( 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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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 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 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 ) .

O O O O O O O O O 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 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

(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 .

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

(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 .

(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 .

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

(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 ) .

O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O 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) 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 .

(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 .

(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 .

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

(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 .

(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 .

(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 .

(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 ) .

(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 .

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 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 B-MEASURED_VAR 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 O O O O O O O O

(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 .

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 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 O O O O O

(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 .

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

(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 .

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

(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 .

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 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 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 O O O O O O O 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-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 .

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

(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 ) .

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

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 .

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

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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

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 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 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 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

(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 .

(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 .

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(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.


B . The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin ( H & amp ; amp ; E ) stained sections ( scale bar 90 µm ) .

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B. The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&amp;E) stained sections (scale bar 90 µm).


C . Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret ' s diameter .

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C. Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter.


D . Average ± SD of minimum Feret ' s diameter in quadriceps ( p = 0 . 26 ) and tibialis anterior ( p = 0 . 33 ) muscles is not significantly different between summer and hibernation .

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D. Average ± SD of minimum Feret's diameter in quadriceps (p = 0.26) and tibialis anterior (p = 0.33) muscles is not significantly different between summer and hibernation.


A . Western blot of quadriceps muscle from summer active ( S ) and hibernating ( H ) squirrels using antibodies against the proteins indicated . An increased abundance of P − Foxo3a ( serine − 253 ) is accompanied by a decrease in P − Akt ( serine − 478 ) . Corresponding densitometry of P − Akt and P − Foxo3a as a function of total Akt and Foxo3a .

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A. Western blot of quadriceps muscle from summer active (S) and hibernating (H) squirrels using antibodies against the proteins indicated. An increased abundance of P−Foxo3a (serine−253) is accompanied by a decrease in P−Akt (serine−478). Corresponding densitometry of P−Akt and P−Foxo3a as a function of total Akt and Foxo3a.


B . Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation .

B. Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation.


C . Western blots analysis of mTOR downstream targets shows significant upregulation of P − P70 and P − 4E − BP1 during hibernation .

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C. Western blots analysis of mTOR downstream targets shows significant upregulation of P−P70 and P−4E−BP1 during hibernation.


A . Western blot analyses and densitometry of quadriceps muscles from non − hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1 . Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation .

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A. Western blot analyses and densitometry of quadriceps muscles from non−hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation.


B . Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation . Densitometric analysis of P ‐ Foxo3a as a function of Foxo3a .

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B. Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P‐Foxo3a as a function of Foxo3a.


A . The morphology of tibialis anterior muscle as revealed by haematoxylin - eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers ( scale bar 90 µm ) .

A.The morphology of tibialis anterior muscle as revealed by haematoxylin-eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers (scale bar 90 µm).


B . Percentage distribution and mean minimum Feret ' s diameter in tibialis anterior muscle ( p = 0 . 02 ) .

B. Percentage distribution and mean minimum Feret's diameter in tibialis anterior muscle (p = 0.02).


C . Increased levels of phosphorylated Akt ( S478 ) in skeletal muscles of sgk1 − / − mice .

C. Increased levels of phosphorylated Akt (S478) in skeletal muscles of sgk1−/− mice.


D . sgk1 − / − mice exhibit decreased running distance after 36 days of exposure to a running wheel , ( p = 0 . 01 ; n = 6 animals per group ) .

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D. sgk1−/− mice exhibit decreased running distance after 36 days of exposure to a running wheel, (p = 0.01; n = 6 animals per group).


E . Isometric force from soleus muscles of WT and sgk1 - / - mice during repetitive electrical stimulations for 500 ms . Amplitudes of isometric force from soleus muscles at two stimulation frequencies . Specific force is plotted in all cases . Mean values ± SD from n = 9 ( sgk1 − / − ) and n = 10 ( WT ) independent animals ( p = 0 . 01 ) .

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E. Isometric force from soleus muscles of WT and sgk1-/- mice during repetitive electrical stimulations for 500 ms. Amplitudes of isometric force from soleus muscles at two stimulation frequencies. Specific force is plotted in all cases. Mean values ± SD from n = 9 (sgk1−/−) and n = 10 (WT) independent animals (p = 0.01).


A . H & amp ; amp ; E staining of tibialis anterior sections from 2 ‐ month old control and transgenic mice shows no differences ( scale bar 90 µm ) .

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A. H&amp;E staining of tibialis anterior sections from 2‐month old control and transgenic mice shows no differences (scale bar 90 µm).


B . Morphometric analyses of minimum Feret ' s diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size .

B. Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size.


C . Western blots and densitometric analysis of muscle from transgenic and control mice . Downstream targets of the mTOR signalling cascade ( p70S6K and 4EBP1 ) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice .

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C. Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice.


D . Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice ( n = 4 ) show an increase of phosphorylation at S315 and T32 .

D. Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n = 4) show an increase of phosphorylation at S315 and T32.


A . H & amp ; amp ; E staining of tibialis anterior sections reveals no changes in muscle architecture ( scale bar 90 µm ) .

A. H&amp;E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90 µm).


B . Morphometric analysis demonstrates a decrease in muscle fiber size of the wild − type control mice compared with sgk1tg transgenic littermates ( p = 0 . 0015 ) .

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B. Morphometric analysis demonstrates a decrease in muscle fiber size of the wild−type control mice compared with sgk1tg transgenic littermates (p = 0.0015).


C . Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg .

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C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.


A . Transfection of constitutively active Sgk1 ( CA ) into immobilized tibialis anterior muscle ( green ) reveals increased fiber size diameter when compared to control , EGFP only transfected muscle fibers of immobilized tibialis anterior muscles ( 100 µm ) . Laminin γ − 1 staining ( red ) outlines the basement membrane and blue staining marks nuclei ( DAPI ) .

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A. Transfection of constitutively active Sgk1 (CA) into immobilized tibialis anterior muscle (green) reveals increased fiber size diameter when compared to control, EGFP only transfected muscle fibers of immobilized tibialis anterior muscles (100 µm). Laminin γ−1 staining (red) outlines the basement membrane and blue staining marks nuclei (DAPI).


B . Representation of the minimum Feret ' s diameter average ± SD ( control and immobilized ; n = 9 - 10 per group ; 800 - 950 fibers were measured ) ( * p = 0 . 029 , * p = 0 . 034 , * p = 0 . 047 , p = 0 . 43 , respectively ) .

B. Representation of the minimum Feret's diameter average ± SD (control and immobilized; n = 9-10 per group; 800-950 fibers were measured) (*p = 0.029, *p = 0.034, *p = 0.047, p = 0.43, respectively).


C . Percentage distribution of the minimum Feret ' s diameter of tibialis anterior muscle immobilized and transfected with eGFP ( GFP ) , wild − type Sgk1 ( WT ) , kinase dead Sgk1 ( KD ) and constitutively active Sgk1 ( CA ) compared to non − immobilized control TA muscle ( black dotted line ) .

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C. Percentage distribution of the minimum Feret's diameter of tibialis anterior muscle immobilized and transfected with eGFP (GFP), wild−type Sgk1 (WT), kinase dead Sgk1 (KD) and constitutively active Sgk1 (CA) compared to non−immobilized control TA muscle (black dotted line).


A - F Immunostaining of primary HL with H2A . B ( brown , indicated by black arrows ) with a nuclear counterstain ( blue ) . Human testis ( positive control ) ( A ) , Human tonsils ( negative control ) ( B ) , Nodular sclerosis classical cHL , double staining with CD15 ( pink ) to identify HRS cells and H2A . B ( brown ) ( C ) , Mixed cellularity cHL ( D ) , Lymphocyte rich cHL ( E ) , Nodular sclerosis HL ( F ) . Scale bar for panels A , B , D - F is 50 µm ; scale bar for panel C is 15 µm .

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A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm.


A Relative mRNA expression levels of H2A . B in L1236 , L428 , HDLM2 HL cell lines and Germinal centre B lymphocytes ( GC ) compared to the Ramos B lymphocyte cell line . The results are presented as the mean ± SD of three biological replicates .

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A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates.


D H2A . B interacts with Pol I . Western blot showing co - immunoprecipitation of RPA194 with H2A . B . The Pol I subunit RPA194 was immunoprecipitated from L1236 ( left panel ) and L428 ( right panel ) nuclear extracts . The pull - down by RPA194 antibody ( IP ) , pull - down by empty beads or IgG isotype control , and 1 % input protein were loaded for Western blot detection using anti - H2A . B and anti - RPA194 antibodies .

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D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.


E H2A . B interacts with RPA194 in live cells . Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells ( left panel ) , and L428 cells ( right panel ) . The samples were prepared using pull - down with streptavidin beads from the cell lysate of the empty vector , BirA * - H2A . B and H2A . B - BirA * transduced cells , which were preincubated with 50 µM biotin for 24 h . The positive control was loaded as 1 % input .

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E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells (left panel), and L428 cells (right panel). The samples were prepared using pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin for 24 h. The positive control was loaded as 1% input.


