EMBO/SourceData huggingface dataset:
figf2(a) Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm.(b) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments; n, total number of structures analysed; ***P0.007, **P0.004, *P0.01.(c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P0.01, **P0.004.(e) Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments; n, total death events analysed; **P0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm.(f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP-mCherry-LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P0.03, **P0.003, ***P0.0004.(g) Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy; **P0.005. (h) Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead).
figf3(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P0.05, **P0.02.(b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P0.004.(c) Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm.(d) Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colony formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments; *P0.03, **P0.004, ***P0.001. (e) Representative wells for data in d. The insets show higher magnifications.(f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.
figf4(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P0.0001, chi-squared test.(c) Apoptotic U937 cells expressing H2B-mCherry phagocytized by J774 macrophages expressing GFP-LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P0.0001, chi-squared test.(e) Correlative light-electron microscopy of an apoptotic phagosome with GFP-LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus).(f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP-LC3 cells; ***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.
figf5(a) Representative images of a J774 macrophage expressing GFP-LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. See Supplementary Movie S9.(c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP-LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P0.0001, chi-squared test.(e) J774 GFP-LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.
figf6(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow).(c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P0.0001, chi-squared test.(e) Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.; ***P0.0001.(g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.
Figure 1Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM).The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).
Figure 2HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria.Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM).
Figure 3The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids.
Figure 4Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).
Figure 5Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).Human striatal organoids were injected with AAV-Con (GFP) or AAV-mtHSF1 at D42. Scatterplot showed the percentage of NeuN+ cells (red) among Flag+ or GFP+ cells (green) after injection for 2 weeks. The data were obtained from 3 independent biological experiments. Con: n=11 organoids; Flag-mtHSF1: n=13 organoids. The scale bar represents 100 µm.Representative images of immunostaining with DARPP32 (red) and Flag (green). The DARPP32 fluorescence density was analyzed in Flag+ or Flag- sections by confocal microscopy and showed by scatterplot. Thickness: 30 µm. The scale bar is 2 mm, and the enlarged image scale bar is 40 µm. (n=12 mice/group).The striatum of AAV-Con- or AAV-mtHSF1-injected mice were harvested. The DARPP32 protein levels were tested by WB analysis. (n=6 mice/group).The percentage of NeuN+ cells in Flag+ or Flag- sections was determined by confocal microscopy and showed by scatterplot. Thickness: 10 µm. (n=9 mice/group).Mouse weight and motor activity were recorded post AAV injection. (n=7-13 mice/group).
Figure 6Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti-HSF1 antibody followed by immunoblotting. (n=3 biological replicates).Drp1 and HSF1 binding in vitro was analyzed by WB analysis. (n=3 biological replicates).Mitochondrial fractions were isolated from WT or Drp1-KO MEFs. The protein levels of Drp1 and HSF1 were tested by immunoblotting. (n=3 biological replicates).GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).HdhQ7 or HdhQ111 were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).
Figure 7Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal  treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.H. PGC1α protein levels were determined by immunoblotting. (n=3 biological replicates).I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).
Figure 2) Animals harboring CRISPR-generated deletion alleles in y48g1c.1 (gg682 or gg683) were treated with dpy-11 RNAi and progeny, grown in the absence of dpy-11 RNAi, were scored for Dpy. Percentage of Dpy animals is shown. N=3, error bar is standard deviation, s.d.E) Fluorescence micrograph of GFP::ZNFX-1 and PGL-1::TagRFP fluorescence in animals harboring a CRISPR-generated deletion allele (gg682) in y48g1c.1. Scale bar, 5μm.
Figure 4E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm.
Figure 5Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig. EV4B and Materials and Methods to determine; the size of P granules (panel B) Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine; the size of P or Z granules (panel C); or the number of P or Z granules surrounding a typical nucleus in wild-type and zsp-1(gg682) animals (panel D). Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.G,H) GFP::ZNFX-1 (panel G) fluorescence or PGL-1::TagRFP fluorescence (panel H) in the anterior (A) or posterior (P) section of a one cell embryo. Anterior and posterior regions were determined based on corresponding DIC images. N=4 embryos for G, N=3 embryos for H. Error bars are +/- s.d.
Figure 7A) Lineages of animals of the indicated genotypes were grown at 25°C or 20°C for the indicated generations. Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny. Data are represented as +/- s.e.m. N=3 for generation 8 or 9, N=6 for all other generations.F) oma-1(zu405) is a temperature sensitive (ts) lethal (embryonic arrest at temperatures > 20°C) allele of oma-1 (R. Lin 2003). oma-1 RNAi suppresses oma-1(zu405ts) lethality. Animals were exposed to oma-1 RNAi and the number of embryos that hatched was scored at 20°C. N=3. Error bars are +/- s.d. Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma-1 dsRNA but fail to inherit this silencing (Wan et al. 2018). zsp-1 mutants fail to respond immediately to direct exposure to oma-1 dsRNA, indicating that ZSP-1 is not specific to inheritance and, therefore, is generally required for RNAi (see also Fig. EV5B-D).
Figure 1Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches.Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls).Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).