B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment . Enrichment over a random value and the p - value ( based on the hypergeometric test ) are shown ( bars corresponding to non - significant enrichment values are semi - transparent ) . Analysis was performed separately for genes up - and down - regulated .

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B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated.


C , D Genomic views of relevant TF binding to Cebpa and Gata3 loci ( supporting direct regulatory interactions identified in Figs 3A , B , C ) . The red arrow indicates a putative enhancer element , which is bound by Ebf1 and flanked by AcK27 - rich regions .

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C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.


C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8 - FL cells . Each quadrant corresponds to a set of target genes with a common regulation by Gata3 / Ebf1 - I - inhibition / inhibition , II - activation / inhibition , III - activation / activation , IV - inhibition / activation . For each group the sums of scaled expression values in mouse LK / LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression . For landscape annotation see Fig 7B andE . . Changes in expression for example genes are provided in Appendix Fig 2G - J . Blue line indicates the linear fit with shaded areas as confidence intervals .

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C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals.


E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8 - FL cells and re - expression of Ebf1 in Ebf1 - / - pre - pro B cells ( Li et al , 2018 ) . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals.


F , G Expression of the key marker genes ( F ) and Ebf1 , Pax5 , Gata3 , Cebpa factors ( G ) along pseudotime corresponding to the B - cell differentiation trajectory in human foetal liver cells , data from ( Popescu et al , 2019 ) .

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F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).


B . Still images of GFP ( syncytia ) and Propidium Iodide ( PI ) ( cell death ) at different time - points . Scale bar : 100 µm .

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B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm.


C . Quantification of U2OS - ACE2 fusion and death by time - lapse microscopy . Results are mean ± sd from 3 fields per condition .

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C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean±sd from 3 fields per condition.


D . S staining of infected U2OS - ACE2 cells analyzed by immunofluorescence . The Hoechst dye stains the nuclei . Scale bar : 40 µm .

D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm.


E . Surface S staining of infected U2OS - ACE2 cells analyzed by flow cytometry . Results are representative of at least three independent experiments .

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E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments.


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . A . TMPRSS2 increases fusion and cell mortality . Right panel : Areas of GFP + cells and PI +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . B . IFITM1 , but not IFITM2 and 3 , inhibits SARS - CoV - 2 - induced syncytia formation . Right panels : Area of GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . C . The fusion of IFITM1 + cells with U2OS - ACE2 syncytia is drastically reduced . Right panel : Area of IFITM + GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . D . IFITM1 decreases the number of infected cells . Right panel : Fraction of cells positive for S , normalized to control cells ( transduced with pQCXIP - empty vector ) .

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector ).


B . Representative images of cell - cell fusion . Scale bar : 100 µm .

B. Representative images of cell-cell fusion. Scale bar: 100 µm.


C . Quantification of GFP + areas . Results are mean ± sd from 5 independent experiments .

C. Quantification of GFP+ areas. Results are mean±sd from 5 independent experiments.


D . TMPRSS2 accelerates fusion . Cells were monitored by video microscopy and the GFP area was quantified over time . Left panel : one representative experiment . Results are mean ± sd from 3 fields per condition . Right panel : Mean ± sd from 7 independent experiments ( at 12h post transfection ) .

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D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean±sd from 3 fields per condition. Right panel: Mean±sd from 7 independent experiments (at 12h post transfection).


E . Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S - expressing 293T cells . One representative out of three independent experiment is shown .

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E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown.


A . S - expressing cells ( Donor cells ) , ACE2 - expressing cells ( Acceptor cells ) were co - transfected with TMPRSS2 , IFITM or control plasmids . Cell fusion was quantified by measuring the GFP + area after 18h . The indicated combinations were tested . The impact of IFITMs was measured in absence of TMPRSS2 ( left panel ) , in presence of TMPRSS2 in donor ( middle panel ) or acceptor cells ( right panel ) . Data are mean ± sd of 4 independent experiments . Statistical analysis : One - Way ANOVA , ns : non - significant , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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A. S-expressing cells (Donor cells), ACE2-expressing cells (Acceptor cells) were co-transfected with TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area after 18h. The indicated combinations were tested. The impact of IFITMs was measured in absence of TMPRSS2 (left panel), in presence of TMPRSS2 in donor (middle panel) or acceptor cells (right panel). Data are mean±sd of 4 independent experiments. Statistical analysis: One-Way ANOVA, ns: non-significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.


Impact of TMPRSS2 on IFITMs levels . B . TMPRSS2 does not decrease IFITM amounts measured by flow cytometry . 293T cells were transfected with the indicated IFITM plasmids , with or without TMPRSS2 , and analyzed 18h later .

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Impact of TMPRSS2 on IFITMs levels. B. TMPRSS2 does not decrease IFITM amounts measured by flow cytometry. 293T cells were transfected with the indicated IFITM plasmids, with or without TMPRSS2, and analyzed 18h later.


