EMBO/SourceData huggingface dataset:
Figure 1Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show: (A) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques. Scale bar = 50 μm. (B) Colocalization of (red) apoE23 and (green) FH around brain capillaries. The blue nuclei were detected using DAPI staining. (C) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer's clinical syndrome (ACS). Colocalization of apoE on Aβ is shown with an arrow. The negative staining control is shown. (D) ApoE-FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient biopsy samples. Six microscope images from Alzheimer's clinical syndrome (ACS) biopsy sample are included in the apoE44 dataset (red dots).Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples (I) MST showing binding of NT647-labeled full-length FH to different apoE isoforms, (J) NT647-labeled apoE2(1-165) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1-4 and 19-20 and (K) NT647-labeled recombinant N-terminal apoE2(1-165) to increasing concentrations of FH5-7 402Y and FH5-7 402H fragments.
Figure 2(B) Different mixtures of apoE isoforms, FH, FH19-20 and Aβ1-42 were incubated for 72 h, and the presence of stable complexes was analyzed by running the sample in Tris-glycine PAGE for (left and middle) 90 or (right) 60 min at 100 V in the presence of 1 Bolt™ LDS Sample Buffer (Thermo Fisher). Gels were subjected to silver staining (left) or WB using anti-Aβ antibody (middle and right). ApoE isoform and incubation time are shown on the top. The presence of each protein is indicated with arrows. Aβ1-42 aggregate and the stable complexes formed between apoE, Aβ1-42 and FH are shown near the top of the gel, while apoE2/Aβ1-42 formed in the absence of FH are shown in several bands below apoE/Aβ1-42/FH complexes.(C) Single-molecule TIRF micrograph of Hylite 488 labeled Aβ1-42 and Alexa Fluor 568 C5 Maleimide labeled apoE2. Scale bar = 5 μm. (D) Jitter plot of proportion of colocalized Aβ1-42/apoE2 and Aβ1-42/apoE3 over multiple fields of view in the presence and absence of FH. Statistics was calculated using Student's t-test from multiple images (n=12) in each sample. Error bars indicate SD values. ((E) Jitter plot of Aβ1-42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH. Statistics of multiple images was calculated using two-sided Student's t-test. Statistics was calculated using Student's t-test from all detected colocalized foci in multiple images (n=12) in each sample. Error bars indicate SD values.
Figure 3(A and B) Immunofluorescence staining of apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH (green) with Iba-1 stained cells (red) and clustering around cells and brain capillaries. Presence of (white) Aβ plaques in apoE44 samples and (arrow) FH in both samples are shown.. C-E (C) Iba-1, (D) Aβ and (E) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient biopsy samples.(F) Flow cytometric experiments showing binding of (left) 405-labeled apoE4 and (right) 405-labeled apoE2 to activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH, FH fragments 19-20 and 5-7, BSA and Aβ1-42.(I) Binding of 488-labeled Aβ1-42 to (left) activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH19-20, BSA, with combinations of apoE4 and FH, apoE2 and FH or apoE3 and FH. The fluorescence intensities of 488-Aβ1-42 in each experiment was normalized against activated U937 empty cells sample.
Figure 4(A) Immunofluorescence staining of iNPH patient biopsy samples (apoE33) showing (arrow) colocalization of complement activator, (red) C1q, and complement regulator (green) FH. (B) (arrow) Colocalization of (green) apoE and (red) C1q on (white) Aβ plaques. Colocalization analysis is calculated from colocalized foci in two separate microscope images.(D) C3a formation in supernatant of C3, factor B (fB), factor D (fD) and factor I (fI) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH complexes. Beads with488-Aβ1-42/BSA complexes shows the level of C3a formation without inhibitor. Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488-Aβ1-42/FH sample.(G) (left) Phagocytosis of complement C3, fB, fD and fI (C) or only buffer (no C) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA (oligomerized Aβ = OAβ) or with non-oligomerized 488-Aβ1-42 (monomeric Aβ = MAβ). Also, samples without apoE or FH were included in the assay (buffer). (right) Cell permeability of C3, fB, fD and fI incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA complexes (OAβ) or with MAβ. Also, cells incubated with only buffer were included in the assay (buffer). Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population. The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with (U937 CR3 cells) or without (U937 empty cells) CR3 expression.
