EMBO/soda-nel huggingface dataset:
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 3

Figure 3. GASZ interacts with itself at mitochondrial outer membrane.

A. Mouse GASZ contained a putative transmembrane domain and a region outside of mitochondria predicted by a bioinformatic software TMHMM.

B. 293T cells with ectopically expressed proteins were extracted for soluble and membrane bound fraction and examined by Western blots.

C. Mitochondrial fraction from 293T cells ectopically expressing GASZ, MitoPLD, AIFM1, or SIRT3 were treated in the presence or absence of Triton X-100 and/or Proteinase K, and examined by Western blots.

D. GST pull-down assay of GST-tagged GASZ with FLAG-GASZ produced from 293T cells.

E. Co-IP assays of FLAG or GFP-tagged GASZ in 293T cells. Antibodies for Co-IP (IP) or Western blots (WB) were listed on the left.

F. BiFC assays in which YFP was detected by flow cytometry in control 293T cells, or 293T cells co-expressed with GASZ fused with N- or C-terminal half of YFP. Ctrl: empty vector control.

G. Co-IP assays of GFP-GASZ with various FLAG-tagged wild-type GASZ or its deletion mutants in 293T cells
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 4

Figure 4. GASZ regulates mitochondrial fusion and metabolism.

A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m.

B. EM on 293T cells with empty vector control (Ctrl), wild-type GASZ, or GASZ with F to P mutation in MLS. Scale bar: 1 m. N: nucleus.

C. Averaged percentage of HeLa cells (determined from 100 cells in each experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with GASZ, its SAM/ZIP deletion mutant, or GASZ with MLS mutation.

D. HeLa cells transfected with COX4-DsRed, mito-PAGFP, and empty vector control, GASZ or GASZ with MLS mutation, followed by photoactivation of PAGFP. Representative images of PAGFP and DsRed were taken at 0, 10, and 20 min post photoactivation. Scale Bar: 10 m. Insets show images with higher magnification. The averaged exponential decay constant (±s.e.m.) of normalized PAGFP fluorescence within the designated region (white circle) at photoactivation area over a period of 10 min was shown as a bar graph below. n>20 cells for each group.

E. Averaged percentage of C18-4 cells (determined from 100 cells in experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with Su9-GFP or GFP-GASZ.

F. Real-time PCR analyses of mtDNA copy numbers on testes at different time points postnatal, relative to a single copy gene, PECAM, encoded by nuclear DNA. n=5 per genotype.

G. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.

(C, E, F) Bars represent as mean ± one s.e.m. calculated from three (C), or five (E, F) experiments or biological replicates.

(C-F): **: p<0.01.
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 5

Figure 5. GASZ promotes MFN-dependent mitochondrial fusion.

A-B. Co-IP assays of ectopically expressed FLAG-GASZ and MFN-HA in 293T cells (A) or endogenous proteins of GASZ and MFNs from adult testes (B).

C. Co-IP assays of HA-tagged MFN1 with various FLAG-tagged wild-type GASZ, its domain deletion mutants, or GASZ with MLS mutations in 293T cells.

D. Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker (Red) staining in wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid.

E. PEG fusion assays of Su9-GFP and COX4-DsRed labeled wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid. Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion.

(D, E) Bars represent as averaged percentage ± one s.e.m. calculated from 100 randomly chosen cells (D) or areas (E) for each assay in three independent experiments. *: p<0.05. **: p<0.01.
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 6

Figure 6. MFN1 deficiency led to male infertility.

A. Gross morphology of testes from MFN1f/f-Cre compared to their wild-type littermates.

B. Histological studies of testis sections from wild-type mice or MFN1f/f-Cre littermates at different time points postnatal. Red stars: tubules with spermatocytes.

C. IHF of PLZF (day 14 postnatal), MILI, MVH, and GASZ/CytC (day 7) of testes sections. Scale bars for PLZF are 50 m and 10 m for others.

D. EM of testical sections from wild-type neonatal mice or MFN1f/f-Cre littermates. N: nucleus.

E. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.
Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite (2020)
Figure 1

Figure 1. Immunoprecipitation of var gene-targeted dCas9.

Schematic of a representative var gene with two exons flanking an intron. Transcription originates from the promoter (sense) and intron (sense and antisense). Specific sgRNAs direct dCas9 to either the promoter region (red) or intron (blue). Antibodies were used to isolate the dCas9 and bound genomic regions via a 3xHA tag (yellow star).

Schematic of var genes throughout the P. falciparum genome targeted with intron- (blue), promoter- (red), or both intron and promoter-targeted (green) dCas9. Chromosomes are represented with grey bars, and chromosome numbers are indicated in roman numerals. var gene ups type is indicated on the chromosome and var gene ID (excluding the preceding chromosome number) is listed to the left of its position on the chromosome. Direction of var gene transcription is indicated with an arrowhead.

Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter-targeted strain at ring stage. Levels of dCas9 and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. dCas9 is enriched in the immunoprecipitated fraction (IP) compared to the unbound supernatant (SN) and input (i.e. chromatin fraction) of the IP. Molecular weights are shown to the right.
Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite (2020)
Figure 2

Figure 2. ChIP sequencing shows specific enrichment of dCas9 at targeted var gene introns and promoters.

Circos plots of ChIP-seq data showing genome-wide enrichment of dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer grey bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment for intron- or promoter-targeted dCas9 (normalized to non-targeted dCas9) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 1,200 RPM for the promoter-targeted dCas9 (rings represent increments of 200) and 400 RPM for the intron-targeted dCas9 (rings represent increments of 66.7). var genes are represented by red bars. An asterisk indicates an unintended binding event. One replicate was performed for each strain. Peak quantification for promoter- and intron-targeted dCas9 ChIP-seq can be found in Tables EV1 and EV2, respectively.

ChIP-seq data show enrichment of dCas9 in strains at ring stage expressing non-targeted "Control" (grey), var intron-targeted (blue), or var promoter-targeted (red) sgRNAs. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted ChIP enrichment. q-values for promoter-targeted dCas9 peaks shown are 2.22x10-289 for PF3D7_0413100 and 7.07x10-257 for PF3D7_1240400. q-values for intron-targeted dCas9 peaks shown are 2.03x10-76 for PF3D7_412700, 6.81x10-46 for PF3D7_0412900, 1.53x10-62 for PF3D7_0413100, and 8.37x10-4 for PF3D7_1240900. One replicate was performed for each strain. Peak quantification for promoter- and intron-targeted dCas9 ChIP-seq can be found in Tables EV1 and EV2, respectively.

ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, grey). One replicate was performed for ChIP-seq and RNA-seq and one replicate was performed for HA and IgG control RIP-seq. ChIP-seq peak quantification can be found in Table EV1 and RIP-seq quantification can be found in Table EV3.
Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite (2020)
Figure 3

Figure 3. Proteomic analysis of dCas9-purified var gene introns and promoters.

Volcano plot representation of label-free quantitative proteomic analysis of P. falciparum proteins present in intron- (left) and promoter-targeted (right) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. For the main volcano plot, x-axis = log2(fold-change), y-axis = -log10(p-value), horizontal yellow line indicates adjusted p-value = 0.05, and vertical yellow lines indicate absolute fold-change = 1.5. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids). dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted dCas9. Fold enrichment and adjusted p-values for each protein highlighted in red (promoter) and blue (intron) can be found in Tables EV5 and EV6, respectively.

Correlation plot of label-free quantitative proteomic analysis of P. falciparum proteins enriched in promoter-targeted (top right quadrant in red) versus non-targeted control (top left quadrant in grey) or intron- (bottom right quadrant in blue) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. The x-axis = log2[fold-change(promoter/non-targeted control)] and y-axis = log2[fold-change(promoter/intron)]. dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted and non-targeted dCas9. Fold enrichment and adjusted p-values can be found for each protein highlighted in red (promoter) in Tables EV5 (intron comparison) and EV7 (control comparison) and for each protein highlighted in blue (intron) in Tables EV6 (promoter comparison) and EV8 (control comparison).

Venn diagram comparing proteins enriched in the ISWI IP LC-MS/MS (red) and the promoter-targeted dCas9 IP LC-MS/MS compared to the intron-targeted dCas9 (blue) or the non-targeted dCas9 (yellow).

Schematic of proteins shared between the ISWI IP LC-MS/MS and the promoter-targeted dCas9 IP LC-MS/MS (overlapping regions shown in Fig 3C): ISWI (PF3D7_0624600), MORC (PF3D7_1468100), ApiAP2 (PF3D7_1107800), MCM2-4,6,7 (PF3D7_1417800, PF3D7_0527000, PF3D7_1317100, PF3D7_1355100, PF3D7_0705400), and acetyl-CoA synthetase (ACS, PF3D7_0627800).
Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease (2008)
figf1