C . Impact of TMPRSS2 on S and ACE2 , measured by Western Blot . 293T cells were transfected with or without IFITM1 , TMPRSS2 , S or ACE2 plasmids , and analyzed 18h later .

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C. Impact of TMPRSS2 on S and ACE2, measured by Western Blot. 293T cells were transfected with or without IFITM1, TMPRSS2, S or ACE2 plasmids, and analyzed 18h later.


Impact of TMPRSS2 on S levels . D . TMPRSS2 decreases S surface levels , measured by flow cytometry . 293T cells were transfected with S plasmid , with or without TMPRSS2 , and analyzed 18h later . Murine polyclonal anti - S antibodies , one serum from a convalescent COVID - 19 patient ( sera n ° 111 ) , and two monoclonal antibodies ( anti - S1 and anti - S2 ) were tested . Data are representative of three independent experiments .

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Impact of TMPRSS2 on S levels. D. TMPRSS2 decreases S surface levels, measured by flow cytometry. 293T cells were transfected with S plasmid, with or without TMPRSS2, and analyzed 18h later. Murine polyclonal anti-S antibodies, one serum from a convalescent COVID-19 patient (sera n°111), and two monoclonal antibodies (anti-S1 and anti-S2) were tested. Data are representative of three independent experiments.


A . Identification of FASTKD2 interacting proteins by AP - MS and BioID . 26 high confidence interactors detected by both techniques are listed on the right .

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A. Identification of FASTKD2 interacting proteins by AP-MS and BioID. 26 high confidence interactors detected by both techniques are listed on the right.


B . Prey specificity graph for BioID interactome of TRUB2 protein . Prey specificity was calculated as the relative enrichment of interaction of individual preys and TRUB2 , compared to their interaction with 138 other baits ( 42 mitochondrial baits , 96 baits from other cellular compartments ) .

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B. Prey specificity graph for BioID interactome of TRUB2 protein. Prey specificity was calculated as the relative enrichment of interaction of individual preys and TRUB2, compared to their interaction with 138 other baits (42 mitochondrial baits, 96 baits from other cellular compartments).


A . Immunoblot analysis of indicated proteins in control and siRNA treated cells .

A. Immunoblot analysis of indicated proteins in control and siRNA treated cells.


B . BN - PAGE analysis of siRNA - mediated depletion shows an OXPHOS defect as revealed by subunit - specific antibodies against individual OXPHOS complexes . C . Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels . The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls . The bars represent the mean ± SEM of 3 - 7 independent experiments . P - values were calculated using paired two - tailed t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. BN-PAGE analysis of siRNA-mediated depletion shows an OXPHOS defect as revealed by subunit-specific antibodies against individual OXPHOS complexes.C. Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels. The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls. The bars represent the mean ± SEM of 3-7 independent experiments. P-values were calculated using paired two-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


D , E . Pulse - labeling mitochondrial translation experiment of the 13 mitochondria - encoded polypeptides ( seven subunits of complex I [ ND ] , three subunits of complex IV [ COX ] , two subunits of complex V [ ATP ] and one subunit of complex III [ cyt b ] ) in control and siRNA treated cells . ( E ) Detail of a mitochondrial pulse - labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells .

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D, E. Pulse-labeling mitochondrial translation experiment of the 13 mitochondria-encoded polypeptides (seven subunits of complex I [ND], three subunits of complex IV [COX], two subunits of complex V [ATP] and one subunit of complex III [cyt b]) in control and siRNA treated cells. (E) Detail of a mitochondrial pulse-labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells.


A , B . Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in control cells ( A ) Individual fractions were separated by SDS - PAGE and immunoblotted with the indicated antibodies . The migration of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in control cells (A) Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


A , B . Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in cells treated with siRNA ( B ) . Individual fractions were separated by SDS - PAGE and immunoblotted with the indicated antibodies . The migration of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in cells treated with siRNA (B). Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


C . Quantification of the levels of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) normalized to the SDHA levels . The graph represents the relative abundance of individual subunits in cells treated with specified siRNA versus controls . The bars represent the mean ± SEM .

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C. Quantification of the levels of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) normalized to the SDHA levels. The graph represents the relative abundance of individual subunits in cells treated with specified siRNA versus controls. The bars represent the mean ± SEM.


D . 2D - immunoblot analysis ( BN - PAGE / SDS - PAGE ) of pseudouridine synthase interacting proteins . The migration ( sizes in kDa ) of known protein complexes in the first dimension is indicated on the top of the blot .

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D. 2D-immunoblot analysis (BN-PAGE/SDS-PAGE) of pseudouridine synthase interacting proteins. The migration (sizes in kDa) of known protein complexes in the first dimension is indicated on the top of the blot.