Figure 5(A) 488-Aβ1-42 phagocytosis in the presence of FH and apoE isoform. Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample.
Figure 6(B) Comparison of mRNA transcripts (two individual samples obtained from three times repeated experiments) and MS1 intensities (from 8 apoE44/apoE43 and 9 apoE33/23 carriers) between genes or proteins that showed differential expression in vitro (Fig. 5B) and in vivo.(D) (left) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers (four markers/patient). (center) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive (n=10) or negative (n=7) for Aβ pathology (four markers/patient). (right) Expression levels of Phosphodiesterase A control, showing similar expression levels in frontal cortex, between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers The data were normalized against neutral apoE33 samples with no apoE association.
Figure 1Representative immunofluorescence images (left) of GAPDH (red) in HeLa cells untreated (Utr), or treated with IR, etoposide (ETO), adriamycin (ADR), and camptothecin (CPT) as indicated. DNA was stained with DAPI (blue). Quantitative analysis (right) of nuclear GAPDH. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001Immunoblot (left) and quantification (right) of endogenous GAPDH in subcellular fractions of HeLa cells treated 4 Gy IR and recovered for 4 h. β-Tubulin and Lamin A/C served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001Immunoblot (upper) and quantification (lower) of phospho-Src-Y529 in HeLa cells collected at the indicated time points after 4 Gy IR treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001HeLa cells were pretreated with or without 10 μM PP2 for 24 h and exposed to 4 Gy IR. After 4 h of recovery, nuclear and cytoplasmic fractions were separated and subjected to immunoblot with indicated antibodies (left). Quantification of nuclear GAPDH is in the right panel. β-Tubulin and Lamin B1 served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001E-F. Immunoblot of anti-GAPDH immunoprecipitates from HEK293T treated with 4 Gy IR and recovered for 4 h (E), or with 5 μM ADR for 2 h (F).
Figure 2Cell viability analysis of HeLa cells stably expressing the sh-NC and sh-GAPDH were treated with the indicated concentration of ADR for 48 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantLeft, representative immunofluorescence images of γH2AX (C, green) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right quantification of γH2AX foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantLeft, representative immunofluorescence images of 53BP1 (D, red) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right, quantification of 53BP1 foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot (left) and quantification (right) of γH2AX in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at indicated points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant
Figure 3Immunoblot (left) and quantification (right) of HR proteins in HeLa cells transfected with scrambled siRNA and si-GAPDH. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantQuantification of RAD51 (left) or γH2AX (right) foci/cell in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at the indicated time points after 4 Gy IR exposure (n=50). Mann-Whitney U-test. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot (left) and quantification (right) of RAD51 in HeLa cells with or without GAPDH knockdown treated with 50 μg/mL CHX at the indicated time points. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot (left) and quantification (right) of RAD51 in GAPDH knockdown HeLa cells and control cells treated with or without MG132 (10 μM) for 10 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant
Figure 4Immunoblot (left) and quantification (right) of RAD51 in HeLa cells at different durations of 50 μg/mL CHX administration with or without TSA (10 μM, 12 h) treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot of cell lysates or anti-RAD51 immunoprecipitates from HeLa cells with scramble siRNA or si-GAPDH transfection treated with MG132 (10 μM) for 2 h.Immunoblot (left) and quantification (right) of Flag-RAD51 in cell lysates from HEK293T cells transfected with indicated Flag-RAD51 mutations. Endogenous RAD51 and Flag-RAD51 are denoted respectively. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with indicated Flag-RAD51 mutations and then exposed to 10 μM MG132. Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with HA-Ubiquitin and indicated Flag-RAD51 mutations and then exposed to 10 μM MG132.
