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

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

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

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 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 .

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

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 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 .

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

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.


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 .

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

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 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 ) .

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

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 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 .

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

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 ) 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 ) .

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

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

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 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 .

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

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.


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 .

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

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.


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 .

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

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 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 .

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

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 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 .

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

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 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 .

O 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 O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD B-ORGANISM 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 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 .

O 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 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 B-GENEPROD B-ORGANISM 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 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 .

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 O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE 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 O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD 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-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 B-SUBCELLULAR 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 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 .

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

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

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

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

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 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 .

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

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 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 .

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

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 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-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 I-EXP_ASSAY O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-GENEPROD O 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 O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O 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 O B-GENEPROD O 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 ) 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 .

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

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 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 .

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 O B-GENEPROD O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O 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 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 ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O 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 O O O O B-TISSUE O B-ORGANISM O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA from 8 week - old ( A ) K5 - R1 / R2 and 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 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 RNA from 5 day - or 9 day - old ( B ) K5 - R1 / R2 and 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 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 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 .

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O 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 RNA from primary keratinocytes of neonatal CTRL and K5 - R1 / R2 mice .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-CELL_TYPE O 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 and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5 - Cre and wild - type ( WT ) mice .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-GENEPROD O 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 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 ) .

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

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

O 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 ) . RNA samples were analyzed by qRT - PCR for IRF7 and 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-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 ) . Protein lysates were analyzed by Western blot for IRF1 , IRF3 , IRF9 and 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 O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-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 ) 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 .

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

O 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 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 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 .

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 O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O 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 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 .

B-CELL_LINE B-CELL_TYPE O 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-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 . RNA samples were analyzed by qRT - PCR for IRF7 and 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-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 and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA 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 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 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 ) ) and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA 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 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 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 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 .

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

O O O B-ORGANISM O B-CELL_TYPE O O O O O 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 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 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 .

O O O B-TISSUE O O B-CELL_LINE O 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 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 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 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 .

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

B-TISSUE O O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O 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 O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O 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 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 ) .

B-TISSUE O O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O 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 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 images of HaCaT cells 48 h post infection ( hpi ) .

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

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


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 .

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

B-ORGANISM O 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 O B-GENEPROD O O O O O B-EXP_ASSAY I-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 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 .

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

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 O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY 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 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 .

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

O O O B-TISSUE O O B-CELL_LINE 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 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 ) . RNA 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 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 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 ) ,

B-TISSUE O O B-CELL_LINE O 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-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 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 ) ,

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

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

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

O O O B-CELL_LINE O O O O B-ORGANISM 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 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 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 .

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

B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O B-ORGANISM B-SMALL_MOLECULE 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 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 .

O O O O O B-CELL_LINE O O O O O B-ORGANISM 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 B-GENEPROD O B-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 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 .

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

B-CELL_LINE O 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 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 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 ) .

B-CELL_LINE O O O O O O O O B-TISSUE O 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 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 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 ) .

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 O B-ORGANISM I-ORGANISM I-ORGANISM O 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 O O B-EXP_ASSAY O B-GENEPROD O 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 ) . 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 ) .

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

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 O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 ) .

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 B-CELL_TYPE 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-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 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 ) 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 ) .

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 B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE 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 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 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 .

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 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 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 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

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 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 ) .

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 B-GENEPROD 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 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 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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

O B-SUBCELLULAR O 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 O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-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 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 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 ) .

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 B-CELL_TYPE 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

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 and DUXAP8 expression in WT and GS ∆ 44 myoblasts ( GM ) and 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 O B-GENEPROD B-EXP_ASSAY O O O O O O B-CELL_TYPE O 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-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 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 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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

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 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 ) .

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

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 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 .

B-EXP_ASSAY I-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 B-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O 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 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 ) .

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

(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 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 .

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


Cells 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.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI . Fluorescence images of tissue after microinjection with dextran - A555 ( magenta ) . The tissue was stained for Tbr2 ( green ) and TuJI ( white ) . Microinjected cell at 0 h ; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2 . Two - daughter cell progeny 24 h after microinjection ; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2 ; the bottom cell is a newborn BP positive for Tbr2 . Both cells are negative for the neuronal marker TuJI ( white ) . Four - daughter cell progeny 48 h after microinjection ; note that daughter 1 and 2 reside in the VZ , while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ . Distribution of microinjected cells and their progeny in the VZ , SVZ and CP ( cortical plate ) . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface ( 0 µm ) and expressed as % of total . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white). Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white). Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI . Expression of Tbr2 in microinjected cells and their progeny at 0 , 24 an 48 h . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h . Expression of TuJI in microinjected cells and their progeny at 0 , 24 an 48 h . n = 147 cells total for 0 h , 151 cells total for 24 h , and 26 cells total for 48 h .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.