PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO in saline ( PMO - S ) , PMO - G ( IV ) or PMO - G ( D ) ( scale bar = 100μm ) . PMO - G ( IV ) refers to intravenous injection of PMO in combination with intravenous injection of 5 % glycine every other day for 3 weeks . PMO - G ( D ) represents intravenous injection of PMO supplemented with 1 % glycine orally .

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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( C ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( IV ) , PMO - G ( D ) or PMO - S . 0 . 5µg , 2 . 5 µg , 5µg , 10µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , D - diaphragm and A - abdominal muscle . ( D ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( i . v . , n = 4 ) , PMO - G ( D25 and D50 , n = 3 ) and PMO - S ( n = 3 ) ( * P < 0 . 05 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle. (D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( A ) Muscle function was assessed to determine the physical improvement with grip strength test ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( B ) Measurement of bodyweight changes of DKO mice treated with PMO - G or PMO - S ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( C ) Measurement of TA muscle mass of DKO mice treated with PMO - G or PMO - S ( n = 3 , * P < 0 . 05 ; one - way ANOVA post - hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( D ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) . TA - tibialis

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). TA-tibialis anterior


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( E , F ) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle and D - diaphragm . 2 . 5 µg , 5µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( B ) Measurement of Ejection Fraction ( EF ) and Fractional Shortening ( FS ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 mice ( n = 6 ) with echocardiography ( * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (B) Measurement of Ejection Fraction (EF) and Fractional Shortening (FS) in treated DKO mouse hearts (n=3) and C57BL/6 mice (n=6) with echocardiography (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( C ) Morphological examination of treated DKO mouse hearts with H & E staining ( scale bar = 800μm ) . Boxed areas are magnified . ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (C) Morphological examination of treated DKO mouse hearts with H&E staining (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test) Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Immunohistochemistry for immunoglobin G ( IgG ) in treated DKO mouse hearts ( D , scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Immunohistochemistry for immunoglobin G (IgG) in treated DKO mouse hearts (D, scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Muscle function was evaluated to determine the physical improvement with grip strength ( E ) or running wheel tests ( F ) for treated DKO ( n = 3 ) or C57BL / 6 mice ( n = 4 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Muscle function was evaluated to determine the physical improvement with grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( G ) Immunohistochemistry for dystrophin expression in body - wide muscles from treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (G) Immunohistochemistry for dystrophin expression in body-wide muscles from treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( H ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 20µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (H) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Survival rate of DKO mice treated with PMO - GM ( n = 6 ) . The number of untreated DKO controls is 15 .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Survival rate of DKO mice treated with PMO-GM (n=6).The number of untreated DKO controls is 15.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Measurement of kyphosis index of DKO mice treated with PMO - GM ( scale bar = 5mm ) and wild - type C57BL / 6 mice ( scale bar = 8mm ) ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Measurement of kyphosis index of DKO mice treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of tidal volume in treated DKO mice with a cardio - respiratory gated technique ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of tidal volume in treated DKO mice with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( E - F ) Examination of EF and FS ( E ) and stroke volume and cardiac output ( F ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 ( n = 5 ) with echocardiography ( * P < 0 . 05 , * * P < 0 . 001 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (E-F) Examination of EF and FS (E) and stroke volume and cardiac output (F) in treated DKO mouse hearts (n=3) and C57BL/6(n=5) with echocardiography (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Masson trichrome ' s staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts ( scale bar = 800 μm ) . Boxed areas are magnified ( n = 3 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Masson trichrome's staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( H ) Measurement of serum creatine kinase - MB ( CK - MB ) levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (H) Measurement of serum creatine kinase-MB (CK-MB) levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( I - J ) Muscle function was assessed to determine the physical improvement with grip strength ( I ) or running wheel tests ( J ) in treated DKO ( n = 4 ) and C57BL / 6 mice ( n = 6 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (I-J) Muscle function was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( A ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (A) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 25µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Re - localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture ( scale bar = 50μm ) . The arrowheads point to identical myofibers .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Re-localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of serum CK levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Measurement of serum indices from treated DKO mice to reflect liver ( E ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of serum CK levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Measurement of serum indices from treated DKO mice to reflect liver (E) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . Measurement of serum indices from treated DKO mice to reflect kidney ( F ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. Measurement of serum indices from treated DKO mice to reflect kidney (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Morphological examination of liver and kidney from treated DKO mice ( scale bar = 50 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Morphological examination of liver and kidney from treated DKO mice (scale bar=50 μm).


e . Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue . As suggested by the consensus statement on the pathology of IgG4 - RD ( Deshpande et al , 2012 ) , three 40x fields with the highest number of IgG4 + and IgG + cells were selected , counted and averaged within these fields . Cell counts as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.


f . C - X - C motif chemokine 13 ( CXCL13 ) concentrations measured by ELISA from inflammatory pseudotumor ( IPT ) CSF , control CSF , and MS patient - derived CSF . Individual values , mean ± SEM ; n = 4 experiment repeats with technical replicates .

