Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones ( Nanogtg ; dTal1del # 1 and Nanogtg ; dTal1del # 2 ) where the binding site for NANOG distal to Tal1 has been deleted ( left ) . On the right , relative expression of Tal1 determined by RT - qPCR for each ES cell line ( ESC ; n = 9 for all three lines ) and EpiL cells without ( EpiLC ; Nanogtg and Nanogtg ; dTal1del # 1 , n = 8 ; Nanogtg ; dTal1del # 2 , n = 6 ) or with dox treatment ( EpiLC + dox ; n = 9 for all three lines ) . The genotype of the cell lines is indicated below . Values were normalized to Nanogtg ESC . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ; ANOVA with Fisher post - test . Horizontal line represents mean values and error bars standard error of the mean ( SEM ) .

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Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).


B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths . Yeast cells expressing clogger proteins ( cytochrome b2 - DHFR and cytochrome b2 ∆ 19 - DHFR ) or cytosolic DHFR for comparison under control of the galactose - inducible promoter were grown to mid - log phase on lactate medium . Ten - fold serial dilutions were dropped on lactate ( no induction ) or lactate with 0 . 5 % galactose medium ( induction ) . MTS , matrix - targeting signal .

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B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.


E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates . Cells were grown in lactate medium and clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before samples were harvested and lysed . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


A The clogger was expressed in wild type ( WT ) and ∆ rpn4 ( ∆ ) cells for 4 . 5 h with medium containing 0 . 5 % galactose . Medium was exchanged for a non - inducing lactate medium . Precursor ( pre ) and mature ( m ) forms of the b2 ∆ - DHFR clogger were visualized by Western blotting using a DHFR - specific antibody .

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A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.


G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆ rpn4 cells . The cytosolic soluble DHFR protein was expressed for control . The strains were grown in lactate medium and clogger expression was induced with 0 . 5 % galactose for 6 h .

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G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.


A Cytosolic DHFR ( control ) or the clogger protein b2 - DHFR were expressed in wild type and ∆ rpn4 cells for 4 . 5 h . The distribution of constitutively expressed Hsp104 - GFP was visualized by fluorescence microscopy . Diameter of Hsp104 - GFP - bound aggregates were quantified . . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates . Significance was assessed using two - sided , paired Student ' s t - test . P values are indicated as asterisks * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . See also Fig . EV4A for quantification of the number of aggregates per cell .

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A Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell.


B Hsp104 - GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures . SN , supernatant representing the non - bound fraction . The signals for Ssa1 and Sod1 are shown for control .

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B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.


A The mitochondrial proteins Pdb1 , Mam33 and Aim17 were co - expressed for 4 . 5 h with clogger as fusions with the red fluorescent protein RFP ( mCherry ) . Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4 . 5 h after induction of the clogger or cytosolic DHFR for control . For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17 - expressing plasmids and grown to mid - log phase in lactate medium . Clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before microscopy was perfomed . Please note that in the absence of Hsp42 and Hsp104 , no Aim17 - RFP - containing granules are formed . C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N - terminally truncated version of Mam33 - RFP ( both under control of GAL promoter ) together with constitutively expressed Hsp104 - GFP ( TPI promoter ) . The cells were grown on lactate plus 0 . 5 % galactose - containing medium for 4 . 5 h . The distribution of the fluorescent proteins was visualized by microscopy .

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A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy.


D Wild type cells expressing Hsp104 - GFP were grown on glucose or lactate medium to log phase . Cells were diluted and visualized by life cell imaging . Please note , that in glucose media , Hsp104 - GFP is initially dispersed in cells and only aggregates when cells become stationary . In contrast , Hsp104 - GFP - containing granules are frequent on lactate medium even at log phase . See movie EV1 for life cell imaging of glucose - grown cells .

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D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells.


E Clogger and Mam33 - HA were co - expressed for 4 . 5 h in wild type and ∆ hsp42 cells . Mitochondria were isolated and subjected to Western blotting to detect mature ( m ) and precursor ( pre ) forms of Mam33 - HA . The matrix protein Tim16 served as loading control .

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E Clogger and Mam33-HA were co-expressed for 4.5 h in wild type and ∆hsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33-HA. The matrix protein Tim16 served as loading control.


F The mitochondria used for E were treated with proteinase K ( PK ) to remove surface - exposed proteins . Mitochondrial membranes were lysed with NP - 40 and soluble ( S ) and aggregated ( P ) proteins were separated by centrifugation . T , total .

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F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.


G To measure the toxicity of clogger expression , clogger proteins were induced for 24 h with lactate medium containing 0 . 5 % galactose in the mutants indicated . Aliquots were removed and the number of living cells were assessed by a plating assay on glucose - containing plates . The resistance of ∆ rpn4 to clogger expression depends on Hsp104 and Hsp42 . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


( B ) Expression of CH25H in heathy donors and COVID - 19 patients . The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors , three moderate COVID - 19 patients and six severe COVID - 19 patients by scRNA - seq analysis ( Liao et al . , 2020 ) .

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(B) Expression of CH25H in heathy donors and COVID-19 patients. The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors, three moderate COVID-19 patients and six severe COVID-19 patients by scRNA-seq analysis (Liao et al., 2020).


Characterization of SARS - CoV - 2 pseudovirus . ( C ) SARS - CoV - 2 , SARS - CoV and VSV pseudovirus ( VSVG ) were concentrated by ultracentrifuge . Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS - CoV and SARS - CoV - 2 . VSV Matrix protein served as a loading control for viral particles . Black arrows indicate full length S and cleaved spike ( S2 subunit ) , respectively .