Figure 5HeLa cells were transfected with or without Flag-GAPDH and HA-HDAC1, then subjected to immunoprecipitation and immunoblots as indicated. Recombinant HDAC1 protein and recombinant GAPDH protein were co-incubated at 4℃ for 2 h. In vitro immunoprecipitation was performed using anti-HDAC1 and subjected to immunoblots as indicated.HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-Maspin (I) Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-GAPDH (J). Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001Co-immunoprecipitation was performed to determine the interaction between endogenous GAPDH and Maspin, full-length Flag-HDAC1, and truncated forms of Flag-HDAC1 in HeLa cells, untreated (L) or treated with 4 Gy IR (M). Asterisks indicate nonspecific bands.
Figure 6Immunoblot (upper) and quantification (lower) of RAD51 in HEK293T cells transfected with empty vector, Flag-HDAC1, Flag-HDAC1-S3 (∆306-353), and Flag-HDAC1-H141A. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantHeLa cells were transfected with empty vector, Flag-HDAC1, and Flag-HDAC1-H141A, then subjected to immunoprecipitation and immunoblots as indicated.Left, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti-RAD51 (red). DNA was stained with DAPI (blue). The number of RAD51 foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantImmunoblot (upper) and quantification (lower) of γH2AX in GAPDH knockdown HeLa cells transfected with or without Flag-HDAC1 at the different time points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significantLeft, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti- γH2AX (green). DNA was stained with DAPI (blue). The number of γH2AX foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant
Figure 1C Similar to CSR-1, the majority of small RNAs interacting with CSR-2 in vivo have 5′PPP ends. The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase, and finally labeled with [γ-32P]ATP and a polynucleotide kinase. RNA products were resolved by 15% sequencing gels.G Three csr-1 hypomorphic mutants showed a diminished RNAi response to exo-dsRNA. The WT, the csr-1(fj150) mutant, and several strains were injected with dsRNA (1.25 µg/µL) targeting the maternal gene pos-1, essential for embryogenesis. The incidence of lethal embryos in the F1 progeny was examined. The number of F1 progeny counted is indicated by n. Error bars represent 95% confidence intervals (CIs).
Figure 2A Nuclei of the WT strain were stained by the immuno-FISH method using an anti-histone H3K9me2 antibody and a probe against the right region of the X chromosome. B Shapes of H3K9me2-positive chromosomes in male testes of the WT and the csr-2 mutant. The relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs.E Immunofluorescence images of H3K9me2 and SYP-1 in male nuclei of the WT and the Argonaute mutants. High-resolution fluorescence images, 3D reconstruction images (3D CG), and maximum intensity projection (MIP) images of segmented H3K9me2 signals are presented. In the male nuclei of the csr-1 mutant and the csr-2; csr-1 double mutant, some H3K9me2 segments showed abnormally large volumes (narrow arrow) and/or overlapped partially with SYP-1 signals (arrowhead). In the double mutant, DAPI signals were often detected in an aggregated state (dashed line).
Figure 3Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. A The affinities of the recombinant chromodomain peptides of CEC-5 and CEC-8 for the N-terminus of histone H3 were assayed by in vitro pull-down experiments. CEC-4 served as a control.Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. C H3K9me2 signals at the pachytene region in the males of cec mutants and cohesin mutants. Worms were cultured at 20°C, except for the smc-1(e870) mutant that was cultured at 23°C. The intensity of the H3K9me2 signal in the him-8 mutant male was similar to that in the WT male.Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. D The nuclear intensities of H3K9me2 signals were measured from wide-field (WF) images. The image quantification was performed E The nuclear intensities of H3K9me2 and H3K9me3 signals were measured from deconvolved (DV) images. The H3K9me2 signals in the major strains were double-checked using deconvolution microscopy.
Figure 4A The meiosis-specific kleisin(s) COH-3/4 was detected on paired chromosomes and the heterochromatinized unpaired X chromosome. The testes of WT males were immunostained with anti-COH-3/4 antibodies and an anti-H3K9me2 antibody.B Immunofluorescence images of COH-3/4 and SYP-1 on paired chromosomes in the gonads of WT hermaphrodites. COH-3/4 kleisin(s) was detected laterally to the string-like signal of SYP-1, which corresponds to the central region of the SC.C SC formation in the WT and multiple mutants were analyzed by immunostaining against SYP-1 and COH-3/4. The SC formed string-like structures in the csr-2; csr-1 double mutant and the ego-1 mutant, but the superstructure of the SC was often abnormally branched.
