Figure 2TUNEL assays on eye and brain sections of stage ( st ) 30 medakafish control and MO - injected embryos reveal a decrease in the number of apoptotic cells in both hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected compared to hccs - MO - and cox7b - MO - injected embryos . Scale bars are 20μm . TUNEL - positive cells / eye . n ≥ 5 eyes for each model . Caspase assays show restored levels of caspase - 3 and caspase - 9 activities in hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected embryos with respect to hccs - MO - and cox7b - MO - injected embryos . n ≥ 5 embryos for each model .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.TUNEL-positive cells/eye. n≥5 eyes for each model.Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.


Figure 3Representative images of st30 medaka embryos injected with hccs - MO ( B ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in both hccs - MO embryos . hccs - MO / miR - 181a / b - MO - b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation HA14 - 1 treatment counteracts the effect of miR - 181a / b downregulation in the hccs - MO / miR - 181a / b - MO model Scale bars are 100μm . Representative images of st30 medaka embryos injected with cox7b - MO ( H ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in cox7b - MO embryos . cox7b - MO / miR - 181a / b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in cox7b - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation Scale bars are 100μm . Percentage of embryos with or without MLS phenotype in the different conditions in hccs - MO embryos . N ≥ 300 embryos / conditions . Percentage of embryos with or without MLS phenotype in the different conditions in cox7b - MO ( N ) embryos . N ≥ 300 embryos / conditions .

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Figure 3Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.


Figure 4Immunofluorescence analysis with anti - NeuN antibody in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . RGCs are preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . Scale bars are 50μm . Number of RGCs / mm ( indicated as % ) at one - and two - weeks post - injection . N = 10 . NADH dehydrogenase histochemical reaction on retinal sections of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . At one week post - injection , NADH dehydrogenase activity is lost in RGCs ( GCL , areas within dashed lines ) of miR - 181a / b - 1 + / + Rotenone - injected eyes , while it is preserved in those of miR - 181a / b - 1 - / - Rotenone - injected eyes . GCL , Ganglion Cells Layer ONL ; INL , Inner Nuclear Layer ; Outer Nuclear Layer . Scale bars are 50μm . Immunofluorescence analysis , with an anti - CoxIV antibody , in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice , injected with either Rotenone or DMSO , showed preserved mitochondria in miR - 181a / b - 1 - / - rotenone - injected eyes at one - week post - injection . Dashed boxes indicate the area of magnifications shown in right panels . Scale bars are 10μm . Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . N = 10 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10.NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.


Figure 5Immunofluorescence analysis with anti - NeuN antibody in the retina of WT , Ndufs4 - / - , Ndufs4 - / - / miR - 181a / b - 1 - / - and miR - 181a / b - 1 - / - mice . RGCs are preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . Scale bars are 50μm . Number of RGCs / mm ( % ) . N = 5 . Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 8 . Electroretinographic analysis reveals amelioration of b - wave patterns in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 5 .

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Figure 5Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm.Number of RGCs/mm (%). N=5.Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8.Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.


Figure 6Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . Scale bars are 1μm . The quantitative increase of mitochondria is reported in C as number of mitochondria / RGC , at p30 and p55 . N ≥ 2 animals / genotype . Ndufs4 - / - / miR - 181a / b - 1 - / - mice show increased mtDNA content vs . Ndufs4 - / - mice as measured by q - PCR . N ≥ 4 animals / genotype . Biochemical activity of MRC complexes I , II and IV normalized by the percentage of citrate synthase ( CS ) activity in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . N ≥ 3 animals / genotype . p ‐ values were calculated by two tailed Student ' s t - test ; Error bars are SEM .

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Figure 6Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype.Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.


Figure 1B . Identification of highly variable genes ( HVGs ) for the time - point ZT2 . The trend for the global relation ( red line ) between CV2 ( variance / mean2 ) and mean expression is defined using all genes , minus small and lowly expressed genes ( see material and methods for more detail ) and used to identify HVG ( blue ) . For each gene , a corrected CV2 is calculated : log2 ( CV2 / trend ) . C . Expression profiles ( top ) in the 14 seedlings over a 24 hour time course ( with 12 time - points ) for a non - variable gene ( left ) , a highly variable gene ( middle ) and a gene with the level of variability changing across the 24 hour ( right ) . Each dot is the mean normalised expression level for a single seedling . Variability profiles ( bottom ) of the log2 ( CV2 / trend ) for the same genes are also shown . Blue stars indicate time - points for which the gene is identified as being highly variable .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend).C. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.


