EMBO/SourceData huggingface dataset:
Figure 6B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).
Figure 1Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 (indicated by asterisk) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate. The predicted molecular weight sizes of putative, unanchored, poly-NEDD8 chains are denoted by N2 through to N5. Unconjugated NEDD8 is denoted by N1.NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.Components of the NEDD8 conjugation machinery are enriched in HALO-NEDP1 pulldowns from NEDP1 KO lysates. Neddylated proteins from HEK293 KO cells were enriched by HALO-NEDP1 CA pulldown, as in (B) but not by the NEDD8 nonbinder mutant, HALO-NEDP1 DAGC (D29W A98K G99K C163A). The NEDD8 E1s, UBA3 and ULA1, are modified in NEDP1 KO cells, as well as E2 UBE2M, and co-E3s DCNL1 and DCNL2. Cul2 and Cul3 are hyper-neddylated in NEDP1 KO cells. CSN components, CSN5 and CSN8, also co-precipitate in HALO-NEDP1 CA pulldowns.Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation/de-conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells. Apart from UBA3, there is no detectable amount of NEDD8-modified enzymes in whole-cell lysates from NEDP1 KO cells.Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.Neddylated species are UBE2M dependent. WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours. Lysed cells were then processed for Western blot analysis. Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment (si2M), but not with control siRNA (siCTRL) or when NEDD8 E2 UBE2F was depleted (si2F). The double knockdown (si2M/2F) does not further reduce NEDD8 modified proteins.
Figure 2(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).
Figure 3Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E).Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression.Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.
Figure 4PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.
Figure 62D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.592D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.
Figure 7In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.
Figure 1A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM). B Same as A for MEF exposed to 50 nM BafA1 for 12 h. C Same as A, 4 h after BafA1 wash-out. D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB). F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05. Data information: Scale bars: 10 μm.G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF. L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.
Figure 2A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies. B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05. WT MEF. Scale bar: 10 μm. D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm. E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001. H Max projection of the same cell as in D after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm.I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. (J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05). K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).
Figure 3A WB analysis showing KO efficiency for CRISPR134B MEF.WB analysis showing KO efficiency for CRISPR62 MEF.C-E CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F Quantification of C-E (n=10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.
Figure 5A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm. B Inset of A. Red asterisks show site of EV:EL membranes fusion C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots ES, ER subdomains. Scale bar: 500 nm.G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.
Figure 6A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CNX as ER membranes marker.Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CLIMP63 as ER membranes marker.G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001. Data information: Scale bars 10 μm.H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001). Data information: Scale bars 10 μm.
Figure 7A KO efficiency in CRISPR17 MEF.KO efficiency in CRISPR8.C-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.Quantification (n=10, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, **** P<0.0001.G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm. K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.
Figure 1A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm. B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003).  C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118).  D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm. E. Temporal expression profile of NDIME by qRT-PCR. mESCs were cultured in differentiation medium alone or with 10% FBS (D3, P = 0.074; D5, P = 0.0048; D7, P = 0.0034).  F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3.  G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.  Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy analyses during 7 days of neural differentiation. Scale bars, 100 µm.  I. Knockout of NDIME strongly inhibited neural differentiation, as shown by FACS analyses during 7 days of neural differentiation. Scale bars, 100 µm.  J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation.  K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm.  L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm. M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).
Figure 2 A, Expression levels of MEF2C (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, following knockout of NDIME at day 5 of neural differentiation.  B. (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, and protein level of MEF2C following knockout of NDIME at day 5 of neural differentiation. C. Western blot analysis of the effects of MEF2C overexpression in NDIME-knockout cells at day 5 of neural differentiation. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy  E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by FACS  F. NESTIN and GFP immunostaining of the cells in (D) at day 5 of neural differentiation. Scale bars, 100 µm.  G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.