EMBO/SourceData huggingface dataset:
Figure 2Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.C) Quantification of eGFP-GAn aggregate velocity within cortical neurons.D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.
Figure 3Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm. B) Kaplan-Meier survival analysis of cortical neurons followed for 13 days post-transfection.C) Kaplan-Meier survival analysis (left) of motor neurons followed for 5 days post-transfection.
Figure 4Cortical 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.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.
Figure 5Primary 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).
Figure 6eGFP-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).
Figure 7i3 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.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.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.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.
Figure 9Spinal 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.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.
Figure 10Muscle 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.
Figure 1(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.
Figure 4(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).
Figure 1(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.(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.(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.(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.
Figure 2(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.(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.(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.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.
Figure 3Serum-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 (ASerum-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 RPLP0Serum-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.
Figure 4(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.
Figure 5Serum-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).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.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.(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.(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.
Figure 6Serum-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),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.(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.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.
Figure 7(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)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).
Figure 1A 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.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).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).