Figure 2A . Number of genes identified as being highly variable for each time - point . These genes are separated between those that are also selected as highly variable in at least one other time - point ( dark blue ) and those highly variable in only one time - point ( light blue ) . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) . B . Distribution of the number of time - points at which genes are identified as highly variable ( blue ) or lowly variable ( green ) . The distribution of average ( black ) and 95 % confidence interval ( dotted grey ) for the thousand random sets is also represented . C . Heatmap of the percentage of HVGs shared between time - points . Red indicates a high percentage of HVGs in common between two time - points . The top and side bars indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) . D . Hierarchical clustering of HVGs based on the log2 ( CV2 / trend ) at each time point . The result is represented as a heatmap where yellow indicates a high log2 ( CV2 / trend ) . The genes were separated into four clusters , indicated by the side colored bar . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) . See Appendix Fig S3G for heatmaps of the log2 ( CV2 / trend ) with the same colour cutoffs for HVGs , LVGs and random genes . E . Heatmap of the mean normalised expression level for the genes in Fig 2D , keeping the same clustering organisation . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Number of genes identified as being highly variable for each time-point. These genes are separated between those that are also selected as highly variable in at least one other time-point (dark blue) and those highly variable in only one time-point (light blue). The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).B. Distribution of the number of time-points at which genes are identified as highly variable (blue) or lowly variable (green). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.C. Heatmap of the percentage of HVGs shared between time-points. Red indicates a high percentage of HVGs in common between two time-points. The top and side bars indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). D. Hierarchical clustering of HVGs based on the log2(CV2/trend) at each time point. The result is represented as a heatmap where yellow indicates a high log2(CV2/trend). The genes were separated into four clusters, indicated by the side colored bar. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). See Appendix Fig S3G for heatmaps of the log2(CV2/trend) with the same colour cutoffs for HVGs, LVGs and random genes. E. Heatmap of the mean normalised expression level for the genes in Fig 2D, keeping the same clustering organisation. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


Figure 4C . Distribution of the number of TFs targeting a gene , based on the DAP - seq available dataset , for LVG ( green ) and HVG ( blue ) . The distribution of average ( black ) and 95 % confidence interval ( dotted grey ) for the thousand random sets is also represented .

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Figure 4C. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


Figure 5A . Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds ( black ) , HVGs ( blue ) , LVGs ( green ) and the average for the thousand random sets ( grey ) . Error bars indicate the 95 % confidence interval for the thousand random sets . Blue and green stars indicate statistical differences in the number of marked genes compare to all genes , for variable and stable genes respectively ( * Indicates a p - value < 0 . 05 , * * * Indicates a p - value < 0 . 001 , Chi - square test ) . B . Average profile for H3K27me3 , H2A . Z , H3K4me3 and H3K23ac at HVGs ( blue ) , LVGs ( green ) and random genes ( grey ) . C . Heatmap of the enrichment for H3K27me3 , H2A . Z , H3K4me3 and H3K23ac for HVGs ( top ) , random genes ( middle ) and LVG ( bottom ) . Red means a high level and blue means a low level for the chromatin marks .

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Figure 5A. Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds (black), HVGs (blue), LVGs (green) and the average for the thousand random sets (grey). Error bars indicate the 95% confidence interval for the thousand random sets. Blue and green stars indicate statistical differences in the number of marked genes compare to all genes, for variable and stable genes respectively (* Indicates a p-value < 0.05, *** Indicates a p-value < 0.001, Chi-square test).B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks.


Figure 1B . Screening results of compounds competing Dvl - CXXC5 interaction . Each 2 μM of the compounds was used for competing as described in Fig 1A . [ n = 3 ] C , D . Primary calvaria cells were treated with 2 μM of each compound for 2 days . The cells were subjected to immunofluorescence analyses to visualize β - catenin ( C ; green ) . The relative numbers of β - catenin - positive nuclei were counted ( D ) . [ n = 3 ] E , F . The calvariae from 4 - day - old mice were cultured ex vivo for 7 days with 2 µM of each compound . Representative calvaria sections were visualized by H & amp ; amp ; E staining ( E ) . The calvaria thicknesses were measured from the images using Image Pro software ( F ) . [ n = 3 ]

