exon 45 skipping . GAPDH was used as loading control . arrow indicates DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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

A GS∆44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al, 2016). Cells were collected after 10 days in differentiation medium (DM) and the RNA was analysed by RT-PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI ( in blue ) of iPSCs obtained from WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) ∆ 44 edited clones ( UP ∆ 44 # 3 . 1 UP ∆ 44 # 3 . 5 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm .

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

B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.


C Histogram represents fusion index . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of biological replicates .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.


D extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones was analysed by RT - PCR for CELF2a , CELF2b , CELF2c DMD exon 45 skipping . GAPDH was used as loading control . arrow indicates DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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

D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


E Western blot on ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For WT 5 % sample 2 μg of were diluted in 38 μg of belonging to UP ∆ 44 # 3 to reach a total amount of 40 μg . Representative results are shown ( n = 3 ) .

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

E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP∆44#3 to reach a total amount of 40 μg. Representative results are shown (n=3).


A iPSCs obtained from a control ( WT # 1 ; ( Lenzi et al , 2015 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) GSM ( GSM # 1 ) were differentiated into myocytes by MYOD / BAF60c overexpression . were collected before induction ( iPSC ) after 9 days in DM ( Myo ) was analysed by RT - PCR for CELF2a , CELF2b , CELF2c DMD expression . GAPDH was used as loading control . Representative results are shown ( n = 3 ) .

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

A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3).


B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI ( in blue ) of iPSCs obtained from WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm . C Histogram represents fusion index . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of biological replicates .

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

B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.


D extracted from myocytes obtained by differentiation of WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) was analysed by RT - PCR for CELF2a , CELF2b , CELF2c DMD exon 45 skipping . GAPDH was used as loading control . arrow indicates DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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

D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


E Western blot on ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 8 ) edited GS ∆ 44 ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) iPSC clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For WT 5 % sample , 2 μg of were diluted in 38 μg of belonging from GS ∆ 44 # 8 to reach total amount of 40 μg . Representative results are shown ( n = 3 ) .

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

E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).


A accessibility ( ATAC - sequencing ) marks ( ChIP - seq ) signals in Celf2 region ( + / - 50kb ) using UCSC genome browser . ATAC - seq peaks called in two conditions are reported below each track . differential peaks specific for one of two conditions are reported in tracks ATACseq _ specific peaks _ WT ATACseq _ specific peaks _ GS ∆ 44 . ChIP - seq signals for H3K4me3 , H3K27ac H3K27me3 in WT GSΔ44 myoblasts ( WT myoblasts in orange ; GSΔ44 myoblasts in blue ) . ChIP - seq for MyoD in control was obtained from ( MacQuarrie et al , 2013 ) ( GSM1218849 ) . Orange boxes highlight regions containing first exon of each isoform ( a , b c ) .

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

A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).


D Chromatins extracted from Control ( WT ) GS ∆ 44 myoblasts were immunoprecipitated with anti - MyoD control IgG antibodies . Regions corresponding to MyoD binding sites present in Peak A ( see Figure EV3D ) in MAFA LSTZ2 genes were analysed by qPCR . Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation ( IP ) are plotted for WT GS ∆ 44 samples . mean ± of triplicates from one representative among three experiment is shown . Statistical significance of differences between means was assessed by a two - t - P < 0 . 05 was considered significant . * P = 0 . 0339 , * * P = 0 . 0067 .

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

D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.


B RT - PCR analyses of indicated RNAs in control ( WT ) , ∆ 44 in GS ∆ 44 myocytes collected after 9 days upon induction of differentiation . GAPDH was used as control . Representative results are shown ( n = 3 ) .

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

B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).


C RT - PCR analyses for CELF2a DUXAP8 expression in WT GS ∆ 44 myoblasts ( GM ) myocytes ( DM ) . GAPDH was used as control . Representative results are shown ( n = 3 ) .

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

C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).


D RT - PCR of DUXAP8 expression in extracted from GS ∆ 44 , GS ∆ 44 ' s mother ( GSM ) GS ∆ 44 ' s father ( F ) fibroblasts . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

D RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


E RT - PCR for DUXAP8 expression in extracted from WT GS ∆ 44 myocytes from WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) GSM ( GSM # 1 ) iPSCs induced to differentiation for 9 days . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

E RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


F RT - PCR for indicated RNAs on extracts from GS ∆ 44 myoblasts treated with either control siRNAs ( siSCR ) or siRNAs against DUXAP8 ( siDUXAP8 ) collected two days after transfection . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


G qPCR of DUXAP8 CELF2a expression in control myoblasts transfected with a plasmid for overexpression of DUXAP8 ( OE DUXAP8 ) or an empty vector ( Empty ) , collected after 96h . Relative mRNA were calculated with delta delta Ct method . qPCR reactions were normalized against an internal control ( GAPDH ) plotted relative to expression level in sample treated with empty vector which was set to a value of 1 . mean ± of triplicates from one representative experiment is shown ( n = 3 ) .

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

G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).


Microscope of tissue microinjection pipette before ( top ) after ( bottom ) annotation of tissue surface microinjection micropipette tip by experimenter . Scalebars : top 100µm , bottom 10µm . Ap indicates apical surface BL indicates basal lamina .

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

Microscope images of the tissue and microinjection pipette before (top) and after (bottom) annotation of the tissue surface and microinjection micropipette tip by the experimenter. Scalebars: top 100µm, bottom 10µm. Ap indicates the apical surface and BL indicates the basal lamina.


( Left ) Confocal image of a section of mouse telencephalon fixed stained immediately after microinjection . Dashed lines indicate locations in slice where microinjections were attempted at different pressures . Microinjections were attempted at pressure of 75 mbar ( left ) , 100 mbar ( center ) , 125 mbar ( right ) . ( Right ) of successful injections achieved by Autoinjector at 75 mbar ( 35 % , n = 314 total , 110 successful ) , 100 mbar ( 35 % , n = 185 total , 65 successful ) , 125 mbar ( 8 % , n = 287 total , 23 successful ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-TISSUE O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Left) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different pressures. Microinjections were attempted at pressure of 75 mbar (left area), 100 mbar (center area), and 125 mbar (right area). (Right) Percentage of successful injections achieved by the Autoinjector at 75 mbar (35%, n = 314 total, 110 successful), 100 mbar (35%, n = 185 total, 65 successful), and 125 mbar (8%, n = 287 total, 23 successful).


( Left ) Confocal of a section of mouse telencephalon fixed stained immediately after microinjection . Dashed lines indicate locations in slice where microinjections were attempted at different depths keeping pressure constant at 75 mbar . Microinjections were attempted at depths of 10 μm ( left ) , 15 μm ( center ) , 25 μm ( right ) into apical surface . ( Right ) of successful injections achieved by Autoinjector at depths of 10 μm ( 68 % , n = 163 total , 111 successful ) , 15 μm ( 22 % , n = 170 total , 37 successful ) , 25 μm ( 11 % , n = 169 total , 19 successful ) into apical surface .

O O O B-EXP_ASSAY O O O O B-ORGANISM B-TISSUE O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O

(Left) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar. Microinjections were attempted at depths of 10 μm (left area), 15 μm (center area), and 25 μm (right area) into the apical surface. (Right) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm (68%, n = 163 total, 111 successful), 15 μm (22%, n = 170 total, 37 successful), and 25 μm (11%, n = 169 total, 19 successful) into the apical surface.


targeted at a constant spacing of 30 μm , scale bar is 10 μm .

Cells targeted at a constant spacing of 30 μm, scale bar is 10 μm.