B Cell proliferation assay in the presence ( shNTC ) or during depletion of H2A . B ( shH2A . B ) . The results are presented as mean ± SD for five biological replicates . The significance of the difference was determined by Student ' s t test and denoted as ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.


E Genome browser screen shots of gene examples ( RPs RPLP1 and RPL23A , and HDAC5 and TAF10 ) that contain H2A . B at their promoters identified by the CUT & RUN peak calling and whose expression decreased significantly following H2A . B knockdown in the RNA - seq analysis . Green and purple indicates L1236 and L428cells , respectively .

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E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.


B Differential exon usage of the CD44 gene . C . Differential exon usage of the RPL17 gene .

B Differential exon usage of the CD44 gene. C. Differential exon usage of the RPL17 gene.


D , E Two examples show increased accessibility at the promoter peak ( NXF1 ) ( D ) and decreased accessibility at the peak across the intron - exon boundary ( KDM4A ) ( E ) , respectively .

D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.


Module - trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples . Gene modules ( y axis ) are denoted by an arbitrary color name . Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype / disease stage ( x axis ) and p values . A value of 1 ( red ) quantifies the strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) the strongest negative correlation ( genes are downregulated ) , and 0 ( white ) no correlation .

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Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.


Microarray analysis was performed following doxycycline - regulated specific AR - V7 depletion ( using a tet - plKO backbone ) in 22Rv1 PC cells compared to doxycycline - treated shGFP controls . The genes that were significantly regulated by shAR - V7 ( in either direction , p value < 0 . 05 ) were distributed among the gene modules defined by WGCNA in panel A . Upregulated genes ( red ) are those in which expression decreased following AR - V7 depletion and conversely downregulated genes ( blue ) are those that increased following AR - V7 depletion .

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Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.


Hornberg et al . , 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases , grouped by their relative levels of AR - VS , mainly AR - V7 . ( High - levels of AR - V7 ( top quartile ) or lower levels of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as the mean ± s . e . m . . Non - parametric Mann - Whitney test was performed ( two - tailed ) . Note that BUB1B expression was not measured in these microarrays . * * Significant at a p value < 0 . 05 . N ( AR - V7 low ) = 20 ; N ( AR - V7 high ) = 10 .

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Hornberg et al., 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases, grouped by their relative levels of AR-VS, mainly AR-V7. (High-levels of AR-V7 (top quartile) or lower levels of AR-V7 (quartiles 1-3). Data are plotted as the mean ± s.e.m.. Non-parametric Mann-Whitney test was performed (two-tailed). Note that BUB1B expression was not measured in these microarrays. ** Significant at a p value < 0.05. N (AR-V7 low) = 20; N (AR-V7 high) = 10.


The Kaplan - Meier curves for Disease - Free Survival ( DFS ) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset ( 465 samples ) ( upper graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of the seven genes were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0009 ; for death : p - value = 0 . 026 . An independent dataset was analyzed ( Prostate Adenocarcinoma MSKCC , Cancer Cell 2010 , 123 samples ) ( lower graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0007 ; for death : p - value = 0 . 00546 .

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The Kaplan-Meier curves for Disease-Free Survival (DFS) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset (465 samples) (upper graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of the seven genes were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0009; for death: p-value = 0.026. An independent dataset was analyzed (Prostate Adenocarcinoma MSKCC, Cancer Cell 2010, 123 samples) (lower graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0007; for death: p-value = 0.00546.


Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls , using shRNA against the coding region for each gene ( shRNA # 2 ) . Cell number was measured using a non - perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System . Data shown are mean ± s . e . m . of 8 to 12 replicates normalized to their shGFP control . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed .

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Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.


Representative images of 22Rv1 stably depleted of BUB1B , or control ( shGFP ) are shown .

Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.


22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription . The assay was conducted in 2 % CSS to measure AR ligand - independent transcriptional activity . Data represent two independent experiments performed in triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The expression of FKBP5 and UBE2C determined by RT - qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes . Cells were cultured in 2 % CSS . Data represent two independent experiments performed in duplicate or triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The CRPC cell line 22Rv1 was cultured in 2 % CSS media and was treated for 72 hours with vehicle ( DMSO ) , doxorubicin ( DOX ) , N9 - isopropylolomoucine ( N - 9 ) , or the combination of DOX and N - 9 at different concentrations . Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each . The data were analyzed using Compusyn software , and a normalized isobologram was built . The table shows the Combination Index ( CI ) for the different drug combinations . CI = 1 represents additivity , CI < 1 synergism , and CI > 1 antagonistic effects .

