EMBO/SourceData huggingface dataset:
Figure 5(a) Total cellular lysates from HEK293T cells transfected with myc-VPS39 or VPS41 were incubated with GST-empty vector (EV), RAB2A-S20N (SN), or RAB2A-Q65L (QL) proteins bound to glutathione sepharose beads. Immunoblotting was performed with anti-myc antibody. Input lysates are also shown. GST proteins loaded in gels were stained with CBB.(b) FLAG-EV or RAB2A were transiently co-transfected with EGFP-VPS39 or VPS41 into HEK293T cells. Immunoprecipitated (IP) fraction from cell lysates with anti-FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right.(c, d) MDA-MB-231 cells were transfected with EGFP-fused empty EV, VPS39, or VPS41. Cells were immunostained with RAB7 (c, red) or anti-MT1-MMP (d) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. Bars, 10 μm, (inset 5 μm).(e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm.(j-l) Inverse invasion assay of MDA-MB-231 cells transfected with siRNA oligonucleotides targeting interest genes. Invasive activity is the numbers of cells/field that migrate through Matrigel further than 50 µm (k). Efficacy of targeted gene silencing was verified by qPCR (l). Data are mean ± SD from 3 different fields. Bar, 250 µm.(m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.
Figure 6(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast light microscopy images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm.(b-d) MCF10.DCIS.com cells (104 cells) transfected with scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A) were plated onto 6-well dish. Representative phase contrast light microscopy images (b) and the distribution of cell numbers per cluster of the indicated cell number (c) are shown. Data are means ± SD of 10 fields (n > 60 cells/field) of independent 4 independent experiments. Bars, 100 μm. Efficacy of RAB2A gene silencing was verified by qPCR (d). Bar, 200 µm.(e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue).(f, g) MCF-10AneoT (f) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.(f, g) MCF10-CA1a (g) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.(h) Total cell lysates from (e), (f), and (g) were immunoblotted by anti-E-cadherin, RAB2A, and anti-actin antibodies. ***, P < 0.001, NS, not significant.
Figure 7(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.(c) CTR and RAB2AMCF10A cells (104 cells) were plated onto 6-well dish. Representative phase contrast light microscopy images (left) and the distribution of cell numbers per cluster composed of the indicated number of cells are shown. Data are means ± SD of 10 fields (n > 20 cells/field) from 3 independent experiments. Bar, 100 μm.(d) The circularity of CTR and RAB2A MCF10A cells and the percentage of cells with protrusive structure were quantified. Data are means ± SD of ten independent fields (n > 100 cells/field). n, number of cells scored for each cell population.(e) Total cell lysate from (c) were immunoblotted with antibodies anti-E-cadherin, which recognize either an epitope in the cytoplasmic domain (IC), an epitope in the extracellular domain of mature E-cadherin (SHE78-7), respectively, or anti-RAB2A, and anti-actin. An arrow indicates the unprocessed form of E-cadherin.(f-h) MCF10A cells were transfected with scrambled siRNA (siNC) or siRNA against Furin, and immunostained with anti-E-cadherin (green) and anti-TGN46 (red) antibodies. Merged images with DAPI (blue) are shown on the right (f). Bar, 20 μm. Total cell lysate were immunoblotted with antibodies indicated on the right (g). Efficacy of Furin silencing was verified by qPCR (h).(i) Representative electron microscopy of Golgi stacks (arrows) of CTR and RAB2A MCF10.DCIS.com cells. Bars, 1 μm.(j) CTR and RAB2A MCF10A cells were transfected with Str-KDEL_SBP-EGFP-E-cadherin (green). At the indicated time points after the addition of biotin, cells were fixed, and stained without permeabilization with anti-GFP antibody, followed by Alexa568-conjugated secondary antibody (red). The cell surface and total levels of SBP-EGFP-E-cadherin at 60 min after the addition of biotin were quantified using ImageJ software on non-saturated images, and surface/total ratio was expressed as a surface GFP index (relative to control cells). Bar, 20 µm. ***, P < 0.001.