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Figure 1B . Screening results of compounds competing Dvl-CXXC5 interaction. Each 2 μM of the compounds was used for competing as described in Fig 1A. [n=3]C, D. Primary calvaria cells were treated with 2 μM of each compound for 2 days. The cells were subjected to immunofluorescence analyses to visualize β-catenin (C; green). The relative numbers of β-catenin-positive nuclei were counted (D). [n=3]E, F . The calvariae from 4-day-old mice were cultured ex vivo for 7 days with 2 µM of each compound. Representative calvaria sections were visualized by H&amp;E staining (E). The calvaria thicknesses were measured from the images using Image Pro software (F). [n=3]


Figure 2B . A competition curve for the Dvl - CXXC5 interaction by KY - 02061 . C - E . NMR titration analyses for Dvl PZD domain with KY - 02061 . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02061 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02061 ) are displayed as red ( 1 : 0 ) , orange ( 1 : 10 ) , purple ( 1 : 20 ) , cyan ( 1 : 40 ) , green ( 1 : 60 ) and blue ( 1 : 80 ) ( C , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 80 ( D , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( E ) . F . Molecular docking of Dvl binding motif ( DBM ) or KY - 02061 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of DBM ( green ) and KY - 02061 ( yellow ) on the surface of Dvl PDZ domain ( gray ) was visualized .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B . A competition curve for the Dvl-CXXC5 interaction by KY-02061.C-E. NMR titration analyses for Dvl PZD domain with KY-02061. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02061. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02061) are displayed as red (1:0), orange (1:10), purple (1:20), cyan (1:40), green (1:60) and blue (1:80) (C, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:80 (D, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (E).F. Molecular docking of Dvl binding motif (DBM) or KY-02061 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.


Figure 3A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with DMSO , KY - 02061 in DMSO , or 5 µM PolyR - DBM for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ n = 3 ] B . Primary calvaria cells were isolated from the calvariae of 4 - day - old mice ( Manton et al , 2007 ) , and treated with DMSO or KY - 02061 in DMSO for 4 days . ALP activity levels were visualized by ALP staining . C . Primary calvaria cells were treated with DMSO or 5 µM KY - 02061 in DMSO for 14 days . The cells were subjected to immunofluorescence analyses to visualize Runx2 ( green ) and β - catenin ( red ) . The cell nuclei were counterstained with DAPI ( blue ) . D , E . Calvariae from 4 - day - old mice were cultured ex vivo for 7 days with KY - 02061 in DMSO ( D ) . The calvaria thicknesses were measured from the stained sections using Image Pro software ( E ) . [ n = 3 ]

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]B . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining.C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue).D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]


Figure 4B . A competition curve for the Dvl - CXXC5 interaction by KY - 02327 . C . Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY - 02327 . From top to bottom final moral ratio between Dvl PDZ and KY - 02327 was , 1 : 0 , 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 6 , 1 : 7 , 1 : 8 , 1 : 9 , 1 : 10 , 1 : 15 , 1 : 20 and 1 : 25 , respectively . D - F . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02327 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02327 ) are displayed as red ( 1 : 0 ) , yellow ( 1 : 5 ) , green ( 1 : 10 ) , and magenta ( 1 : 20 ) ( D , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 20 ( E , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( F ) . G . Molecular docking of KY - 02061 or KY - 02327 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of KY - 02061 ( yellow ) and KY - 02327 ( cyan ) on the surface of Dvl PDZ domain ( gray ) was visualized .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.C. Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY-02327. From top to bottom final moral ratio between Dvl PDZ and KY-02327 was, 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 and 1:25, respectively.D-F. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02327. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02327) are displayed as red (1:0), yellow (1:5), green (1:10), and magenta (1:20) (D, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:20 (E, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (F).G. Molecular docking of KY-02061 or KY-02327 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.