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The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.


The non - tumorigenic prostate epithelial cell line RWPE1 , the AR - null PC cell line PC3 , the androgen dependent cell line LNCaP , and the CRPC cell lines C4 - 2B and 22Rv1 were treated for 72 hours with vehicle ( DMSO ) , DOX ( 100 ng / mL [ 184 nM ] ) , N - 9 ( 200 ng / mL [ 613 nM ] ) , or the combination of DOX ( 100 ng / mL [ 184 nM ] ) and N - 9 ( 200 ng / mL [ 613 nM ] ) . C4 - 2B and 22Rv1 cells were kept in 10 % CSS media , and the other cell lines in 10 % FBS . Cell confluence was monitored using the Incucyte Zoom System . Data represent two independent experiments , with four to six replicates each , showing the mean ± s . e . m . , and normalized to vehicle controls ( Kruskal - Wallis test , P value < 0 . 0001 , two - tailed ) . * Significant at a p value < 0 . 05 , * * p value < 0 . 01 , * * * p value < 0 . 001 .

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The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.


The non - tumorigenic prostate cell line RWPE - 1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle ( DMSO ) or the combination of DOX and N - 9 at 100 ng / mL and N - 9 200 ng / mL , respectively . Cell confluence was monitored using the Incucyte Zoom System .

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The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.


Sashimi plots of representative SRSF3 - dependent exons . C2C12 cells were treated with control siRNA ( siCont ) , Srsf3 siRNA ( siSrsf3 ) , Hnrnpk siRNA ( siHnrnpk ) , or both Srsf3 and Hnrnpk siRNAs ( siSrsf3 + siHnrnpk ) , and RNA - seq was performed . Lines indicate junction - spanning reads . Gene structures are shown below the Sashimi plots .

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Sashimi plots of representative SRSF3-dependent exons. C2C12 cells were treated with control siRNA (siCont), Srsf3 siRNA (siSrsf3), Hnrnpk siRNA (siHnrnpk), or both Srsf3 and Hnrnpk siRNAs (siSrsf3+siHnrnpk), and RNA-seq was performed. Lines indicate junction-spanning reads. Gene structures are shown below the Sashimi plots.


Volcano plot showing fold changes versus P - values of the normalized MASCOT scores ( see Materials and Methods ) of identified proteins between 3XFLAG - SRSF3 - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) . Volcano plot showing fold changes versus P - values of the normalized MASCOT scores of identified proteins between 3XFLAG - HNRNPK - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) .

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

Volcano plot showing fold changes versus P-values of the normalized MASCOT scores (see Materials and Methods) of identified proteins between 3XFLAG-SRSF3-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).   Volcano plot showing fold changes versus P-values of the normalized MASCOT scores of identified proteins between 3XFLAG-HNRNPK-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).


mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA . * * p < 0 . 01 by CuffDiff ( Trapnell et all . , 2010 ) .

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mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA. **p < 0.01 by CuffDiff (Trapnell et all., 2010).


Western blots showing protein expression levels of CPSF6 and β - actin ( ACTB ) . Whole cell lysates were harvested 42 h after siRNA transfection .

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Western blots showing protein expression levels of CPSF6 and β-actin (ACTB). Whole cell lysates were harvested 42 h after siRNA transfection.


SRSF3 - responsive alternative 3 ′ ends of the Med1 gene . ( Top panel ) Disorder analysis for MED1 using PONDR VL - XT ( deep blue ) and IUPred2 ( light blue ) . The scores in the ordinate indicate disordered tendencies between 0 and 1 ( a score of more than 0 . 5 indicates disordered ) . ( Middle panel ) Protein and gene structures of MED1 . The protein structure was segmented into individual exonic regions . The SRSF3 - responsive last exon is indicated in pink . ( Bottom panel ) Sashimi plots of RNA - seqs from Srsf3 - and / or Hnrnpk - silenced cells .

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SRSF3-responsive alternative 3′ ends of the Med1 gene. (Top panel) Disorder analysis for MED1 using PONDR VL-XT (deep blue) and IUPred2 (light blue). The scores in the ordinate indicate disordered tendencies between 0 and 1 (a score of more than 0.5 indicates disordered). (Middle panel) Protein and gene structures of MED1. The protein structure was segmented into individual exonic regions. The SRSF3-responsive last exon is indicated in pink. (Bottom panel) Sashimi plots of RNA-seqs from Srsf3- and/or Hnrnpk-silenced cells.