Figure 1 A, B. Coronal CT image of the chest at baseline with pleural fluid and intralobar septal thickening (arrow) (A), and 3 months after starting trametinib (B).  C, D. MR T2 weighted coronal slice of the chest at baseline demonstrating bilateral pleural effusions (arrow heads) and pulmonary interstitial edema (arrows) (C), and 3 months on trametinib therapy (D).  IN-DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD (arrow) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion (arrowheads) (E), and 6 months after trametinib therapy demonstrating dilated and tortuous TD (arrow) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary perfusion  IN-DCMRL coronal slice of the chest at the level of the carina demonstrating extensive mediastinal and pulmonary interstitial perfusion (arrows) at baseline (G), and after 6 months of treatment with trametinib (H).
Figure 2 A. Deep coverage exome sequencing Integrative Genomics Viewer (IGV) view of CBL c.2322T>G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid. The alternate allele fraction was 4% (20/493). B. Droplet digital polymerase chain reaction (ddPCR) result with 5 mutant positive droplets (circle) from a low concentration left over sample which resulted in ~0.005% fractional abundance.
Figure 1(B) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a single growing germling, at 70, 80, 100, 120, 140, 160 and 170 min after derepression of transcription via its native uapA promoter (for details see text and Materials and methods). Notice that in the growing germling tip UapA localizes in a membranous mesh and some cytosolic puncta whereas in more tip-distal parts of the germlings UapA is in PM-localized puncta (see white arrows), which progressively become more abundant so that at more posterior areas the PM is homogeneously labeled.(C) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a germling maturing to young hypha at 180, 240, 300, 360 and 400 min after derepression of transcription via its native uapA promoter. During this developmental transition, UapA apical localization is gradually diminished. As the cell tip acquires a faster growth rate, UapA is completely retracted from the hyphal apex (see zoon-in panels on the right and white arrows). (D) In a mature hyphal cell, UapA is no longer found in the PM of the extreme apical compartment, but in a cytoplasmic a membrane network resembling the ER. As the distance from the apex increases, UapA progressively populates the PM as cortical puncta (see white arrows). (F) In vivo epifluorescence microscopy following neosynthesized alcAp-UapA-GFP in a single hypha at 120, 130, 140, 150, 160 and 170 min, under overexpressing (derepression/ethanol-induction) conditions, as described in Materials and methods. Notice that UapA labels the perinuclear ER rings (white arrows).(G) In vivo epifluorescence microscopy following de novo expressed alcAp-GFP-SynA in a single young hyphal cell, at 60, 70, 80, 90 and 100 min after transcriptional derepression (for strain details see Materials and methods). SynA is a standard polar membrane cargo that traffics to the hyphal tip via Golgi-dependent secretion. Notice the very distinct GFP fluorescent signals obtained using UapA (non-polar membrane network and PM) versus SynA (Golgi-like puncta and polar depositioning at the tip; see also alter).(H) Similar experiment as in (B) performed in an isogenic strain where ArtA, the arrestin required for UapA endocytosis, is genetically depleted.
