EMBO/soda-nel huggingface dataset:
Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 5

Figure 5. Super-resolution microscopy demonstrates formation of a F-actin cage around the nucleus during invasion.

A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). 3D rendering is presented in Movie EV8.

B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole.

C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion.

D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate.

E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). These images are expanded with additional panels in Appendix Figure S5. 3D rendering is presented in Movie EV9.

H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location.

K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ.

Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images and 1 µm for 3D models. White arrow points to direction of invasion.
Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 6

Figure 6. Plasmodium F-actin localisation during invasion

A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus.

B. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus.

C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus.

D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total.

Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum and 3 µm for Plasmodium knowlesi.
ER stress in antigen presenting cells promotes NKT cell activation through endogenous neutral lipids (2020)
Figure 1

Figure 1: iNKT cell activation by antigen presenting cells undergoing UPR

A-B: Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) or with (B) a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).

C-D: Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells or (D) human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.

E: Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).
ER stress in antigen presenting cells promotes NKT cell activation through endogenous neutral lipids (2020)
Figure 2

Figure 2: Autoreactivity of iNKT cell is CD1d dependent

A: Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.

B: Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

C: Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).
ER stress in antigen presenting cells promotes NKT cell activation through endogenous neutral lipids (2020)
Figure 3

Figure 3: Autoreactivity of iNKT cell requires both the IRE1α and PERK pathway

A-B: qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine and human macrophage cell lines J774.2 (A), U937 (B) stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).

C: Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.

D: Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene. E: Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.
ER stress in antigen presenting cells promotes NKT cell activation through endogenous neutral lipids (2020)
Figure 4

Figure 4: Fractionated neutral lipid from stimulated macrophages activates iNKT cells:

A: Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

B: Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).

C: Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.

D: Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.
ER stress in antigen presenting cells promotes NKT cell activation through endogenous neutral lipids (2020)
Figure 5

Figure 5: UPR-induced neutral lipids require CD1d endosomal/ lysosomal recycling:

A: Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

B: qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

C: Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.

D: L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

Expanded View Figure legends:
Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites (2013)
f1

a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ–Flag–LC3 in normal medium or Earle’s balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins. b–d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin. e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells. f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3. g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with &bgr;-tubulin. Similar results were observed in three independent experiments.
Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites (2013)
f2

a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker &ggr;-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 h serum starvation (SS). b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 h serum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satellite OFD1 for 100 cells per well in triplicate samples. ***P  0.001, two-tailed unpaired student’s t-test. Similar results were observed in three independent experiments.
Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites (2013)
f3

a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student’s t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.
Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites (2013)
f4

a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with &bgr;-tubulin. b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P  0.001, two-tailed unpaired student’s t-test. c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation. d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples. f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 1

Figure 1. Tf2 retrotransposons are upregulated in Fft mutants. Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). A) Transcription in fft2Δ relative to WT over chromosome 1. The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands, respectively. Coordinates are shown in the middle panel; genes more than 2-fold upregulated are marked: Tf2 retrotransposons (black); antisense to Tf2 elements (gray); coding genes (green); noncoding RNAs (blue). B) Northern blot for the Tf2 ORF; rRNA staining of the same membrane by methylene blue is shown below. Two different exposures of the molecular size marker are shown (left). C) qPCR scheme. Amplicon 1 spans the end of the LTR and into the ORF. Amplicon 2 does not include the LTR but covers the translation start site. Amplicon 3 is entirely within the ORF. D) Bar graph representing RNA levels for the amplicons depicted in (C), relative to a control locus (SPAC1F8.07c) and to WT levels, as measured by qPCR. Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates. E) 5’RACE products run on a 1.5% agarose gel. F) The sequence of a Tf2 retrotransposon transcript from the ‘R’ sequence of the upstream LTR to the start codon of the coding region. The long and short RACE products (E) were sequenced and the transcript start sites are highlighted (yellow). Orange text, R. Light blue, U5. Dark blue, self-primer and primer binding site. Green, start codon.
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 2

Figure 2. Transcription initiation occurs at alternative TSS in the U3 LTR region in Fft mutants.

Top panel: Y-axis shows the CAGE signal intensity (tag-per-million normalized number of mapped CAGE tags on plus strand). X axis shows LTR and 5’ of Tf2 ORF. Three biological replicates are shown for each strain: fft2Δfft3 and WT. Bottom: LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all Tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements. The black square indicates the 5’ end of the Tf2 mRNA in WT and the green square indicates the 5’ end in fft2Δ fft3Δ detected by 5’RACE.
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 3

Figure 3. Fft remodelers bind to and affect nucleosome occupancy at Tf2 LTRs. A) Fft2 and Fft3 are both enriched at LTRs, while Fft2 is also enriched at the tf2 coding region. Fft2 LTR enrichment increases in fft3Δ cells, while Fft3 LTR enrichment decreases slightly in fft2Δ cells. Top: Fft2 –myc enrichment in WT (solid line) and in fft3Δ (dashed line). Bottom: Fft3 –myc enrichment in WT (solid line) and in fft2Δ (dashed line). ChIP-chip signal is normalized against no-epitope control arrays. B) Nucleosome occupancy over the LTR U3 is significantly reduced (average reduction ~50%; average p-values at each position <0.001, Poisson test) in fft2Δ fft3Δ cells. MNase-seq data aligned at the start of tf2 5’LTRs is shown for WT (black line), and fft2Δ fft3Δ (dark gray line). Light gray line indicates significance of difference between WT and mutant signals: amplitude reflects significance (p-value) and sign reflects the sign of occupancy change in mutant vs. WT cells. Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements. LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements, while the green dot indicates the TSS in fft2Δ fft3Δ.
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 4

Figure 4. LTR-proximal genes are upregulated in fft2Δ fft3Δ A) Mean nucleosome occupancy over solo LTRs (n=236) and tf2-associated LTRs (n=25), in WT and in fft2Δ fft3Δ. B) Mean ratio of expression (fft2Δ fft3Δ /WT) for each of the following categories: non-coding with LTR (n=69) / without LTR (n=1665) (p=0.008); protein coding with LTR (n=246) / without LTR (n=4887) (p<0.001); Tf2s (n=13). A and B) Data shown as box plots, significance of difference between categories was assessed by Wilcoxon-Mann-Whitney test (** p<0.001).
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 5

Figure 5. Tf2 elements undergo an Fft remodeler-dependent TSS shift when stressed. A) Heat and oxidative stress cause a Tf2 TSS shift in WT cells, resulting in the production of reverse transcription competent Tf2 transcripts. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates. B) Heat shock-associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy. The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells, where nucleosome occupancy is already very low (Fig 2B, Fig S3). LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF +1 nucleosome, measured by MNase-qPCR. C) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 7

Figure 7. Fft2 and Fft3 are required for Tf2 element clustering in tf bodies A) Fluorescence in situ hybridization with a probe against tf2 elements (green), and DAPI staining (blue) in WT and fft2Δ fft3Δ cells. B) The number of tf2 element signal clusters is significantly elevated (p=9.8x10-18, χ2-test) in fft2Δ fft3Δ cells, indicating a loss of retrotransposon clustering. Tf2 clustering was counted in wt cells (n=663), fft2∆ (n=556), fft3∆ (n=493), fft2∆ fft3∆ (n=636). Significance of difference between strains was assessed by Chi-square test.