EMBO/SourceData huggingface dataset:
Figure 4(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.(B Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm.(D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).(E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments.
Figure 1A) GFP tagged Syb protein expressed in Hh-receiving cells co-localizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal planes (arrows), and can be detected along cytonemes marked with Syb-GFP (arrowhead).B) Expression of the GFP tagged Syt1 also co-localizes with inmmuno-labelled endogenous Ptc at vesicles (lateral plane, arrows point at vesicles with co-localization) and decorates the cytoneme membranes (basal plane, arrowheads indicate cytonemes).C) GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1-10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. Note the fluorescent discrete punctae (arrows) along Hh producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area.D) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal planes, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytoenemes).
Figure 2A) Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc (arrowheads) when knocking down Syb or Syt1.B) Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh producing cells in wild type receiving cells (left panel), or B') when either blocking exocytosis by down-regulating Syb (middle panel) or B'') blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrowheads), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows).C) Confocal apical (left) and basal (right) images of a shits mutant wing disc expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis.D) Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (-1=basal, +1=apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N=7-16, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis.
Figure 3A) Electron Microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 hours in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium a) anti-GFP gold-labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. b) Ptc immuno-gold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), c1) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV).B) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side.accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side. C) Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire.
Figure 4A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation.B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh receptionC) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition.D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).
Figure 5A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes.B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames).C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures).D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows).E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures).F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames).G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures).H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.
Figure 6A) 3D reconstruction of wing disc immuno-labelled for endogenous Ptc and dorsally over-expressing the Tsp96F-HA construct, keeping the ventral side of the disc in wild type condition as internal control. the dorsal accumulation of Ptc in very large punctae across the apico-basal axis, but mainly at the basal side of the epithelium. On the right, graphs represent fluorescence values from apical to basal, comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96-HA expression.B) Confocal image of a wing disc with a clone expressing Tsp96F-HA and immuno-labelled with anti-Ptc and anti-HA antibodies, which co-localize in the large basolateral punctae (yellow arrows).C) Electron microscopy image from immunolabelled (anti-GFP) Hh-receiving cells of a wing disc expressing Ptc-GFP and Tsp96F-HA. The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc.
Figure 7A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells.B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70.C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs.E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.
Figure 8A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization.B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates using apical projection images; central horizontal lines show median values of N=8-12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression.C) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).
Figure 9A) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition.B) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient.C) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β−galactosidase (Ptc-LacZ). Middle graph shows the extension covered by signal normalized to wing pouch size, in wild type condition (N=6) or after ectopic expression of the Tsp96F-HA construct (N=9). Green shaded areas show data variability and dotted line gives the average values. Note the greater distance covered after Tsp96F-HA transient expression. Boxplot (right) shows distance quantifications with central horizontal lines showing median values of N=6-9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers. Note a significant extension under Tsp96F-HA expression, suggesting again flattening of the signal gradient.
Figure 1BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD.Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 (loading control).LC-MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC-MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.
Figure 2BACE1 from temporal lobe membrane fractions of NC, eAD, or AD patients was immunoprecipitated and blotted with E4-PHA (upper) or anti-BACE1 (lower). The signal intensity of E4-PHA relative to that of BACE1 was calculated (n = 10). The graph shows means ± SEM (*P < 0.05; one-way ANOVA with post hoc Tukey-Kramer test. P = 0.014 for NC versus eAD, P = 0.028 for NC versus AD).
Figure 3APP metabolites from 3-month-old mouse brain membrane (A) or soluble (B) fractions were immunoblotted. The signal intensity was quantified (n = 4-5). All graphs show means ± SEM (*P < 0.05; Student's t-test for (A) βCTF and (B) sAPPα, and Mann-Whitney U-test for the others. P = 0.386 for APP, P = 0.602 for αCTF, P = 0.045 for βCTF, P = 0.218 for sAPPα, P = 0.022 for sAPPβ).