B . Quantification of Δ ( the difference between the rate of deletions in CMC treated vs un - treated cells ) for individual ψ sites in control and siRNA treated cells . The bars represent the mean ± SEM of two independent experiments . P - values were calculated using a one - tailed t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. Quantification of Δ (the difference between the rate of deletions in CMC treated vs un-treated cells) for individual ψ sites in control and siRNA treated cells. The bars represent the mean ± SEM of two independent experiments. P-values were calculated using a one-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for 4 h ( as described in the Materials and Methods ) , and then incubated for 24 h ( left ) or the indicated periods of time ( right ) . Cells were fixed , stained with DAPI to visualize nuclei ( blue ) , and immunolabeled with an anti - LC3 antibody . Primary antibody was detected using an Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( green ) . Left : representative immunofluorescence images of LC3 punctae ; right : quantification of data ( LC3 - punctated cells were counted manually ) . * * * p < 0 . 001 , vs . Mtb - WT - infected condition . Scale bars , 5 µm . (

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. (


( B ) BMDMs were infected with Mtb - Δeis in the absence or presence of 3 - methyladenine ( 3 - MA ; 10 mM ) and subjected to confocal analysis as described in Figure 1A . LC3 - punctated cells were counted manually . Each condition was assayed in triplicate , and at least 250 cells were counted in each well . * * * p < 0 . 001 , vs . SC .

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(B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.


( C ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . Experimental conditions were identical to those outlined in panel A . Gel images representative of three experiments are shown .

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(C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown.


( D ) Electron micrographs of Mtb - Δeis - infected BMDMs under low ( left ) and high ( right ) magnification show the accumulation of autophagic vesicles ( black arrow , initial autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left ) , 0 . 5 µm ( right ) .

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(D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs were infected with Mtb - Δeis in the presence or absence of 3 - MA ( 10 mM ) or bafilomycin A1 ( Baf - A1 ; 100 nM ) . Gel images representative of three experiments are shown . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane ( C and E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % distilled water ( B ) , 0 . 1 % DMSO ( E ) ) .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).


( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis or Mtb - c - eis at different MOIs ( 0 . 1 , 1 and 10 ) for 18 h ( A ) or for the indicated periods of time ( B ; MOI = 10 ) . Supernatants were assessed by ELISA for levels of TNF - α and IL - 6 . Data ( A and B ) are presented as the mean ± SD of five experiments .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments.


( C and D ) BMDMs were stimulated with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 30 min . Cells were then incubated with 10 µM DHE or 5 µM DCFH - DA for 15 min , washed thoroughly , and immediately analyzed for superoxide or H2O2 production by flow cytometry ( C , Left ) . Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry ( D , top ) . Quantitative analysis of ROS generation ( C , right ; D , bottom ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A - C ) BMDMs were infected with Mtb - Δeis ( MOI = 10 ) for 18 h in the presence or absence of DPI ( 10 µM ) , NAC ( 20 mM ) , catalase ( Cat , 1 mU / mL ) , or tiron ( 5 mM ) . ( A ) Representative immunofluorescence images ( top ) ; percentage of endogenous LC3 - punctated cells ( bottom )

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(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)


( B ) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β - actin . Gel images are representative of three experiments . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane .

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(B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane.


( C ) Experimental conditions were identical to those outlined in Figure 3A . Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data represent the mean ± SD of five experiments .

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(C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments.


( D - F ) BMDMs from WT and NOX2 KO mice were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 18 h . ( D ) Numbers of LC3 - punctated cells ( counted manually ) are shown ( at least 250 cells were counted in each well ) .

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(D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs from WT and NOX2 KO mice were infected with Mtb - Δeis for 18 h . Gel images representative of three experiments are shown .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown.


( F ) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data are presented as the mean ± SD of at least three separate experiments , each performed in triplicate . * * * p < 0 . 001 , vs . SC ( A and C ) ; WT mice ( D and F ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for the indicated periods of time , washed to remove unbound mycobacteria , and then incubated in complete DMEM at 37 ° C in 5 % CO2 . Cell viability was assessed by PI staining and then examined by fluorescence microscopy . ( B and C ) Experimental conditions were identical to those outlined in panel A . BMDMs were infected with the three strains of mycobacteria for 36 h .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h.


B ) Apoptosis was assessed using a TUNEL / apoptosis detection kit , according to the manufacturer ' s protocol . Cells were then examined under a laser - scanning confocal microscope ( LSM 510 ; Zeiss ) . Percentages of TUNEL - positive , PI - positive , and TUNEL - / PI - double - positive cells were calculated . Data are representative of three separate experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.


( C ) Morphological changes in BMDMs infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis at MOIs of 1 and 10 . Representative images are shown . Scale bars : 50 µm ( low magnification ) , 20 µm ( high magnification ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification).


