EMBO/SourceData huggingface dataset:
figf4(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel.(b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).
figf5(a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment.(b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).
figf6Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285.(d) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.
figf7(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.
figf8(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test).(b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.
Figure 1(A) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba. (B-C) Quantitative representation of LC3-II and p62 protein levels during ZIKV infection at indicated time points. Mean ± SEM, n=3 biological replicates. (D) GFP-LC3 puncta of mock- and ZIKV-infected hNSCs. Scale bar = 100 µm.(E) Tandem EGFP-LC3 and mCherry punctae co-localization in control and ZIKV infected hNSC. Scale bar = 100 µm, Boxed magnified images with scale bar = 50 µm(F) Number of GFP-LC3 puncta in MR766 and Paraiba infected hNSCs, n =>50 cells. Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.(G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test. (H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.  (I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test.  (L) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains. Mitochondria (MT), endoplasmic reticulum (ER), Golgi bodies (GB), ribosomes (RS), phagophores (PH), lipid droplet (LD). Scale bars, first row, 1 μm (left), 500 nm (control right); 200 nm (Right).
Figure 2 Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone.  Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the hippocampus of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. (E) qRT-PCR for ZIKV RNA expression in brain, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.(F-G) Western blot for autophagy protein LC3-II, p62 and quantification normalized with GAPDH, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.
Figure 3(B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test.(C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test.(E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection.(F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm.(G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm. (H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm, (J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells.(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.(L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.
Figure 4(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm. (B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.
Figure 5Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the SVZ of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.(C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(D) RT-qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. Mean ± SEM of n=3 biological replicates, *p < 0.05 and ns by Student's t test.(E) RT-qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock- and ZIKV Paraiba infected Ifnar-/- newborn mice. Mean ± SEM of n=3 biological replicates, ***p < 0.001 by Student's t test.(F) Western blot analysis of Fancc protein in brain regions from mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(G) Bar graph showing the quantification of Fancc proteins, mean ± SEM of n=3 biological replicates, *p<0.05 and ns by Student's t test.(H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(I) qRT-PCR for Ifnab gene expression in wild type and Fancc KO mice, Mean ± SEM of n=3 biological replicates, ns by Student's t test.
Figure 6(A) Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Bottom row shows enlargements of the hippocampus region. GCL = granular cell layer, SGZ = subgranular zone, DG = dentate gyrus, InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the olfactory bulb of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.(C-E) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post-infection of ZIKV (MR766). Mean ± SEM of n=3 biological replicates, *p<0.05, **p<0.005, by Student's t test. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(F) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker (Sox2) with apoptosis marker (Cleaved-caspase-3, CC3) in hippocampus region of Fancc KO mice. Scale bar=50µm. (G) Quantitative analysis of number of Sox2+ cells and Sox2/CC3+ co-labelled cells in Fancc KO mice hippocampal brain region. Mean ± SEM, n=3 biological replicates, *p < 0.05 by Student's t test.
Figure 1Micrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIα), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 μm.
Figure 3A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.D MitoSox fluorescence following treatment for 1-3 days with dsRNA against the genes indicated.E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated.F, G Relative mtDNA level (F) and MitoSox fluorescence (G) after 5 days of treatment with oligomycin.
Figure 4A‐D Relative mtDNA level (A, B) and MitoSox fluorescence (C, D) following 5 days of treatment with dsRNA against the genes indicated.E‐G Relative mtDNA level (E), MitoSox fluorescence (F) and TMRM fluorescence (G) of cells stably expressing AOX or empty vector, as indicated, after 5 days of treatment with dsRNA against CG2968, as shown. Note that these cells were cultured in the presence of hygromycin to maintain the AOX‐expressing or control plasmid.H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.
Figure 5A. LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes, normalized against the values for untreated cells. Mean ± SD for four independent experiments; asterisks indicate significant differences from untreated, P < 0.05.B. Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red. The scale bar is 10 μm.C. Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes, or with chloroquine. Mean ± SD for three independent experiments, in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane. Analysis of z−stacks in ≥ 30 cells gave similar results. Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes. Asterisks indicate significant differences from untreated, P < 0.01.D. Western blot of total protein extracts from control S2 cells and cells treated with dsRNAs against GFP or CG2968 (cV), probed for NDUFS3 (cI), ATP synthase subunit α (cV) and GAPDH (loading control).E. Relative mtDNA level after 5 days of treatment with dsRNAs against the indicated genes. Mean ± SD for five independent experiments; other data classes were significantly different from untreated, P < 0.05.
Figure 1 (B) Genotyping of KLF4loxp/loxp coupled AAV-Cre inducible mouse. 2x1011 particles of AAV7-Cre-mCherry or AAV7-mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6~8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel.  Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot  Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by immunohistochemistry  (E) Kaplan-Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7-Cre-mCherry or AAV7-mCherry followed by the treatment with 8-Gy (total-body) γ-irradiation 5-weeks after then. AAV7-mCherry, n=10 per group; AAV7-Cre-mCherry, n=12 per group. p = 0.0023, log-rank test.  (F) Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7-mCherry or AAV7-Cre-mCherry intraperitoneal injection followed by 8-Gy (total-body) γ-irradiation 5-weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ-irradiation. Scale bars, 100 μm.  (G-H) Immunofluorescent staining of γ-H2AX, 53BP1 and Cleaved Caspase-3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7-mCherry or AAV7-Cre-mCherry followed by treatment of γ-irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ-irradiation and then staining with indicated antibodies. (G) Quantification of γ-H2AX, 53BP1 and active Caspase-3 -positive cells based on the Immunofluorescent staining results presented in H. Data are mean ± SEM; n=4 per group; p = 0.0023 (r-H2AX), p = 7.9x10-4 (53BP1) p = 5.5x10-3 (Active Caspase-3). one-way ANOVA was used for the statistical analysis. Scale bars, 60 μm.