Figure 5A Multicolor FISH for the 5S rRNA region (rDNA, green) of autosome V and the right region of the X chromosome (LGX-R, magenta) in nuclei of the WT strain, coh-3/4 double mutant, csr-1 mutants, csr-2; csr-1 double mutant, and ego-1 mutant. Arrows indicate occasional colocalizations of the 5S rDNA and X-R loci detected in the mutant strains.D Simultaneous staining of the 5S rRNA region (rDNA, green) by FISH, and SYP-1 (magenta) by immunofluorescence.E csr-1(fj67) and csr-1(fj126) in-frame mutations reduced the abundance of CSR-1 at the chromosomal regions in pachytene nuclei. The WT strain and the csr-1 mutants were stained by anti-CSR-1 antibodies.
Figure 6A, B ChIP-seq data for CSR-1, SMC-1, COH-3/4, and histone H3K9me2 were compared with sub-populations in CSR-1-interacting small RNAs. Adult worms were used for experiments. ChIP signals on autosome III (A) and the X chromosome (B) from the WT hermaphrodites were visualized by aggregation plots with a bin size of 20 kbp. H3K9me2 ChIP signals derived from the him-8 mutant population rich in X0 males are shown in the lower part. The common ChIP regions computed from the technical duplicates were used for panels A, B, In the adult body, about half of the nuclei correspond to germ cells. Most H3K9me2 ChIP signals from the WT hermaphrodites were likely to originate in somatic cells because staining signals of H3K9me2 in adult hermaphrodites were detected abundantly in somatic cells and very weakly in the germline. Most meiosis-specific COH-3/4 ChIP signals were thought to originate in the germline. For comparison, aggregation plots were also constructed from the following sub-populations of CSR-1-interacting small RNAs: small RNAs mapped in sense orientation to coding genes (exons and introns), and repeat-derived small RNAs. The small RNA data merged from the technical duplicates were used for panels A and B. Densities of protein-coding exons, all non-simple repeats, and five major repeats on the reference genome are attached. The CeRep55 sequences were partially degenerate, and no clear ChIP signal for CSR-1 was detected in CeRep55 cluster #4 on the X chromosome in the ce10 reference genome.G Frequencies of small RNAs expressed from the major non-simple repeats were counted in the CSR-1 and CSR-2 complexes and the input cell lysates. The small RNA reads obtained by the experiments in were analyzed. The data from technical duplicates (#1 and #2) are individually presented. The y-axis indicates reads per million (rpm). Error bars represent 95% CIs. The red border indicates the expected value of non-specific recovery, which was predicted with the recovery rate of miRNAs.
Figure 7F The CeRep55 lncRNA signals partially colocalized with those of COH-3/4 in hermaphrodites. Pachytene nuclei in the WT strain and the csr-2; csr-1 double mutant were stained.H The CeRep55 lncRNA signal was also detected at a region of the unpaired X chromosome where the CEC-5 and COH-3/4 signals overlapped. The pachytene chromosome showing the strongest CEC-5 enrichment was considered to be the unpaired X chromosome in males. The RNA-directed in situ hybridizations were performed with stringent conditions that detected only long RNAs.
Figure 8Pachytene nuclei were analyzed. B Multicolor FISH using the 5S rDNA and LGX-R probes in the csr-1(fj150) mutant and the csr-1(fj150); CeRep55_X(Q) mutant.Pachytene nuclei were analyzed. D Nuclei of male testes carrying an unpaired X chromosome were immunostained with an antibody against histone H3K9me2. H3K9me2-positive chromosomes showing an extended state (arrow) were observed in the males of the CeRep55_X(Q) deletion mutant more frequently than in WT males.Pachytene nuclei were analyzed. E In male pachytene nuclei, the relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs. P-value (two-sided) was determined by two-proportion z-test.