( D ) Macrophages were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis in the presence or absence of zVAD - fmk ( 20 µM ) or 3 - MA ( 10 mM ) . Staurosporine ( STS ; 500 nM ) was used as a positive control . After 36 h , cells were stained with PI and then examined by fluorescence microscopy . Data are presented as the mean ± SD of at least three separate experiments , each performed in duplicate . * p < 0 . 05 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( A and B ) ; Mtb - Δeis - infected condition without inhibitors ( D ) . UI , uninfected .

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(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.


( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 1 ) for the indicated periods of time , and then subjected to Western blot analysis using Abs raised to p - ERK1 / 2 , p - p38 , p - JNK , and β - actin . Data shown are representative of three independent experiments that all yielded similar results ( A ) . Expression of phospho - MAPK / β - actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically ( B ) .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B).


( C and D ) BMDMs were pretreated with U0126 ( 5 , 10 , 20 µM ) , SB203580 ( SB ; 1 , 5 , 10 µM ) , or SP600125 ( SP ; 5 , 10 , 20 µM ) for 45 min , and then infected with Mtb - Δeis for 30 min ( C ) or 36 h ( D ) . ( C ) Cells were then incubated with DHE for 15 min , washed rapidly and thoroughly , and analyzed immediately for superoxide levels by flow cytometry . Quantitative DHE fluorescence data represent the mean ± SD of four experiments . ( D ) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy . (

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (


( E ) Raw264 . 7 cells were transfected with siRNA specific for JNK1 ( siJNK ) or a non - specific control siRNA ( siNS ) . At 24 h after transfection , cells were infected with Mtb - Δeis for 36 h . Cell death was then assessed by PI staining , and then examined by flow cytometry . Transfection efficiency was assessed by RT - PCR ( inset ) . Data represent the mean ± SD of five random fields and are representative of three independent experiments ( D and E ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( B ) ; SC ( C and D ) ; siNS ( E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).


C57BL / 6 mice were challenged , by aerosol , with 10 - 30 CFU Mtb - WT , Mtb - Δeis , or Mtb - c - eis and sacrificed 4 weeks post - infection . ( A ) High - and low - magnification electron micrographs of lung tissue sections from mice infected with Mtb - Δeis show accumulation of autophagic vesicles ( black arrow , bacteria in autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left upper ) , 0 . 5 µm ( right ) . Numbers of autophagic vacuoles per cell in each TEM section ( left lower ) ( mean ± SD ; n = 50 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50).


( B ) Quantitative RT - PCR analysis of lung tissue from Mtb - WT - , Mtb - Δeis - , and Mtb - c - eis - infected mice . Total RNA was extracted from paraffin - embedded lung tissue sections , as described in the Materials and Methods .

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(B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods.


( C ) To assess in vivo cell death , bronchoalveolar lavage fluid cells from Mtb - WT - , Mtb - Δeis - , and Mtb - c - eis - infected mice were subjected to PI staining , and analyzed by flow cytometry . Data are presented as the mean ± SEM ( n = 4 ) .

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(C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4).


( D ) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb - WT , Mtb - Δeis , or Mtb - c - eis . Data are presented as log10 CFU ± SEM ( n = 4 ) .

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(D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4).


( E ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis and then analyzed by CFU assay . CFU data represent the mean ± SD of four individual experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A ) Intracellular superoxide production was analyzed by flow cytometric analysis . BMDMs were infected with Mtb - Δeis ( MOI = 10 ) in the presence or absence of recombinant Eis protein ( rEis ; 5 , 10 , 20 µg / mL ) or 30 kDa Mtb antigen ( 30 k ; 5 , 10 , 20 µg / mL ) . Upper , representative flow cytometric analysis ; lower , quantitation of superoxide generation .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation.


( B ) THP - 1 cells were transfected with mock , Eis - WT , or Eis - ΔAT constructs , and infected with Mtb - Δeis for 30 min . Cells were stained with DHE ( for superoxide ) or DCFH - DA ( for H2O2 ) and subjected to flow cytometric analysis . Inset , transfection efficiency .

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(B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency.


( C and D ) Experimental conditions were identical to those outlined in panels A and B , respectively . Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF - α and IL - 6 . Data are presented as the mean ± SD of five experiments .

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(C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments.


( E and F ) THP - 1 cells transfected with mock , Eis - WT , or Eis - ΔAT constructs were pretreated with SP600125 ( SP ; 20 µM ) or SB203580 ( SB ; 5 µM ) for 45 min before infection with Mtb - Δeis for 30 min ( E ) or 18 h ( F ) . E , Intracellular superoxide production was analyzed by flow cytometric analysis . F , ELISA analysis for TNF - α and IL - 6 levels . Data are presented as the mean ± SD of three experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . SC ( A , C , E , and F ) ; mock control ( B and D ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).