Figure 5A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with indicated dose of KY - 02327 for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ n = 3 ] B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot . B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot ( B ) or immunofluorescence ( C ) analyses . Nuclei were counterstained with DAPI ( C , blue ) . D . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 14 days . The mRNA level of collagen 1a ( Col1a ) was measured by quantitative real - time qRT - PCR . [ n = 3 ] E . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 21 days . The mRNA level of osteocalcin ( OCN ) was measured by qRT - PCR . [ n = 3 ]

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue).D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]


Figure 6A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . After calcein injection , the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein ( A , green , arrows ) . Nuclei were counterstained with DAPI ( A , blue ) . Osteoblast numbers ( B ) and calcein double labelled surfaces ( C ) were measured in the femoral sections . A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The mouse femurs were analyzed using micro - CT and BMD ( D ) and BV / TV ( E ) were calculated from the micro - CT data . A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The three - dimensional images of femoral trabecular bone were reconstructed ( F ) , and trabecular number ( G ) and trabecular separation ( H ) were calculated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections.A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data.A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.


Figure 1HEK293 cells were treated for 3 hours with 150 nM of LY411575 , 1 µM of MLN4924 , 10 µM of Roscovitine , 10 µM DRB or 10 µM XAV939 . DMSO served as vehicle control . Transfection with pCS2 - NICD vector served as positive control . Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine , DRB , XAV939 , or MLN4924 . NICD is undetectable following LY411575 treatment . NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform . β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) using ImageJ software . Data are expressed as fold changes compared to DMSO treatment . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed with * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant . iPS cells were treated for 3 hours with 150 nM of LY411575 , 1 µM of MLN4924 , 10 µM of Roscovitine , 10 µM DRB or 10 µM XAV939 . DMSO has been used as vehicle control . Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine , DRB , XAV939 , or MLN4924 . NICD is undetectable following LY411575 treatment . NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform . β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) using ImageJ software . Data are expressed as fold changes compared to DMSO treatment . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed with * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 and ns = not significant . HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos - tag assay . DMSO served as vehicle control . NICD phosphorylation profile varies following Roscovitine , DRB , XAV939 or MLN4924 treatment . Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1HEK293 cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO served as vehicle control. Transfection with pCS2-NICD vector served as positive control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01 and ns=not significant.   iPS cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO has been used as vehicle control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (C) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01, ***p≤0.001 and ns= not significant.   HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos-tag assay. DMSO served as vehicle control. NICD phosphorylation profile varies following Roscovitine, DRB, XAV939 or MLN4924 treatment. Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms.


Figure 2NICD interaction with FBXW7 at endogenous levels in HEK293 cells . 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 . All data represent the mean ± SEM from three independent experiments . Student t - test was used to determine p values , with * * p ≤ 0 . 01 . Roscovitine treatment reduced the NICD - FBXW7 interaction . 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.   Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.


Figure 3LC - MS - MS analysis of in - gel - digested HEK293 cells transfected with hNICD - GFP , and subjected to immunoprecipitation using GFP antibody , identified multiple phosphorylation sites in NICD , highlighted in green . Phosphorylation of serine 2513 , but not serine 2516 , appears to be essential for the NICD - FBXW7 interaction . hNICD - GFP phospho - mutant peptides encoding non - phosphorylatable residues at S2513 and / or 2516 ( serine to alanine ) were expressed in HEK293 cells . The exogenously expressed protein was subsequently immunoprecipitated with anti - GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody . Wild - type hNICD - GFP and GFP only vectors were included as positive and negative controls , respectively . Western blot using GFP antibody served as immunoprecipitation efficiency control . β - Actin has been used as loading control for the input lanes .

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Figure 3LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.


Figure 4HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK2 ( + ) followed by western blot for NICD , CDK2 , CDK1 , CDK4 and CDK8 . β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 . HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK1 ( + ) followed by western blot for NICD , CDK1 , CDK2 , CDK4 and CDK8 . β - Actin has been used as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 . Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here . At both 1 and 10µM , Roscovitine is able to inhibit more than 95 % of CDK2 activity , but is far less effective against other kinases . Purvalanol B ( 0 . 1 and 1µM ) inhibits more than 94 % of CDK2 activity . At both 1 and 10 µM , GSK650394A is able to inhibit more than 98 % of CDK2 activity . Source : The Kinase Profiling Inhibitor DatabaseHEK293 cells were treated with three highly selective CDK2 inhibitors ( 10 µM of Roscovitine , 0 . 1µM of Purvalanol B and 10µM of GSK650394A ) for 3 hours . Endogenous levels of NICD were detected by western blot . β - Actin served as loading control . Quantification of the density of western blot bands in ( F ) using ImageJ software . Data are expressed as fold changes compared to DMSO . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed , with * p ≤ 0 . 05 , * * p ≤ 0 . 01 , and ns = not significant . HEK293 cells were treated with RO - 3306 , a specific CDK1 inhibitor , for 3 hours at 10 µM . Levels of NICD , FBXW7 , phospho - Histone H3 and Histone H3 were detected by western blot . β - Actin was used as loading control . Quantification of the density of western blot bands in ( H ) using ImageJ software . Data are expressed as fold changes compared to DMSO . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.   HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.   Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor DatabaseHEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.   HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.


