EMBO/soda-nel huggingface dataset:
LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 9

Figure 9. LRRK2 phosphorylation of Rab10 promotes CCL5-induced Akt activation and chemotaxis.

A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown.

B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01.

C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown.

D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001.

E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown.

F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05.
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation (2015)
Figure 1

Figure 1. siRNA screen identifies BRPF3 as novel regulator of DNA replication. A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging. B, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher’s analysis. Selected hits are highlighted. C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown. D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl. The average and standard deviation from three biological replicas is shown. n>2000. ANOVA t-tests: **** p<10-4, *** p<10-3, ** p<10-2, * p≤0.05, n.s. non-significant.
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation (2015)
Figure 2

Figure 2. BRPF3-HBO1 regulates DNA replication and acetylates H3K14ac. A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas. B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2. C, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot. D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown. E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes. F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively. G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates. H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation (2015)
Figure 3

Figure 3. BRPF3 is required for replication origin firing. A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann–Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann–Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann–Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown. D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit. E, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant. G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation (2015)
Figure 4

Figure 4. BRPF3-HBO1 acetylates H3K14 at replication origins. A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HU chromatin. B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells. C, Heatmaps of BRPF3, HBO1 and H3K14ac ChIP-seq signal on ± 5kb surrounding the TSS of genes. Genes were sorted by BRPF3 abundance from high to low. A.U. arbitrary unit. D, Average profiles of BRPF3 and H3K14ac ChIP-seq signal on ± 2.5kb around the centre of (top) BRPF3 and (bottom) ORC1 genome binding sites. E, ChIP-qPCR analysis of H3K14ac on well-characterized replication origins. H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells. Error bars, range; n= 2 biological replicas. F, Expression levels of DNA replication factors were not altered by the lack of BRPF3. Each point represents the expression levels of a gene in control and BRPF3 depleted cells (GSE65065). Replication factors were classified as described previously (Alabert et al, 2014). Total genes (grey), DNA replication factors (colors) and BRPF3 (black) expression levels are shown. Continuous black lines mark 1.5 fold change in expression levels.
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation (2015)
Figure 5

Figure 5. Lack of BRPF3 improves recovery after replication stress. A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown. B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.
DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 1

Figure 1. DnaJC5 overexpression facilitates tau release into the extracellular space.

A. Schematic illustrating the preparation of samples for media analysis by dot blot/Western blot or mass spectroscopy.

B. Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG-DnaJC5 compared to empty vector and tau. Intracellular protein levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=8, ***p<0.001.

C. Light/heavy ratio of tau peptides in label-free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau. Light peptides represent endogenous tau peptides. Data are mean ± SEM, n=3 biological repeats, ***p<0.001.
DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 2

Figure 2. Release of tau is regulated by DnaJC5.

A. Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG-DnaJC5 or empty vector. Media was concentrated prior to analysis. Intracellular DnaJC5 was detected with FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

B. Western blot tau levels from concentrated media from ex-vivo organotypic slice cultures of wildtype mice overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. DnaJC5 is detected by FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

C. Western blot tau levels from concentrated media from ex-vivo organotypic slice cultures of Cspa+/+ or Cspa-/- mice. DnaJC5 was detected by DnaJC5 antibody (Synaptic Systems). Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

D. Schematic showing lack of DnaJC5 (yellow circles) reduces extracellular tau release, whereas overexpression of DnaJC5 results in enhanced release of tau.
DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 3

Figure 3. DnaJC5 facilitates the release of neurodegenerative-associated proteins into the extracellular space.

A. FLAG-DnaJC5 overexpression facilitates extracellular release of wildtype (WT), P301L and R406W tau by dot blot in HEK293T cells. Intracellular protein levels are shown by Western blot; DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

B. Overexpression of FLAG-DnaJC5 induces extracellular release of wildtype (WT) and A53T α-synuclein in HEK293T cells. Extracellular protein levels shown by dot blot (quantification below, mean ±SEM, n=3 ***p<0.001, ** p<0.01 by one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis) and intracellular proteins levels were analyzed by Western blot, DnaJC5 was detected by FLAG antibody.

C. DnaJC5 overexpression induces extracellular release of GFP-tagged wildtype (WT), A315T and Q343R TDP-43 in HEK293T cells. Intracellular DnaJC5 was detected by FLAG antibody. Extracellular protein levels are analyzed by dot blot (quantification below, mean ±SEM, n=4) ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.
DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 4

Figure 4. DnaJC5 participates in an alternative exocytosis pathway important for extracellular release.

A. Dot blot of tau levels in media of cells overexpressing the phospho-deficient FLAG-DnaJC5 S10A compared to FLAG-DnaJC5 WT. Intracellular levels shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

B. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated tau release in HEK293T cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.

C. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated α-synuclein release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 by one-way ANOVA with a Tukey’s post-hoc analysis. DnaJC5 is detected by FLAG antibody.

D. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated TDP-43 release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.

E. Schematic representation of Hsp70 control of DnaJC5-mediated tau release. DnaJ selection on Hsc70 dictates client fate: if a pro-degradation DnaJ binds to Hsc70, clients are sent for degradation. If DnaJC5 binds to Hsc70, client proteins are sent for release by a SNAP23-dependent pathway.
DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 5

Figure 5. Tau release is controlled by chaperones.

