EMBO/SourceData huggingface dataset:
Figure 9(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.
Figure 1A, B DNA methylation level of NTN1 (A) and DAPK1 (B) 5' regions (Illumina's HumanMethylation450K Array (HM450) from The Cancer Genome Atlas breast cohort) in paired breast tissues (normal: green circles, tumor: red circles), n = 92, Wilcoxon matched-pairs signed rank test, P = 0.004 and P = 0.0008.C, D NTN1 (C) and DAPK1 (D) gene expression in paired breast tissues (normal: green bars, tumor: red bars, RNAseq from TCGA breast cohort, n = 112 and 114, respectively.E, F Correlation between NTN1 (E) and DAPK1 (F) gene expression and DNA methylation in the breast cancer cohort (TCGA, n = 807). Pearson correlation, P = 6.7.10-5, r = -0.14 for NTN1 (A) and P<10-16, r = -0.32 for DAPK1 (B), respectively.G, H Tumor/Normal DNA methylation ratio of NTN1 (G) and DAPK1 (H) in humanbreasttumors (data extracted from TCGA cohort, paired samples) according to gene expression (downregulated FC ≤ 0.5, down, n = 33, or upregulated FC ≥ 1.3, up, n = 16). P = 3.10-2 and P = 3.10-4 two-sided Mann Whitney test, for NTN1 and DAPK1, respectively.
Figure 2A Methyl-Cap-seq read density profiles of the 5' end of DAPK1, UNC5B, and NTN1in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite-PCR-sequencing; dark gray boxes the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes the exons and UTR. Chromosome coordinates of each gene are given (black lines).B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student's t-test.D, E Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.
Figure 3A, B Cells were treated with DAC (10 μM, 72 h), and/or net1-mAb (10 μg/ml, 48 h) and/or recombinant netrin-1 (5 μg/ml, 48 h). TUNEL assays from 3 independent experiments. *** P<0.0001 one-way ANOVA.C Cellular mortality was assessed using the toxilight assay. Data represent mean ± s.e.m. from 3 independent experiments. **** P<0.0001, one-way ANOVA.D Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated tumors, compared with PBS treated tumors. >1700 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.E, F After one week of treatment, tumors from xenografted mice were fixed in formalin, embedded in paraffin, and sliced into 4 μm sections. (E) Levels of DAPK1, UNC5B, and netrin-1 were measured in 4 independent tumors per treatment group by immunohistochemistry staining, and expressed as a percentage of total tumor surface normalized against the control PBS-group. Data represented as means ± s.e.m. **** P<0.0001, two-way ANOVA and post-hoc Tukey-test. (F) Representative tumor sections corresponding to MDA-MB-231xenografts, scale bars = 50 μm.G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.
Figure 4A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.C Levels of DAPK1, UNC5B, and netrin-1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry. Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS-group. **** P<0.0001, one-way ANOVA. Error bars = s.e.m.D Cleaved caspase-3 and DNA fragmentation (TUNEL) median number of cells per mm2 were measured in treated xenografts. Data represent mean ± s.e.m. from at least 3 tumors per group. **** P<0.0001, one-way ANOVA.E Representative sections of the xenograft PDX analyzed in (D). Scale bars = 50 μm. Arrows indicate positive staining.F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.
Figure 1A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel).C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.
Figure 2A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).
Figure 3H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).
Figure 4I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.
Figure 1Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).
Figure 2Effects from depleting chromatin remodelers and histone chaperones, as indicated (wide range shown in A, on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. Different OSA RNAi stocks used in C are denoted by (1) and (2). In all conditions except Moira RNAi (A) nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C), on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. w RNAi is a control and BrmK804R is expression of dominant-negative Brm. Different OSA RNAi stocks used in C are denoted by (1) and (2). , Snr1 RNAi and BrmK804R expression (C), nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.
Figure 3Chromatin accessibility in Notch-OFF (B) and Notch-ON (NΔECD expression, C) salivary gland nuclei measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Expression of dominant-negative Brm, BrmK804R, led to reduced accessibility of E(spl)mβ-HLH and E(spl)m3-HLH enhancer regions in Notch-OFF conditions, and to a more widespread reduction in accessibility in Notch-ON conditions. "Eip78C enh" corresponds to the ecdysone receptor-binding region of the Eip78C enhancer, which is highly accessible but not Notch-responsive; "Rab11 tr" and "Mst87F tr" represent highly and lowly-expressed control genes respectively. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test comparing LacZ and BrmK804R samples.Chromatin accessibility in salivary gland nuclei depleted for Su(H) by RNAi, measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Accessibility is increased across most of the E(spl)-C compared to controls expressing LacZ. Control primer regions are as in B and C. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test compared to LacZ controls.
Figure 4Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively, measured by reverse transcription-qPCR in Kc167 cells; percentage cDNA compared to GFP RNAi. The knockdowns are highly effective, with only 1-2% of brm and Snr1 cDNA remaining detectable. Mean +/- SEM; n = 3.Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.
Figure 5Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi.Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars).brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.
Figure 1.(B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot.(C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown.(D) Time spent on a rotarod. The horizontal lines indicate mean times.(E) Epg5−/− males show gradually worsening limb-clasping reflexes.(F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age.(G) Spinal deformation in an Epg5−/− male at 10 mo of age.(H) Epg5−/− females gradually develop the limb-clasping phenotype.
Figure 2.(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.(C and D) H&amp;amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.(E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.(I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice.(J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice.(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.(L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice.(M) Eosinophilic spheroids are swollen degenerating axons (arrow).(N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice.(O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.
Figure 3.(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord.(B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice.(C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice.(D-F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice.(G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.