Figure 4Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from (A) 3-month-old or (B) 12-month-old hAPP/Mgat3+/+, hAPP/Mgat3+/−, or hAPP/Mgat3−/− brains (n = 5). P = 0.015 for TBS Aβ40, P = 0.034 for TBS Aβ42, P = 0.001 and 0.012 for Gn-HCl Aβ40, P = 0.024 for Gn-HCl Aβ42 in (A), P = 0.040 for Gn-HCl Aβ40, P = 0.047 for Gn-HCl Aβ42 in (B).Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from 3- to 4-month-old Mgat3+/+ or Mgat3−/− brains (n = 7-8). An outlier value was rejected by the Smirnov-Grubbs' test (P < 0.05). P = 0.045 for Gn-HCl Aβ40.Immunostaining of Aβ plaques in 12-month-old mouse brain (left). Scale bar, 300 μm. The number of FSB-stained Aβ plaques in brain sections prepared from 12-month-old male mice was quantified (right) (n = 6). P = 0.004 for hippocampus, P = 0.0003 for cortex.The Y-maze test was performed using 12-month-old male Mgat3+/+, Mgat3−/−, hAPP/Mgat3+/+, or hAPP/Mgat3−/− mice (n = 8-10). **P = 0.004 for Mgat3+/+ versus hAPP/Mgat3+/+, *P = 0.022 for hAPP/Mgat3+/+ versus hAPP/Mgat3−/−.
Figure 5BACE1 from 2-week-old mouse brains was immunoprecipitated and immunoblotted (left). Immunoprecipitated BACE1 activity was measured in vitro (right, n = 3). The values of Bace1−/− samples were subtracted as a background. N.S., P = 0.101.MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, APP, rab5, or rab7. EE, early endosome; LE, late endosome.Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, rab5, or rab9. EE, early endosome; LE, late endosome. Signal intensities were quantified and are shown in the right graphs.Immunostaining of 12-month-old mouse cerebral cortex for BACE1 and APP. A typical image in the vicinity of plaque-forming area is shown for hAPP/Mgat3+/+ brain. The area of co-localization was quantified using random images of cerebral cortex (n = 10). *P = 0.015.Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ).Immunostaining of 12-month-old mouse cerebral cortex for BACE1 (F), or nicastrin (G), and Lamp1. LE, late endosome. Scale bar, 10 μm. The area in which co-localized staining was observed was quantified as a percentage of the total BACE1-positive area (right, n = 10). **P = 0.007.
Figure 6Immunostaining of 12-month-old brain sections (cerebral cortex) with anti-BACE1. Scale bar, 100 μm.Proteins from the membrane fractions of 12-month-old mouse brains (n = 4-5) were immunoblotted for BACE1, and BACE1 intensity was quantified. *P = 0.044.Lysates from MEFs were immunoblotted for APP, BACE1, or GAPDH (loading control), and BACE1 intensity was quantified (n = 4).Amount of Aβ40 in the culture medium of immortalized MEFs (n = 3). *P = 0.045.MEFs were transfected with control siRNA or GGA3-siRNA and then immunoblotted for BACE1, GGA3, or GAPDH (loading control) (n = 6).
Figure 7Membrane fractions from 3-week-old Mgat3+/+, Mgat3−/−, and Bace1−/− mice were immunoblotted for CHL1 (upper), contactin-2 (middle), or syntaxin 6 (lower) (n = 4-5). The graphs show means ± SEM (**P < 0.01; two-way ANOVA with post hoc Tukey-Kramer test, P = 0.006 for Mgat3+/+ versus Bace1−/−, P = 0.008 for Mgat3−/− versus Bace1−/− in CHL1/syn6, P = 0.001 for Mgat3+/+ versus Bace1−/−, P = 0.002 for Mgat3−/− versus Bace1−/− in Contactin-2/syn6).Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice.
Figure 1(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.
Figure 3(B) Distribution of GPMs with mutation load lower or greater than 10%, in control and MELAS groups. The majority of GPMs are present at levels lower than 10% in either group.(C) Location of GPMs greater than 10%, in mtDNA.