Immunofluorescence images of MED1 in C2C12 cells treated with siCont ( C ) , siSrsf3 ( S ) , siHnrnpk ( K ) , or siSrsf3 + siHnrnpk ( S + K ) . A white dotted contour outlines the nucleus . Box plots ( right ) show quantification of nuclear ( NUC ) and cytoplasmic ( CYT ) localization of MED1 . The average intensity of one nucleus or a cytoplasmic segment is individually plotted .

Immunofluorescence images of MED1 in C2C12 cells treated with siCont (C), siSrsf3 (S), siHnrnpk (K), or siSrsf3+siHnrnpk (S+K). A white dotted contour outlines the nucleus. Box plots (right) show quantification of nuclear (NUC) and cytoplasmic (CYT) localization of MED1. The average intensity of one nucleus or a cytoplasmic segment is individually plotted.


The intrinsically disordered region ( IDR ) in MED4 . Disorder analysis , protein and gene structures , and Sashimi plots are shown in ( B ) . Splicing of these genes was not affected by the siRNA treatment .

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The intrinsically disordered region (IDR) in MED4. Disorder analysis, protein and gene structures, and Sashimi plots are shown in (B). Splicing of these genes was not affected by the siRNA treatment.


Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs . Box plots show quantification of nuclear localization of MED4 . Each dot represents one nucleus .

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Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs. Box plots show quantification of nuclear localization of MED4. Each dot represents one nucleus.


Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta , including both MED1 and MED4 .

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Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta, including both MED1 and MED4.


The number of MED1 puncta ( left ) and MED4 puncta ( right ) per nucleus in cells treated with the indicated siRNA .

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The number of MED1 puncta (left) and MED4 puncta (right) per nucleus in cells treated with the indicated siRNA.


MED1 signals on MED4 puncta normalized to nuclear MED1 signals . MED1 signal intensity on each MED4 punctum was individually plotted .

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MED1 signals on MED4 puncta normalized to nuclear MED1 signals. MED1 signal intensity on each MED4 punctum was individually plotted.


B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS ( top right ) and OS ( bottom right ) of breast cancer patients in the METABRIC dataset . Genes located at the HER2 amplicon were nominated according to hazard - ratio levels ( all P - values < 0 . 01 ) . The HER2 co - amplification percentage of the indicated genes is represented as bar graphs ( left ) .

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B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard-ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left).


C The frequency of CDK12 amplification in HER2 - amplified - and HER2 - nonamplified breast cancer . The bar graphs indicate the frequency of HER2 and CDK12 co - amplified cases among the patients with HER2 gene amplification ( left ) . Tables show the percentage of CDK12 amplification in the indicated cohorts ( right ) . Amp , amplification ; non - amp , non - amplification .

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C The frequency of CDK12 amplification in HER2-amplified- and HER2-nonamplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification.


D Scatter plot showing the correlation between CDK12 expression and its copy number alteration ( CNA ) in METABRIC ( left ) . The r value was calculated as the Pearson ' s correlation coefficient . Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC ( right ) . P - values were calculated using one - way ANOVA , followed by post - hoc LSD test .

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D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.


E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC ( top ) and KM plotter ( bottom ) using the Kaplan - Meier method with the log - rank test .

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E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.


A FACS analysis of the percentages of CD44 + / CD24 − / ESA + breast CSC - like populations in CDK12 - overexpressing ZR - 75 - 30 and HCC - 1419 cells and CDK12 - siRNA - transfected BT474 and HCC - 1954 cells . Top , representative dot plot of CD44 - APC versus CD24 - PE expression for viable cells ( red ) and ESA - positive cells ( blue ) from the viable cells . Bottom , the percentage of CD44 + / CD24 - / ESA + cell population ( blue dots at lower right quadrant in the plots ) was quantified in the indicated cell lines . Mean ± s . d . of three independent samples . P - values were based on two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) and one - way ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . Amp , amplification ; non - amp , non - amplification ; Trz , trastuzumab .

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A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.


B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines . Cells either treated with ALDH inhibitor DEAB ( with DEAB ; negative control ) or untreated ( without DEAB ) were incubated with ALDEFLURO substrate ( BAAA ) . In the FACS analysis , the percentage of ALDEFLUOR - positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB . Mean ± s . d . . P - values were calculated based on a two - tailed Student ' s t - test cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive .

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B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


C The self - renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere - formation assay . The number of tumorsphere cells ( > 100 μm in diameter ) was counted after 3 , 5 , and 7 days . Data represent the mean ± s . d . ( n = 3 ) . P - value was calculated based on a two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) or ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.