( A ) Crystal structure of a dynamin monomer ( PDB ID : 3SNH ) and corresponding color - coded linear domain architecture of dynamin . The PHD is shown in both the closed ( 3SNH , solution structure ) and open ( 3ZYS , cryo - EM reconstruction of membrane - bound dynamin ) states . The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads . The positions of the L1SP and L2SP loops are identified by red arrowheads .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Crystal structure of a dynamin monomer (PDB ID: 3SNH) and corresponding color-coded linear domain architecture of dynamin. The PHD is shown in both the closed (3SNH, solution structure) and open (3ZYS, cryo-EM reconstruction of membrane-bound dynamin) states. The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads. The positions of the L1SP and L2SP loops are identified by red arrowheads.


Changes in HDX behavior for dynamin analyzed ( B ) in solution the presence GMPPCP , ( C ) in solution in the presence of GDP • AlF4 - , ( D ) membrane - bound , or ( E ) membrane - bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers . Color coding ( see scale below ) shows the extent of solvent exchange relative to apo dynamin in solution . All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics ( red being the highest D2O uptake ) and cool colors representing decreased conformational dynamics ( blue being the lowest D2O uptake ) . Grey indicates no statistically significant change between the two states . Quantitative data on the individual peptides used to generate this map are provided in Appendix ( Figure S1 - S4 ) .

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Changes in HDX behavior for dynamin analyzed (B) in solution the presence GMPPCP, (C) in solution in the presence of GDP•AlF4-, (D) membrane-bound, or (E) membrane-bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers. Color coding (see scale below) shows the extent of solvent exchange relative to apo dynamin in solution. All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics (red being the highest D2O uptake) and cool colors representing decreased conformational dynamics (blue being the lowest D2O uptake). Grey indicates no statistically significant change between the two states. Quantitative data on the individual peptides used to generate this map are provided in Appendix (Figure S1-S4).


( A ) Changes in solvent exchange superimposed onto the structure ( PDB ID : 2SNH ) of the G domain or ( B ) the stalk / PHD of dynamin in the GMPPCP or lipid bound states , respectively . Key structural elements are identified and the color - coding shows the magnitude of solvent exchange relative to the apo state ( see scale below ) .

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(A) Changes in solvent exchange superimposed onto the structure (PDB ID: 2SNH) of the G domain or (B) the stalk/PHD of dynamin in the GMPPCP or lipid bound states, respectively. Key structural elements are identified and the color-coding shows the magnitude of solvent exchange relative to the apo state (see scale below).


( C ) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP • AlF4 - - bound states . The peptides are mapped to secondary structure elements on the polypeptide sequence . Differences in kinetics of solvent exchange of select peptides measured ( D ) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes ( E ) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP . HDX data are presented as an average of three independent triplicates and color coded as in Figure 1 .

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(C) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP•AlF4--bound states. The peptides are mapped to secondary structure elements on the polypeptide sequence. Differences in kinetics of solvent exchange of select peptides measured (D) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes (E) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP. HDX data are presented as an average of three independent triplicates and color coded as in Figure 1.


( A ) Design of a functional dynamin mutant to probe PHD stalk interactions . Minimal structure of the PHD and the PHD - Stalk interface showing the location of important residues discussed in this study . The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans ( W525 and W542 ) in the PHD . The distance between two introduced cysteines ( S357C and native C602 ) used to construct a double cysteine Dyn1 is also shown .

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(A) Design of a functional dynamin mutant to probe PHD stalk interactions. Minimal structure of the PHD and the PHD-Stalk interface showing the location of important residues discussed in this study. The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans (W525 and W542) in the PHD. The distance between two introduced cysteines (S357C and native C602) used to construct a double cysteine Dyn1 is also shown.


( B ) Comparison of emission spectra of various IAEDANS - labeled Dyn1 RCL variants ( D352C , Y354C , S357C , I365C , H367C ) , upon Trp excitation at 295 nm .

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(B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm.


( C ) FRET transfer upon excitation is contributed by two neighboring Trps ( W525 and W542 ) ; Substitution by Phe resulted in significant loss of FRET .

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(C) FRET transfer upon excitation is contributed by two neighboring Trps (W525 and W542); Substitution by Phe resulted in significant loss of FRET.


( D ) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4 , 5P2 ( ex = 295 ) . The arrow indicates addition of lipids .

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(D) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4,5P2 (ex=295). The arrow indicates addition of lipids.


( B ) Histogram shows basal and assembly - stimulated GTPase activities of non - crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT . Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C / S607C ( Dyn1CC ) with the bifunctional crosslinker , MTS - 2 - MTS , to yield Dyn1Closed , which migrates more slowly on SDS - PAGE , as well as liposome binding of Dyn1CC compared to Dyn1Closed ( Bound , B ; Unbound , UB ) .

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(B) Histogram shows basal and assembly-stimulated GTPase activities of non-crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT. Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C/S607C (Dyn1CC) with the bifunctional crosslinker, MTS-2-MTS, to yield Dyn1Closed, which migrates more slowly on SDS-PAGE, as well as liposome binding of Dyn1CC compared to Dyn1Closed (Bound, B; Unbound, UB).