A. Confocal micrographs of tau (red pseudocolor) and Hsp70 (green pseudocolor) colocalization (merged image) levels in wild type primary neurons overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. Boxed area are 5X digital zoom on a 60X lens shown below. Scale bars are 20 µm and 5 µm as indicated. Only neurons overexpressing virus were imaged.

B. Quantification of tau-Hsp70 co-localization (mean Pearson’s coefficient ± SEM, n=18, *p <0.05, one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis).

C. Western blot of immunoprecipitated tau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70. DnaJC5 was detected with FLAG antibody.

D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody.

E. The DnaJC5/Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant-negative point mutation, E175S. Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70. DnaJC5 is immunopreicipitated by DnaJC5 antibody (Synaptic Systems). DnaJC5 was detected on Western blot by FLAG antibody.

F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody.

G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody.

H. Schematic illustrating the Hsc70/DnaJC5/tau complex can be disrupted by both chemical (YM-01) inhibition.
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 2

Figure 2. Downregulated TFAM significantly contributes to the metastasis of HCC cells in vitro and in vivo.

(A and B) qRT-PCR and Western blot assays for the expression level of TFAM in ten HCC cell lines, one normal liver cell line (LO2) and two non-tumor liver tissues. The protein level was quantified as ratios between TFAM and GAPDH (n = 3 independent experiments).

(C) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells (n = 3 independent experiments). shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM.

(D, E) Wound-healing migration assay (D) and Transwell migration and invasion assays (E) for HCC cells with treatment as indicated (n = 3 independent experiments). Scale bars, 100μm.

(F) SNU-368 cells with TFAM knockdown or HLE cells with TFAM overexpression were infected with firefly luciferase expressing lentivirus and transplanted into livers of nude mice. Representative bioluminescent imaging for each group (n =10) is shown.

(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm.

(H, I) Number of metastatic nodules per lung (H) and the incidence of lung metastasis (I) in mice used in (F).

(J) Kaplan-Meier survival curves for mice inoculated with orthotopic HCC cells as indicated. n=10 per group.

Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 3

Figure 3. Downregulated TFAM induced polymerization of nuclear actin in HCC cells.

(A) Representative confocal microscopy images of F-actin and 3D reconstruction of z-stacks from nuclear region with IMARIS software in Phalloidin-stained HCC cells (A) and LifeAct-mCherry Venus expressing cells with treatment as indicated (B) (n = 3 independent experiments). Red: Phalloidin or LifeAct, Blue: DAPI. shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.

(C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.

(D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 5

Figure 5. Downregulated TFAM induced the expression of metastatic genes in a nuclear F-actin dependent way.

(A) Expression heatmap of ATAC-DEGs in TFAM knockdown and control SNU-368 cells (n=3 per group). Up- and down-regulated ATAC-DEGs in TFAM knockdown HCC cells were annotated.

(B and C) Top 20 GO terms and enriched KEGG pathways enriched by ATAC-DEGs. Rich factor represents the ratio of the number of mapped genes to the number of all the annotated genes located in the pathway.

(D) ATAC-seq signal tracks around the FN1, IL-6, TGFB1 and ITGB3 genes in HCC cells with treatment as indicated. Blue box indicates promoter region with increased accessibility.

(E and F) qRT-PCR and Western blot assays for mRNA and protein expression levels of metastasis related gene in HCC cells (n = 3 independent experiments) and tumor tissues from mice (n =4 per group) with treatments as indicated.

(G) ELISA analysis for the levels of IL-6 and TGFB1 in HCC cells and tumor tissues from mice with treatments as indicated (n = 3 independent experiments).

(H and I) Western blot assays and ELISA analysis for the levels of FN1, IL-6, TGFB1 and ITGB3 in HCC cells with treatment as indicated (n = 3 independent experiments).

Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 6

Figure 6. Downregulated TFAM induced nuclear translocation of mDia2 to promote nuclear actin polymerization.

(A) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated. Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus, respectively (n = 3 independent experiments).

(B) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt/β-catenin (n = 3 independent experiments).

(C) Representative immunofluorescence images of HCC cells co-transfected with mDia2-GFP and other constructs as indicated (n = 3 independent experiments). Scale bars, 20μm.

(D) Representative images of immunofluorescence assay for mDia2 and phalloidin staining for HCC cells with treatment as indicated (n = 3 independent experiments). WT-mDia2: expression vector encoding wild type mDia2. mDia2-NLS del.: expression vector encoding truncated mDia2 lacking NLS peptide. Scale bars, 20μm.

(E-G) Representative IHC images of mDia2 nuclear translocation in human (E) and mouse (G) HCC tissues. NT, non-tumor liver tissues, n=100; MT, metastatic tumor tissues, n=58; NMT, non-metastatic tumor tissues n=42. Scale bars, 25μm.

(F) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in (E). Graphs show mean ± s.e.m. One-way ANOVA. **P < 0.01.

(H and I) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level (or positive rates of nuclear F-actin) based on the IHC assay in (E).