(a) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6.0 array reveals a correlated pattern of variation in intensity across six copy-number probes spanning the region upstream of IRGM, suggesting the existence of a common copy-number polymorphism. (Darker shades of red represent reduced hybridization intensity.) Quantitative PCR indicated that the copy-number-variable region was an insertion/deletion (Supplementary Fig. 1). (b) Mapping of the deletion breakpoints by microarray and PCR. Red indicates evidence for deletion; black indicates evidence to the contrary; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele. (c) Sequence of the reference and deletion alleles. The CD-associated SNP rs13361189 is indicated in boldface red type. Physical coordinates are on the reference human genome (build 35/36); allele frequencies are for extended HapMap analysis panels, including 540 samples. (d) CD association of typed and imputed polymorphisms in the IRGM region in the NIDDK IBDGC genome scan. Blue trace indicates recombination map. The deletion polymorphism and rs13361189 are indicated by red arrows. rs13361189 (which is in perfect linkage disequilibrium with the deletion, r2 = 1.0) was also the most strongly associated SNP in the combined WTCCC genome scan and replication study.
Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease (2008)
figf2

(a) The exonic SNP rs10065172, which is in strong linkage disequilibrium with the 20-kb insertion/deletion polymorphism upstream of IRGM, can be used to distinguish IRGM transcripts that arise from the deletion haplotype from IRGM transcripts that arise from the reference haplotype. This makes it possible to measure the relative expression of the two haplotypes in heterozygotes by measuring the relative abundance of the two rs10065172 alleles in cDNA. (Yellow and green rectangles indicate transcribed sequence; green rectangles indicate protein-coding sequence.) (b–g) Differential expression of IRGM from the two structural haplotypes in heterozygotes. The relative abundance of the C and T alleles of rs10065172 in cDNA (colored circles) and genomic DNA (black squares) from human tissues and cell lines were measured. Gray squares indicate control measurements for genomic DNA from 48 HapMap YRI individuals, identifying the three reference genotype classes CC, CT and TT. Genomic DNA from all other samples was heterozygous for the C and T alleles (black squares); HeLa genomic DNA (b,c) showed a 2:1 allelic ratio (CCT) reflecting HeLa's triploid 5q karyotypic status. In e, cDNA and genomic DNA from lymphoblastoid cell lines from ten different heterozygous individuals were analyzed: the ten different colors represent the ten individuals; technical replicates from the same cell line are connected by line segments. HeLa, SNU182 and the lymphoblastoid cell lines expressed the C allele of rs10065172 more strongly than the T allele (b–e); HCT116 and primary bronchial cells expressed the T allele much more strongly than the C allele (f,g).
Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease (2008)
figf3

(a) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six- to eightfold in HeLa cells. Cells were transfected with control (siControl) or IRGM-targeted (siIRGM1, siIRGM2) siRNA duplexes and assayed 48 h later by quantitative RT-PCR. Error bars, s.d. (b) IRGM knockdown reduces the efficiency of anti-bacterial autophagy. HeLa cells stably expressing LC3-GFP (a marker for autophagic vesicles) were transfected with control or IRGM-directed siRNA oligos, then infected with S. typhimurium after 48 h. The percentage of bacteria encapsulated in LC3-positive vesicles was determined by microscopy. Reductions in IRGM expression resulted in a significant loss of anti-bacterial autophagy compared to control cells. (c) Microscopic examination reveals a decreased number of autophagically encapsulated bacteria in IRGM knockdown cells. Control siRNA-treated (siControl) and IRGM siRNA-treated (siIRGM1, siIRGM2) cells were infected with S. typhimurium SL1344 and imaged by confocal microscopy. Control cells show numerous bacteria (red in merged image) surrounded by LC3-GFP membranes (green in merged image). Such bacteria-containing autophagosomes (indicated by dashed boxes) were almost completely absent in IRGM-deficient cells. The actin cytoskeleton was visualized using phalloidin (blue in merge). Images are flat projections of confocal z-stacks. Scale bars, 10 μm. (d) HeLa cells stably expressing LC3-GFP were transfected with increasing amounts of an IRGM expression construct and infected 24 h later with S. typhimurium. The percentage of bacteria found within LC3-positive vesicles increased with increasing amounts of exogenous IRGM. Protein expression levels were confirmed by blotting lysates for Flag-IRGM expression, using an antibody to Flag to detect exogenous IRGM and actin as a loading control (lower panel). Error bars in b and d, s.e.m.
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 1.

Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P < 0.01 (OIS), P < 0.002 (RS). Assays were performed 10 d after induction with tamoxifen (OIS) or 4 wk after the last passage and >90% SA β-gal+ (RS). (C) CCFs in OIS cells are lamin A/C negative. (D) Cells from C were scored for lamin A/C+ and lamin A/C− CCFs. Note that the majority of micronuclei in control cells are lamin A/C positive. Mean ± SEM, n = 3; P < 0.0006 for lamin A/C negative CCF con vs. OIS; P < 0.6 for lamin A/C positive CCF con vs. OIS. (E) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac. (F) Cells from E were scored for indicated histone modifications in CCFs. A single representative experiment is shown and at least 100 cells were scored per slide. Note the majority of micronuclei in control cells are positive for both marks. (G) CCFs in RS contain γ-H2AX but not 53BP1 (top). Intranuclear γ-H2AX foci colocalize with 53BP1 (bottom). (H) CCF+ and CCF− cells from G were scored for colocalizing intranuclear γ-H2AX and 53BP1 foci. Mean ± SEM, n = 3; P < 0.019. Bars (in IF panels), 10 µm.
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 2.