( C ) Ability of Dyn1WT , Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates .

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(C) Ability of Dyn1WT, Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates.


( D ) Size exclusion chromatographic profiles of Dyn1CC , Dyn1Closed at room temperature or after incubation for 10 min at 37 ° C .

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(D) Size exclusion chromatographic profiles of Dyn1CC, Dyn1Closed at room temperature or after incubation for 10 min at 37°C.


( E ) Oligomeric state of Dyn1ΔΔ at room temperature .

(E) Oligomeric state of Dyn1ΔΔ at room temperature.


( F ) Negative - stain electron micrographs of Dyn1WT , Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes . Scale bar = 200

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(F) Negative-stain electron micrographs of Dyn1WT, Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes. Scale bar = 200 nm


( G ) Curvature dependent liposome - stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ .

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(G) Curvature dependent liposome-stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ.


( H ) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates .

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(H) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates.


( A ) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L .

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(A) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L.


( B ) Temperature sensitive membrane fission activity of Dyn1WT , Dyn2WT and Dyn2S619L measured using SUPER templates .

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(B) Temperature sensitive membrane fission activity of Dyn1WT, Dyn2WT and Dyn2S619L measured using SUPER templates.


( C ) Detection of Dyn2 PHD - stalk interactions by FRET using IAEDANS - labeled Dyn2L354C .

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(C) Detection of Dyn2 PHD-stalk interactions by FRET using IAEDANS-labeled Dyn2L354C.


( D ) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C - IAEDANS and Dyn2L354C - IAEDANS .

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(D) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C-IAEDANS and Dyn2L354C-IAEDANS.


( E ) Differential temperature dependence of PHD - stalk FRET for WT and S619L mutant Dyn2IAEDANS . Data are presented as relative FRET distances assuming a single donor / acceptor pair .

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(E) Differential temperature dependence of PHD-stalk FRET for WT and S619L mutant Dyn2IAEDANS. Data are presented as relative FRET distances assuming a single donor/acceptor pair.


( F ) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions . Yellow coloring indicates a significant increase in HDX . Quantitative data on the individual peptides used to generate this map are provided in ( Appendix Fig . S5 ) .

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(F) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions. Yellow coloring indicates a significant increase in HDX. Quantitative data on the individual peptides used to generate this map are provided in (Appendix Fig. S5).


( A , B ) Transferrin receptor ( TfnR ) uptake , a measure of CME , shown as % uptake of total steady - state surface TfnR for control H1299 cells , and H1299 cells stabily expressing low amounts of siRNA - resistant Dyn2WT - EGFP ( A ) or Dyn2S619L - EGFP ( B ) with or without treatment of siRNA - directed towards endogenous Dyn2 .

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(A,B) Transferrin receptor (TfnR) uptake, a measure of CME, shown as % uptake of total steady-state surface TfnR for control H1299 cells, and H1299 cells stabily expressing low amounts of siRNA-resistant Dyn2WT-EGFP (A) or Dyn2S619L-EGFP (B) with or without treatment of siRNA-directed towards endogenous Dyn2.


( C ) Fixed cell TIRF - M images of Dyn2WT - EGFP or Dyn2S619L - EGFP expressing H1299 cells with or without Dyn2 - siRNA treatment showing co - localization with clathrin light chain immunostained rabbit anti - CLC and Alexa 547 - conjugated secondary antibodies .

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(C) Fixed cell TIRF-M images of Dyn2WT-EGFP or Dyn2S619L-EGFP expressing H1299 cells with or without Dyn2-siRNA treatment showing co-localization with clathrin light chain immunostained rabbit anti-CLC and Alexa 547-conjugated secondary antibodies.


( D ) Percentage of Dyn2 - EGFP positive clathrin coated pits detected by live cell TIRF - M and automated master / slave image analysis ( Aguet et al , 2013 ) .

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(D) Percentage of Dyn2-EGFP positive clathrin coated pits detected by live cell TIRF-M and automated master/slave image analysis (Aguet et al, 2013).


( E ) Lifetime analysis of CCPs which are positive for EGFP - Dyn2WT or EGFP - Dyn2S619L with or without siRNA - mediated specific knockdown of endogenous Dyn2 .

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(E) Lifetime analysis of CCPs which are positive for EGFP-Dyn2WT or EGFP-Dyn2S619L with or without siRNA-mediated specific knockdown of endogenous Dyn2.