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Characterization of SARS-CoV-2 pseudovirus. (C) SARS-CoV-2, SARS-CoV and VSV pseudovirus (VSVG) were concentrated by ultracentrifuge. Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS-CoV and SARS-CoV-2. VSV Matrix protein served as a loading control for viral particles. Black arrows indicate full length S and cleaved spike (S2 subunit), respectively.


25HC inhibits membrane fusion mediated by SARS - CoV - 2 S ( F ) 293FT or Vero cells were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding SARS - CoV - 2 S ( F ) in the absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by fluorescence microscopy . White arrows indicate syncytia . Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells .

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25HC inhibits membrane fusion mediated by SARS-CoV-2 S (F) 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding SARS-CoV-2 S (F) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


25HC inhibits membrane fusion mediated by , SARS - CoV S ( G , left ) , and MERS - CoV S ( G , right ) . 293FT or Vero cells were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding , SARS - CoV S ( G , left ) , and MERS - CoV S ( G , right ) in the absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by fluorescence microscopy . Scale bar , 50 μm . White arrows indicate syncytia . Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells .

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25HC inhibits membrane fusion mediated by , SARS-CoV S (G, left), and MERS-CoV S (G, right). 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding , SARS-CoV S (G, left), and MERS-CoV S (G, right) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. Scale bar, 50 μm. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


( B - C ) 25HC depletes accessible cholesterol on the plasma membrane . ( B ) Calu - 3 cells were treated with indicated concentrations of 25HC for 1h , and subsequently incubated with ALOD4 for 30 min . Cell were washed twice with PBS , lysed , and the amounts of bound ALOD4 were examined by western blotting . ( C ) Calu - 3 cells were treated with 5 µM 25HC for 1h , and subsequently incubated with AF488 labeled ALOD4 for 30 min . Cells were washed twice and examined by fluorescence microscopy .

(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.


( D ) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC - treated cells . Calu - 3 cells were pretreated with ethanol or 5 µM 25HC for 16h . Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h . After further incubation with ALOD4 , cells were washed with PBS twice and lysed . The amount of bound ALOD4 was examined by western blotting .

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(D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus into 25HC - treated Calu - 3 cells . Cells were pretreated with ethanol or 5 µM 25HC for 16h , washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . Entry of SARS - CoV - 2 pseudovirus ( E and F ) was assessed by luciferase assay or by fluorescence microscopy at 24h post - infection .

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Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus , and MERS - CoV pseudovirus into 25HC - treated Calu - 3 cells . Cells were pretreated with ethanol or 5 µM 25HC for 16h , washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . , SARS - CoV pseudovirus ( G ) , and MERS - CoV pseudovirus ( H ) was assessed by luciferase assay or by fluorescence microscopy at 24h post - infection .

Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


( C ) SZ58 - 035 rescues surface accessible cholesterol in 25HC - treated cells . Calu - 3 cells were treated with vehicle or 40 µM SZ58 - 035 for 16 h , prior to treatment with ethanol or 2 . 5 µM 25HC for 1h . Cells were then incubated with ALOD4 for 30 min . The amount of bound ALOD4 was examined by western blotting .

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(C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting.


E Reproducibility of 2 independent experiments - correlation of observed changes in expression . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Reproducibility of 2 independent experiments - correlation of observed changes in expression. Blue line indicates the linear fit with shaded areas as confidence intervals.


A Number of target genes identified using differential expression for each assayed TF . * genes identified as essential for self - renewal of Hoxb8 - FL cells either in ( Basilico et al , 2020 ) .

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A Number of target genes identified using differential expression for each assayed TF. *genes identified as essential for self-renewal of Hoxb8-FL cells either in (Basilico et al, 2020).


A A representation of the pairwise degree of overlap in targets ( size of the circle ) and correlation in gene expression changes ( colour ) among overlapping targets for indicated TF perturbations . Red indicates positive correlations and blue negative correlations .

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A A representation of the pairwise degree of overlap in targets (size of the circle) and correlation in gene expression changes (colour) among overlapping targets for indicated TF perturbations. Red indicates positive correlations and blue negative correlations.


D Identification of target gene modules - groups of genes with common regulatory patterns by TFs . Colour indicates the fold change ( adjusted for significance ) of gene expression following each TF perturbation . Rows ( perturbed TFs ) and columns ( target genes ) are hierarchically clustered . 44 modules for target genes are shown . Below examples . Selected modules with highlighted overall regulation pattern and example genes listed below .

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D Identification of target gene modules - groups of genes with common regulatory patterns by TFs. Colour indicates the fold change (adjusted for significance) of gene expression following each TF perturbation. Rows (perturbed TFs) and columns (target genes) are hierarchically clustered. 44 modules for target genes are shown. Below examples. Selected modules with highlighted overall regulation pattern and example genes listed below.


D Changes in expression for genes co - regulated by separate Cebpa / Meis1 / Spi1 and Hoxb8 perturbations ( FDR < 0 . 1 and | log2 ( FoldChange ) | > 0 . 2 ) . The interaction row indicates changes beyond simple additive effect ( white = additive effect ) . Each gene was annotated with an interaction class ( int . class ) as explained in ( C ) , using the | log2 ( FoldChange ) | > 0 . 2 ) threshold to assign change direction . E Fractions of genes in each interaction class showed in ( D ) .

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D Changes in expression for genes co-regulated by separate Cebpa/Meis1/Spi1 and Hoxb8 perturbations (FDR <0.1 and |log2(FoldChange)| > 0.2). The interaction row indicates changes beyond simple additive effect (white = additive effect). Each gene was annotated with an interaction class (int. class) as explained in (C), using the |log2(FoldChange)| > 0.2) threshold to assign change direction. E Fractions of genes in each interaction class showed in (D).


A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8 - FL cells .

A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells.


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 .

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 .

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(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 .

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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 ) .

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(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 . (

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(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 ) .

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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 .

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(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 .

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(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 ) .

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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).