Figure 5Cell cycle profile for HEK293 cells released from synchronization after double thymidine block . Cells were released and harvested at the indicated time points ( AS = asynchronous ) . Analysis of cell cycle arrest and release was performed using propidium iodide ( PI ) staining and flow cytometry . A representative experiment of three performed is shown . Expression of the indicated proteins in HEK293 cells was examined by western blotting , and β - Actin was used as loading control . This summary is a representation of three independent experiments . Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 ( ns = not significant ) . Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 and * * p ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant).Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.


Figure 6iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU ( marked in red within the NICD panel ) . EdU incorporation occurs during S phase ( marked by yellow in the NICD panel ) . Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase . M phase cells are distinguishable by their chromatin condensation , observable in the DAPI panel ( dividing cells are indicated with white in the NICD panel ) . G1 cells do not have any of the features described above ( blue in NICD panel ) . Scale bars are 20 µm . G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells ( p ≤ 0 . 001 ) . All groups were compared against each other with a one - way ANOVA analysis , followed by Turkey test . M phase cells have the lowest NICD . Each point in the box - plot represents the normalized intensity of a single cell . Data from three different experiments ( n = 3 ) was randomised collated and plotted .

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Figure 6iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.


Figure 7Purvalanol B treatment reduced the NICD - FBXW7 interaction . 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0 . 1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin has been used as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 . E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of Purvalanol B ( 1 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin was used as loading control . E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of RO - 3306 ( 10 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin has been used as loading control . Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the " - " explant . n = 18 . Scale bar is 100 µm . Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 10 µM of RO - 3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . RO - 3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the " - " explant . n = 10 . Scale bar is 100 µm . The mean log2 fold change in successive somite length at position + 1 ( last formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 0437 for RO - 3006 and p = 0 . 0524 for PurB , compared to DMSO . n = 14 . The mean log2 fold change in successive somite length at position + 2 ( penultimate formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 6409 for RO - 3006 and p = 0 . 4065 for PurB , compared to DMSO . n = 14 .

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Figure 7Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.   E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.   The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.


Figure 1A Speed of propidium iodide ( PI ) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal . N = 3 independent experiments . Glyoxal fixation enables PI to penetrate far more rapidly into the cells . B Speed of FM 1 - 43 penetration in similar experiments . The arrowhead points to one example of ongoing endocytosis during PFA fixation . N = 3 - 4 independent experiments . The general pattern of FM 1 - 43 entry was similar to that of propidium iodide . Only the first 10 minutes are shown , to enable an optimal observation of the kinetics of the first stages of FM 1 - 43 entry . The results parallel those obtained with PI : faster penetration during glyoxal fixation . Scale bar = 40 µm , * p < 0 . 05 , * * p < 0 .

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Figure 1A Speed of propidium iodide (PI) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal. N = 3 independent experiments. Glyoxal fixation enables PI to penetrate far more rapidly into the cells.B Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3-4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 minutes are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Scale bar = 40 µm, * p < 0.05, ** p < 0.01


Figure 2 Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal . The changes were visualized by DIC images taken at 5 minute intervals during fixation . The graph shows the correlation of each image to the first frame . N = 50 ( PFA ) and 54 ( glyoxal ) cellular regions analyzed , from 3 independent experiments . The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA . Scale bar = 20 µm , * p < 0 . 05 , * * p < 0 . 01

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Figure 2 Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal. The changes were visualized by DIC images taken at 5 minute intervals during fixation. The graph shows the correlation of each image to the first frame. N = 50 (PFA) and 54 (glyoxal) cellular regions analyzed, from 3 independent experiments. The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA. Scale bar = 20 µm, * p < 0.05, ** p < 0.01