( A ) Colocalization of LacCer and LysoTracker ( LyTr ) in different CTR and LSD patient fibroblasts . Mander ´ s coefficients were calculated using the Fiji JACoP plugin . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s ( A , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(A) Colocalization of LacCer and LysoTracker (LyTr) in different CTR and LSD patient fibroblasts. Mander´s coefficients were calculated using the Fiji JACoP plugin. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's (A, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( B - C ) Confocal images ( B ) and statistical analysis ( C ) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 16h ) . ( D - E ) Confocal images ( D ) and statistical analysis ( E ) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 48h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s C , E , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(B-C) Confocal images (B) and statistical analysis (C) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts, treated with TPC2-A1-P (30 µM, 16h). (D-E) Confocal images (D) and statistical analysis (E) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts, treated with TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's C, E, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( F - G ) Confocal images and statistical analysis showing LacCer / LyTr colocalization in MLIV patient fibroblasts which were mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 16h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s G , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(F-G) Confocal images and statistical analysis showing LacCer/LyTr colocalization in MLIV patient fibroblasts which were mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector and treated with either DMSO or TPC2-A1-P (30 µM, 16h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's G, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( H - I ) Ca2 + chelation ( BAPTA - AM ) dose - dependently impairs LacCer trafficking in CTR fibroblasts . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s I ) multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(H-I) Ca2+ chelation (BAPTA-AM) dose-dependently impairs LacCer trafficking in CTR fibroblasts. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's I) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( J - K ) Confocal images ( J ) and statistical analysis ( K ) of NPC1 patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO or TPC2 - A1 - P ( 30 µM ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , Tukey ' s ( K ) multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(J-K) Confocal images (J) and statistical analysis (K) of NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, Tukey's (K) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( A - B ) Confocal images ( A ) and statistical analysis ( B ) of cholesterol accumulation in human CTR , MLIV , JNCL , and NPC1 fibroblasts . Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts , but not for JNCL fibroblasts . The images show filipin staining to visualize cholesterol accumulation and TO - PRO3 as nuclear staining . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test ( B , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(A-B) Confocal images (A) and statistical analysis (B) of cholesterol accumulation in human CTR, MLIV, JNCL, and NPC1 fibroblasts. Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts, but not for JNCL fibroblasts. The images show filipin staining to visualize cholesterol accumulation and TO-PRO3 as nuclear staining. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test (B, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( C - D ) TPC2 - A1 - P ( 30 µM , 48h ) rescued NPC1 and MLIV cholesterol accumulation . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test D , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(C-D) TPC2-A1-P (30 µM, 48h) rescued NPC1 and MLIV cholesterol accumulation. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test D, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images ( E - F ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 48h ) .

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Confocal images (E-F) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h).


statistical analysis ( G ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 48h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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statistical analysis (G) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) two - tailed Student ' s t - test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells) two-tailed Student's t-test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human NPC1 patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO or TPC2 - A1 - P ( 30 µM ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; two - tailed Student ' s t - test K ) . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images and statistical analysis of human NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); two-tailed Student's t-test K). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Statistics ( L ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test , L ) * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Statistics (L) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test , L) *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


electron microscopy images ( M ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells .

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electron microscopy images (M) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells.


( A ) Confocal images of CTR and JNCL fibroblasts . Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength , respectively , corresponding to the lipofuscin autofluorescence spectrum . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) following cell cycle arrest ( 2h mitomycin C treatment ) . ( B ) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength ( used for LAMP1 staining ) .

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(A) Confocal images of CTR and JNCL fibroblasts. Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength, respectively, corresponding to the lipofuscin autofluorescence spectrum. Cells were treated with DMSO or TPC2-A1-P (30 µM) following cell cycle arrest (2h mitomycin C treatment). (B) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength (used for LAMP1 staining).


( C ) Mean autofluorescence intensity in LAMP1 + area . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(C) Mean autofluorescence intensity in LAMP1+ area. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Mean intensity of shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean intensity of shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images of Gb3 accumulation stained with shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest .

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Confocal images of Gb3 accumulation stained with shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( C - D ) Western blot analysis of cathepsin B ( CtsB ) in CTR and MCOLN1IVS3 - 2A > G neurons treated with TPC2 - A1 - P ( 30 µM ) or DMSO . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; two - tailed Student ' s t - test ( C ) . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (C-D) Western blot analysis of cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons treated with TPC2-A1-P (30 µM) or DMSO. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker ( LyTr ) . Endolysosomal expansion was observed in MCOLN1IVS3 - 2A > G , CLN3D416G and CLN3ΔEx4 - 7 neurons , which was ameliorated by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker (LyTr). Endolysosomal expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( H - I ) Electron microscopy analysis of neuronal rosettes ( neuronal progenitor cells , NPC ) treated with DMSO or TPC2 - A1 - P . TPC2 - A1 - P treatment significantly decreased the number of inclusion bodies ( black arrow heads ) in MCOLN1IVS3 - 2A > G . CLN3D416G and CLN3ΔEx4 - 7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies . However , CLN3ΔEx4 - 7 NPC showed significantly more mitochondria with aberrant cristae numbers ( white arrow heads ) , a phenotype which was rescued by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (H-I) Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies. However, CLN3ΔEx4-7 NPC showed significantly more mitochondria with aberrant cristae numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( A ) Confocal images of plasma membrane ( PM ) LAMP1 immunofluorescence in CTR fibroblasts . LAMP1 on the PM is expressed as fold - change relative to DMSO - treated cells . ( B ) Statistical analysis of lysosomal exocytosis data as shown in A . ( C ) Lysosomal exocytosis in CTR , MLIV , NPC1 and JNCL human fibroblasts . PM - localized LAMP1 was measured by flow cytometry , expressed as % of CTR DMSO - treated cells . Ionomycin in A - C ( 4 µM ; 10 min treatment ) was used as positive control . TPC2 - A1 - P and ML - SA1 ( 30 µM , each ; 90 min treatment in A - C ) . Statistics ( B - C ) : Shown are mean values ± SEM . n > 3 for each tested condition ( in B each dot represents an imaged frame containing several cells , in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1 * 104 events ) ; two - way ANOVA , post hoc Dunnett ' s ( B ) or Tukey ' s ( C ) multiple comparisons test , * p - value < 0 . 05 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Confocal images of plasma membrane (PM) LAMP1 immunofluorescence in CTR fibroblasts. LAMP1 on the PM is expressed as fold-change relative to DMSO-treated cells. (B) Statistical analysis of lysosomal exocytosis data as shown in A. (C) Lysosomal exocytosis in CTR, MLIV, NPC1 and JNCL human fibroblasts. PM-localized LAMP1 was measured by flow cytometry, expressed as % of CTR DMSO-treated cells. Ionomycin in A-C (4 µM; 10 min treatment) was used as positive control. TPC2-A1-P and ML-SA1 (30 µM, each; 90 min treatment in A-C). Statistics (B-C): Shown are mean values ± SEM. n > 3 for each tested condition (in B each dot represents an imaged frame containing several cells, in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1*104 events); two-way ANOVA, post hoc Dunnett's (B) or Tukey's (C) multiple comparisons test, *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001.


Immunoblot analysis of endogenous LC3 ( LC3I - II ) following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patientfibroblasts .

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Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts.


analysis of endogenous LC3 following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patientfibroblasts . Graphs show densitometry of LC3II bands normalized to actin . Statistics ( G , Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - tailed Student ' s t - test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

analysis of endogenous LC3 following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts. Graphs show densitometry of LC3II bands normalized to actin. Statistics (G, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


( H ) Immunoblot analysis of endogenous LC3 ( LC3I - II ) following TPC2 - A1 - P ( 30 µM ) or DMSO treatment , under fed ( complete Neurobasal / B27 ) or starvation ( DMEM / F12 free ) conditions in CTR and MLIV iPSC derived cortical neurons . Graphs show densitometry of LC3II bands normalized to actin . Statistics H , Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - tailed Student ' s t - test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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(H) Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P (30 µM) or DMSO treatment, under fed (complete Neurobasal/B27) or starvation (DMEM/F12 free) conditions in CTR and MLIV iPSC derived cortical neurons. Graphs show densitometry of LC3II bands normalized to actin. Statistics H, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


Immunoblot analysis of endogenous SQSTM1 ( P62 ) upon TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patient fibroblasts . Statistics J ) : Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - tailed Student ' s t - test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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Immunoblot analysis of endogenous SQSTM1 (P62) upon TPC2-A1-P or ML-SA1 (30 µM, each) treatment, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patient fibroblasts. Statistics J): Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


( D ) A cDNA array was used to map TPC2 transcripts in the human brain .

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(D) A cDNA array was used to map TPC2 transcripts in the human brain.


( A ) TPC2 - A1 - P was injected intravenously , and mice sacrificed at the indicated time . TPC2 - A1 - P was measured in plasma and brain by LC - MS / MS . TPC2 - A1 - P was rapidly eliminated , being undetectable by 240 min .

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(A) TPC2-A1-P was injected intravenously, and mice sacrificed at the indicated time. TPC2-A1-P was measured in plasma and brain by LC-MS/MS. TPC2-A1-P was rapidly eliminated, being undetectable by 240 min.


( B ) Elimination rate constants were determined using a semi - log plot . A two - phase decay model could fit the obtained data points in plasma , while a one - phase decay model fit the data in brain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Elimination rate constants were determined using a semi-log plot. A two-phase decay model could fit the obtained data points in plasma, while a one-phase decay model fit the data in brain.


( C ) Brain [ TPC2 - A1 - P ] was simulated for various injected doses . 20 mg / kg TPC2 - A1 - P was chosen to avoid off - target activity while providing a therapeutic dose for > 20 min .

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(C) Brain [TPC2-A1-P] was simulated for various injected doses. 20 mg/kg TPC2-A1-P was chosen to avoid off-target activity while providing a therapeutic dose for >20 min.


A . Left column , an active summer squirrel ; right column , a torpid squirrel .

A. Left column, an active summer squirrel; right column, a torpid squirrel.