Figure 2(A, B) Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHosbp) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization was performed by calculating Pearson's Correlation Coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHosbp. For the definition of the two categories of replicates see Materials and methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC=0.25±0.09, P<0.0001) or with PHOSBP (PCC=0.34±0.06, P<0.0001), as PCC values close to 0.2-0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm.(C, D) Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHospb) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant colocalization of SynA with the late Golgi marker (PCC = 0.67±0.04, P<0.0001), but not with early Golgi marker (PCC=0.37±0.06, P<0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies
Figure 5(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P<0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain.(B) In a similar experiment, the apical polarized localization of de novo made SynA, used as a control of conventional secretion, is shown to be abolished in all three strains where RabE, AP-1σ or ClaH are repressed. Scale bars: 5 μm. mCherry-SynA PM/cytosolic intensity ratios are plotted with values being 0.52±0.02 for wild-type, 0.14±0.02 for thiAp-rabE, 0.24±0.02 for thiAp-ap1σ and 0.16±0.02 for thiAp-claH. The statistical evaluation was performed as in (A). There is a significant difference (****P<0.0001) of mCherry-SynA fluorescence in the apical PM membrane between the wild-type and the three strains lacking the post-Golgi proteins. Biological/technical replicates: 2/15 for each strain.(C) Co-localization analysis and relevant quantification of strains co-expressing de novo made UapA-GFP (120 min after initiation of transcription) with RabE-mRFP, AP-1-mRFP or ClaH-mRFP. Scale bars: 2 μm. Quantification by calculating Pearson's Correlation Coefficient (PCC) shows clear non-colocalization of UapA with all the Post-Golgi markers tested, as confirmed by one sample t-test (PCC=0.31±0.08, 0.29±0.04 or 0.07±0.01, with ****P<0.0001, for RabE, AP-1 or ClaH, respectively). Biological/technical replicates: 3/7 for each strain.
Figure 6(A) Time course of treatment with the actin polymerization drug latrunculin B for 2, 5, 10, 30 or 50 minutes of a strain expressing neosynthesized UapA-GFP under conditions of derepression compared to an untreated strain included as control (150 min). In all cases latrunculin B was added at 100 min of UapA derepression, so that total time of UapA-GFP expression was 102, 105, 110, 130, or 150 minutes in the different samples. Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min, respectively. Scale bar: 5 μm.(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm.(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.(D) Effect of latrunculin B or benomyl on COPII vesicle formation, followed through the subcellular localization Sec24-GFP. Notice that 40 min of latrunculin B led to loss of the wild-type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm. In contrast, benomyl treatment did not affect the wild-type punctuate localization of Sec24. Scale bar: 5 μm.
Figure 7(A) Subcellular localization of de novo made alcAp-GFP-SsoA in the PM in a single hypha. Notice the ER-like membranous network (white arrows) and several cytosolic puncta labelled before SsoA reaches eventually the PM. Scale bar: 5 μm.(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P<0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition.
Figure 8(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.(B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.
Figure 1CD4 and CD8 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28 beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots depict one representative donor out of three.CD4 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28; and human IFNβ (top) and IP10 levels (bottom) in the supernatant were analyzed by ELISA at the indicated time points. Data panels depict mean ± SD of biological duplicates from three independent donors.CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by quantitative PCR for IFNB1 and OASL expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP and αCD3/CD28 treated cells without CHX only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.
Figure 2CD4 T cells were stimulated with the indicated stimuli for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of three is shown.CRISPR/Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h. Human IFNβ levels were analyzed by ELISA. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ns, not significant.
Figure 3CD4 T cells were pre-treated with Dasatinib (100, 10, 1 and 0.1 nM), Tacrolimus (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.
Figure 4Freshly isolated CD4 T cells were treated with cGAMP, αCD3/CD28 beads and ABT737/S63845 (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.CD4 T cells were treated with indicated concentrations of cGAMP for 18 h, and ABT737/S63845 for 2 h. Lysates and supernatant precipitations were analyzed by immunoblotting. One representative donor out of two is shown.CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).
Figure 5CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells were activated with αCD3/CD28 beads, stimulated with cGAMP for 96 h and analyzed by flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28 (3/28) for 48 h and analyzed by Seahorse XF mito stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.
Figure 1Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.
Figure 2Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes Scale bar: 25 μm.
Figure 3Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled.
Figure 4Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones.
Figure 5Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA (D) and in 2iL (E). Average and SD of at least three independent clones.
Figure 1Heatmap showing the normalized expression of marker genes for the different clusters (Methods). Expression is normalized by the maximal expression among all cell types.