EMBO/soda-nel huggingface dataset:
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 5

Figure 5 - PI3KC2β depletion disturbs endosomal trafficking with increase of VEE (APPL1+) and recycling (Rab11a+) endosomes.

A Quantification of internal transferrin in sh-Control or sh-PI3KC2β cells at different incubation time points (ImageJ software). Results are expressed as fold change of internalized Alexa Fluor®546-transferrin. Data represent mean ± SEM of cell fields randomly selected (dots n=13-20). Data are collected from 4 independent experiments. ***p<0.001 Mann-Whitney test.

B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI).

C, D Quantification was performed to determine number (C) and average size (D) of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 6

Figure 6 - PI3KC2β depletion increases the levels of VE-cadherin at the plasma membrane.

(A) Representative VE-cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field (DAPI labelling) in sh-Control and sh-PI3KC2β hCMEC/D3 in resting condition and after 24 hours activation with 25 ng/ml of TNFα. Control normalized to 100%. Scale bar, 15 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=14-19). Data are collected from 4-5 independent experiments; ***p<0.001, unpaired t-test.

(B) Graph quantification of fluorescence intensity of VE-Cadherin normalized to total cell number per field (DAPI labelling) in control condition and after 5 hours in oxygen-glucose deprivation (OGD) condition. Control normalized to 100%. Data represent mean ± SEM of cell fields randomly selected (dots n=16). Data were collected from 5 independent experiments);***p<0.001; **p<0.01, Mann-Whitney test.

(C) Representative western blot and graph quantification of VE-cadherin from resting or activated (TNFα) cells. Lanes were run on the same gel but were noncontiguous. Densitometric measurements (mean ± SEM) normalized to actin; control normalized to 1 (n=7-8 independent experiments); **p<0.01; *p<0.05, one sample t-test.

(D) Gene expression of VE-cadherin (CDH5) from sh-Control and sh-PI3KC2β hCMEC/D3 cells in resting condition or after 6 hours of stimulation with TNFα. The mRNA levels are given as fold change to resting control hCMEC/D3 cells normalized to PGK1. Data represent mean ± SEM of n=3 independent experiments in duplicate; for Sh-PI3KC2β+TNFα, n=3 independent experiments with only two experiments in duplicate. No significant changes between conditions were noted (Mann-Whitney test).
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 7

Figure 7 - PI3KC2β-knockdown reduces VE-cadherin level in a Rab11a- dependent manner

A Representative images of PI3P and PI(3,4)P2 labeling and quantification using mCherry-FYVEHRS and GFP-PH(TAPP1) probes respectively in sh-Control or sh-PI3KC2β hCMEC/D3. Scale bar, 10 µm. Nuclei are shown in blue (DAPI). Staining intensity was quantified by ImageJ software and normalized to total number of cells (DAPI labelling) per field. Data represent mean ± SEM of cell fields randomly selected for PI3P (dots n=21) and for PI(3,4)P2 (dots n=14-16). Data were collected from at least 5 independent experiments; ***p<0.001, unpaired t-test with Welch's correction.

B Quantification of total cellular PI3P levels in sh-Control or sh-PI3KC2β hCMEC/D3 cells by mass assay. Results are expressed as pmoles of PI3P/nmoles of total phosphore. Data represent mean ± SD (n=2 independent experiments in triplicate).

C Graph quantification of APPL1-, EEA1- and Rab11- endosomes colocalized with mCitrin-VE-cadherin in sh-Control and sh-PI3KC2β hCMEC/D3 cells. Data are shown as percentage of total count endosomes per cell. Data are expressed as mean ± SEM (n=33-35 cells for APPL1 and EEA1 endosomes and n=16-18 cells for Rab11 endosomes; 3 independent experiments); **p<0.001, Mann-Whitney test or unpaired t-test, as accordance.

D Representative confocal microscopic images of VE-cadherin immunostaining in sh-Control cells, sh-PI3KC2β cells or sh-PI3KC2β cells transduced with Rab11S25N (Rab11-DN). Graph quantification of VE-cadherin; control normalized to 100%. Scale bar, 10 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=6-9). Data are collected from at least 3 independent experiments; **p<0.01 Mann-Whitney test.