Figure 3A SDS - PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives . The graph shows the summed intensity of the bands in each lane . Fixed proteins either no longer run into the gel or form only smears . To compare the efficiency of fixation , the bands that survive fixation were summed and were expressed as % of an unfixed control . The intensity of PFA - fixed samples was significantly higher than that of glyoxal - fixed samples ( N = 5 independent experiments ) . Glut = 0 . 2 % glutaraldehyde . B Staining of nucleic acids after fixation . The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 ( N = 6 - 8 ) . To test whether the fixed nucleic acids were still available for specific detection , we performed FISH for GAPDH in cultured neurons , using a standard protocol provided by the company Affymetrix . The fluorescence signal of the samples fixed with glyoxal ( pH 4 ) was significantly higher than for PFA - fixed samples ( N = 5 - 6 ) . Scale bar = 10 µm , * p < 0 . 05 , * * p < 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A SDS-PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives. The graph shows the summed intensity of the bands in each lane. Fixed proteins either no longer run into the gel or form only smears. To compare the efficiency of fixation, the bands that survive fixation were summed and were expressed as % of an unfixed control. The intensity of PFA-fixed samples was significantly higher than that of glyoxal-fixed samples (N = 5 independent experiments). Glut = 0.2% glutaraldehyde.B Staining of nucleic acids after fixation. The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 (N = 6-8). To test whether the fixed nucleic acids were still available for specific detection, we performed FISH for GAPDH in cultured neurons, using a standard protocol provided by the company Affymetrix. The fluorescence signal of the samples fixed with glyoxal (pH 4) was significantly higher than for PFA-fixed samples (N = 5-6). Scale bar = 10 µm, * p < 0.05, ** p < 0.01


Figure 4 Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal . Strong differences in labeling patterns can be observed . The images are brighter and less " spotty " for the glyoxal - fixed samples . Structures such as filaments or organelles are more easily detected . Quantification of the fluorescence signal ( fold over background ) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples . N = 35 - 132 objects . Scale bar = 6 µm for β - actin and α - tubulin , and 3 µm for the other proteins . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 4 Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal. Strong differences in labeling patterns can be observed. The images are brighter and less "spotty" for the glyoxal-fixed samples. Structures such as filaments or organelles are more easily detected. Quantification of the fluorescence signal (fold over background) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples. N = 35 - 132 objects. Scale bar = 6 µm for β-actin and α-tubulin, and 3 µm for the other proteins. ** p < 0.01, *** p < 0.001


Figure 5 Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal . HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA , glyoxal pH 4 or glyoxal pH 5 . Fluorescence intensities ( fold over background ) were compared and are shown in the graph . The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings . N = 73 - 156 cells per condition analyzed . Scale bar = 10 µm . * * * p < 0 . 001

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Figure 5 Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal. HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001


Figure 6 Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal . AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B , were compared with regard to the fluorescence intensity ( fold over background ) of the stainings . Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins . LC3B staining is brighter in PFA fixed cells . N = 9 - 20 cells per condition . Scale bar = 5 µm . * p < 0 . 05 , * * * p < 0 . 001

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Figure 6 Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal. AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B, were compared with regard to the fluorescence intensity (fold over background) of the stainings. Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins. LC3B staining is brighter in PFA fixed cells. N = 9 - 20 cells per condition. Scale bar = 5 µm. * p < 0.05, *** p < 0.001


Figure 7A Primary hippocampal neurons were stained for Actin and Ankyrin G . A comparison between PFA - and glyoxal - fixed samples shows that actin staining with phalloidin works as least as well in both , showing the prominent actin rings . Ankyrin G staining is brighter in PFA - fixed cells . B Primary hippocampal neurons were stained for pan - Nav and Kv7 . 2 . Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons . Staining of K - channels shows a slightly more regular pattern in glyoxal fixed neurons . C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin . While the spectrin staining seems to be equally well in both fixation conditions , neurofilament staining is brighter in glyoxal fixed cells . D Growth cones of hippocampal neurons were stained for actin and βIII - tubulin after either glyoxal or PFA + glutaraldehyde fixation . The latter is a standard fixation used for the co - labeling of tubulin and actin , and is a stronger fixation than normal PFA fixation , which is incompatible with many organelle immunostainings ( unlike glyoxal fixation ) . The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal , whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures . Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde . Scale bars = 1 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells.B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons.C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells.D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.


Figure 8 Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal . Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide . A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal . The former appear broken and swollen , while the glyoxal - fixed ones appear complete . The effect is presumably due to the different speed of penetration into tissue and / or fixation . Scale bar = 5 µm .