Nuclear-to-cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1. (A) Extrusion of GFP-H2B–positive chromatin fragment from nonmitotic RS nucleus. Confocal time-lapse microscopy of transiently transfected GFP-H2B–expressing cell, 60× original magnification, 150 nm optical section, time in mins. Bars: 10 µm; (insert) 2 µm. (B) Chromatin fragments blebbing out of nucleus are γ-H2AX positive. Bars: (top) 10 µm; (bottom) 5 µm. (C) Down-regulation of lamin B1 and lamin B receptor (LBR) in RS cells, assayed by Western blot. See Materials and methods for time-course details. (D) Depletion of lamin B1 from OIS cells, assayed by immunofluorescence. (E) A line-scan of lamin B1 fluorescence intensity along the arrows in D. Data shown are from a single representative experiment out of three repeats. (F) Depletion of lamin B1 in BRAFV600E-induced OIS melanocytes in vitro. Bars, 10 µm. (G) Quantitative immunofluorescence of lamin B1 in melanocytes from F. A representative experiment out of two repeats is shown. At least 150 randomly selected cells were assessed. (H) Senescent nevus melanocytes (n) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis (e). White dotted line shows the basement membrane between the dermis and epidermis. Bottom panels (Epi and Nevus) are expanded from white boxed areas on top. Confocal microscopy for DAPI (blue), MelanA (red), and lamin B1 (green) stained tissue. Bars, 20 µm. (I) Quantitation of results from H for three different nevi. Epifluorescence images of lamin B1– and MelanA-stained nevi were obtained and epidermal or nevus MelanA–positive cells were scored for the presence (positive) of a characteristic lamin B1 “ring.” At least 50 MelanA-expressing epidermal melanocytes and at least 100 nevus melanocytes were scored. Average of three different nevi ± SEM; P < 0.0005.
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 3.

Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity. (A) Herniation of H3K27me3-positive chromatin into the cytoplasm of OIS cells. Yellow boxed area is magnified in bottom panels. Bars: (top panels) 10 µm; (bottom panels) 5 µm. (B) Quantitation of lamin B1 gaps or herniations in OIS. Mean ± SEM, n = 2; P < 0.01. (C) Isolated nuclei of senescent cells are permeable to both 70- and 500-kD dextrans, indicating impairment of the barrier function of nuclear envelope. Dark nuclei exclude fluorescent dextran. Bars, 10 µm. (D) Quantitation of results from C. Mean ± SD, n = 4; P < 0.000001 for both 70- and 500-kD dextrans. (E) Representative OIS SAHF+ nucleus showing dextran permeability. Bars, 10 µm. (F) Quantitation of dextran 70 uptake in SAHF+ and SAHF− OIS cells. P < 1.9 × 10−5. (G) In individual nuclei, chromatin extrusion and dextran intrusion occurred in close physical proximity. Right-hand panels are expanded from yellow boxed areas on the left. Bars: (left) 10 µm; (right) 2 µm.
Lysosome-mediated processing of chromatin in senescence. (2013)
Figure 4.

Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62+) or not (p62−). Mean ± SEM, n = 3. (C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62+) or not (p62−) and protein ubiquitination (FK2+) or not (FK2−). Mean ± SEM, n = 3; P < 0.0008 for FK2+/p62+ CCF, comparing proliferating and RS cells. (D) Ubiquitinated proteins appear to line chromatin surface in CCF. Yellow boxed areas are magnified in right-hand panels. Bars: (left panels) 10 µm; (right panels) 1 µm. (E) Apparent activation of cathepsin L in RS IMR90 cells. In contrast to RS cells, the majority of cathepsin L in proliferating control cells exists in its inactive pro-cathepsin L form. Loading was normalized by cell number and lamin A/C was used as a loading control. See Materials and methods for details of time course. (F) Accumulation of H3cs.1, a specific N-terminal cleavage product of H3, in OIS cells. See Materials and methods for details of time course. Western blotting was performed using the same cellular lysates as in Fig. S4 B, and the same lamin A/C panel is shown in both figures. (G) Accumulation of H3cs.1 and corresponding high mobility H3 in OIS. (H) Predominant cytoplasmic localization of H3cs.1 in senescent cells. α-Tubulin and lamin A/C were used as fractionation controls for cytoplasmic and nuclear fractions, respectively.