EXPANDED VIEW FIGURE LEGENDS
Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species-specific barrier (2021)
Figure 6

Figure 6. P. yoelii and P. falciparum sporozoites engage with and enter blood vessels in skin. (A) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).
Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species-specific barrier (2021)
Figure 7

Figure 7. Time-lapse microscopy of P. falciparum sporozoites moving in human dermis grafted on NSG mice. (A) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites. See Movie EV9. (E) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks. (F) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft, see movie EV11.
Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species-specific barrier (2021)
Figure 8

Figure 8. Reduced motility of P. falciparum sporozoites in mice passively immunized with CSP-specific monoclonal antibodies. Mice were injected intravenously with 150 μg of anti-PfCSP or control mouse or human monoclonal antibodies 16-24 h prior to intradermal inoculation with P. falciparum sporozoites. Imaging was performed 10 min after sporozoite inoculation. (A) Activity of mAb 2A10 on sporozoite speed and displacement: Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median. For the displacement data, percentage values indicate the fraction of tracks displacing over 75 microns. Statistical analysis: Mann-Whitney, p=0.0001 (speed data) and p=0.0098 (displacement data). Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions, which generated a total number of 217 (naïve) and 136 (2A10) sporozoite tracks. (B) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163 (C) Activity of human mAbs 10 and CIS43, specific for P. falciparum CSP, and VRC01, an isotype control human mAb specific for HIV-1, on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted. Data are pooled from 7 biological replicates, naïve and VRC01 (n=2), naïve and mAb 10 (n=2), VRC01 and CIS43 (n=3). No comparisons were statistically significant (Mann-Whitney for percent motile and Fisher's exact test for blood vessel entry).

Expanded View Figure Legends
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 1

Figure 1 - Post-translational modifications of Egg in OSCs.

A Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.

B Western blotting of Myc-Egg expressed in OSCs. Anti-Myc (Myc) and anti-β-Tubulin (Tub) antibodies were used. Tub was detected as a loading control. Control: empty vector was used as a negative control.

C Schematic of Egg domain structure. The predicted regions and sites for SUMOylation (SUMO: 1-202 a.a.), phosphorylation (Ser125 and Thr217), and ubiquitination (Lys1085) are shown.

D Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.

E Western blotting of Egg immunopurified from total OSC lysates before (-) and after (+) phosphatase treatment. Anti-Egg antibodies were used for both immunoprecipitation (IP) and western blotting.

F Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.

G In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.

H Rescue assays. Myc-Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting (WB) was performed using anti-Egg and anti-Myc antibodies. RT-qPCR was used to quantify Stalker2 transposon expression level. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). n.s.: not significant.
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 2

Figure 2 - Egg monoubiquitination occurs in the nucleus Ubc2-dependently.

A Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

B Western blotting of fractionated OSC materials. Anti-Egg, anti-β-Tubulin (Tub), and anti-histone H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.

C Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).

D Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

E Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

F Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).

G Rescue assays. Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. Stalker2 transposon expression was quantified using RT-qPCR. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).

H In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).

I Rescue assays. Myc-Egg WT, Myc-Egg K1085R, and Myc-SV40-NLS-K1085R were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of the Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 3

Figure 3 - Wde controls the abundance and monoubiquitination level of Egg in the nucleus.

A Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

B Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates.

C Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

D Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

E Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.

F The schematic drawing of domain structures of Wde WT and its ∆CC mutant. The ∆CC mutant lacks the Egg-binding domain (842−907 a.a).

G Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.

H Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 4

Figure 4 - Determination of Egg-NLS.

A Schematic of the domain structure of Egg. The amino acid sequence alignment of the predicted Egg-NLS in fly, humans and mice is also shown. Asterisks, colons, and periods indicate identical, highly conserved, and weakly conserved residues, respectively. The letters in red indicate basic residues.

B Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

C Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used.

D Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

E Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 5

Figure 5 - Wde-mediated Egg recruitment to the chromatin leads to H3K9me3 deposition.

A Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).

B Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction.

C Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.

D Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.
Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing (2019)
Figure 6

Figure 6 - Wde controls Egg function by recruiting the protein to the chromatin in OSCs.

A Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples and used for analyses in Figure 6 and EV Figure 6.

B Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.

C Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.

D Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.

E Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.

F ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.

G Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.

Expanded View

Expanded View Figure legends