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Figure 8 Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal. Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide. A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal. The former appear broken and swollen, while the glyoxal-fixed ones appear complete. The effect is presumably due to the different speed of penetration into tissue and/or fixation. Scale bar = 5 µm.


Figure 9 Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal . Freshly isolated murine ventricular myocytes were either fixed with 4 % PFA or 3 % glyoxal and immunostained for caveolin - 3 or ryanodine receptor type 2 . Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes . The graph shows mean values and error bars represent standard deviations . N = 10 ( RyR2 ) and 12 ( Cav3 ) myocytes per condition . Scale bar = 2 µm . * * * p < 0 . 001

Figure 9 Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal. Freshly isolated murine ventricular myocytes were either fixed with 4% PFA or 3% glyoxal and immunostained for caveolin-3 or ryanodine receptor type 2. Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes. The graph shows mean values and error bars represent standard deviations. N = 10 (RyR2) and 12 (Cav3) myocytes per condition. Scale bar = 2 µm. *** p < 0.001


Figure 10 Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal . Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins . The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins ( CtBP2 , Calretinin and Homer1 ) fixed with glyoxal . None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation . Representative images show maximum intensity projections from z - stacks of inner hair cell ribbon synapses . N = 5 independent stainings from two animals per condition ( PSD95 , CtBP2 , Otoferlin , Calretinin ) and 10 - 15 images per condition ( CaV1 . 3 and Homer1 ) . Scale bar = 2 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 10 Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal. Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001


Figure 11 Figure 11 Comparison of stained vimentin and α - synuclein in human neuroglioma cells after fixation with either PFA or glyoxal . Cells were fixed with PFA or glyoxal for 10 minutes , and were stained for endogenous vimentin , or for expressed alpha - synuclein . A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha - synuclein , but that PFA was superior for endogenous vimentin ( leftmost graph ) . The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal ; the latter allowed more mOrange2 fluorescence to be detected ( rightmost graph ) . N = 29 - 81 cell regions per condition . * p < 0 . 05 , * * * p < 0 . 001 Scale bar : 10 µm .

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Figure 11 Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal. Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm.


Figure 12 Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal . Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B , COA6 , NDUFA9 and TIM23 . Quantification of the staining intensity shows a significant increase of fluorescence ( signal over background ) for 2 markers ( ATP5B and Mitotracker ) after fixation with glyoxal , whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA , albeit the differences are small . N = 18 - 128 cells per condition . Scale bar = 5 µm . * * * p < 0 . 001

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Figure 12 Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal. Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001


Figure 13 Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal . The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal . Quantification is shown as percentage of signal derived from PFA fixed cells . 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal . Only 4 proteins show significantly reduced staining intensity . For LC3B the intensity of less than 5 cells was quantified , therefore single data points were plotted in addition to the bars . N = 3 - 44 cells per condition . Scale bar = 10 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 13 Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal. The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001


Figure 14 Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation . Immunostaining of the Na / K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples . While in PFA - fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm ( 100 % of the 82 cells we analyzed ) , in nucleus - fixed neurons the U2OS is devoid of signal , and the membrane appears to be correctly labeled ( arrowheads ; 100 % of the 60 cells we analyzed ) . mitochondria cells were immunostained for KDEL ( Tom20 ) and ER ( EGFP - KDEL ) . The fixation and / or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells . The staining of the ER shows an improved signal to noise ratio . The signal appears de - localized from the ER for multiple PFA - stained cells ( 25 % of 36 analyzed cells , see arrows ) , while this is rare for the glyoxal - stained cells ( 3 . 4 % of 58 analyzed cells ) . Scale bar = 10 µm .

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Figure 14 Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation. Immunostaining of the Na/K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples. While in PFA-fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm (100% of the 82 cells we analyzed), in nucleus-fixed neurons the U2OS is devoid of signal, and the membrane appears to be correctly labeled (arrowheads; 100% of the 60 cells we analyzed). mitochondria cells were immunostained for KDEL (Tom20) and ER (EGFP-KDEL). The fixation and/or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells. The staining of the ER shows an improved signal to noise ratio. The signal appears de-localized from the ER for multiple PFA-stained cells (25% of 36 analyzed cells, see arrows), while this is rare for the glyoxal-stained cells (3.4% of 58 analyzed cells). Scale bar = 10 µm.