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f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates.


g . Stacked bar chart depicting mean relative expression levels of T helper cell - associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in ( a ) . TEM , T effector memory ; TCM , T central memory ; Treg , regulatory T cell .

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

g. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell.


h . Violin plot depicting relative expression levels of IL4R ( left ) and IL10RA ( right ) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF .

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h. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.


a . Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient - derived CSF . Boxplot depicting 25th - 75th percentiles with median shown as central band and whiskers extending from minimum to maximum values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient-derived CSF. Boxplot depicting 25th-75th percentiles with median shown as central band and whiskers extending from minimum to maximum values.


A . Confocal images of live control ( top ) and U18 - treated ( bottom ) tsA201 cells expressing P4M - YFP . Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo / lysosomal compartments , respectively . B . Quantification of perinuclear P4M - YFP mean fluorescence intensity normalized to the cytoplasm . Control : n = 19 ; U18 : n = 16 .

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 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

A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.


K . Heat map of PtdIns , PtdInsP and PtdInsP2 levels from 4 different murine brain regions . The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild - type brains ; n = 3 for both WT and NPC1I1061T animals .

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

K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.


C . Confocal images of control and U18 - treated tsA201 cells fixed and stained for OSBP ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of OSBP and TGN46 regions . D . Quantification of OSBP signal in TGN46 regions ( OSBPTGN46 ) . Control : n = 40 ; U18 : n = 39 .

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

C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39.


J . Confocal images of control and U18 - treated GFP11 - SAC1 HEK293 cells ( top ) and control and NPC1I1061T patient fibroblasts immunolabeled with anti - SAC1 antibody ( bottom ) . Insets show an enlarged view of SAC1 centered on a perinuclear region . K . Quantification analyses of GFP11 - SAC1 ( top ; Control : n = 10 ; U18 : n = 10 ) and anti - SAC1 ( bottom ; Control : n = 7 ; NPC1I1061T : n = 8 ) mean fluorescence intensities in Golgi normalized to the cytoplasm .

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

J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.


C . Left : Confocal images of control ( left ) and U18 - treated ( right ) tsA201 cells expressing PI4KIIα - GFP . Bottom panels show an enlarged view of the probe centered on a perinuclear region . Right : Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm . Control : n = 12 ; U18 : n = 14 . D . Same as ( C ) only PI4KIIIβ . Control : n = 12 ; U18 : n = 15 .

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

C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15.


E . Confocal images of control and U18 - treated tsA201 cells fixed and stained for PI4KIIα ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of PI4KIIα and TGN46 regions . F . Quantification of PI4KIIα signal in TGN46 regions ( PI4KIIα TGN46 ) . Control : n = 36 ; U18 : n = 36 .

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

E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.


B , Confocal images of wild - type and PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups .

O O O O O O O O O B-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 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

B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.


H . Control or GFP - DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . Quantification of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) intensity relative to cytoplasm .

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

H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.


M . Confocal images of control ( top row ) , NPC1I1061T ( middle row ) , and NPC1I1061T treated with sgDHHC3 ( bottom row ) patient cells expressing P4M , LAMP1 and CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . Quantification of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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

M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


C . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3 . Black and orange squares show expanded views of anti - ACBD3 in Golgi and endomembrane compartments , respectively . D . Analyses of ACBD3 intensity distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 cells ) , normalized to the cytoplasm .

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

C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.


I . Confocal images of control and sgACBD3 HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments . J . Quantitative analyses of P4M - YFP mean fluorescence intensity at Golgi ( left graph ) or endomembranes ( right graph ) normalized to the cytoplasm . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups . For left graph ; Control : n = 19 ; U18 : n = 13 ; sgPI4K3B : n = 12 ; sgPI4K3B + U18 : n = 11 ; sgPI4K2A + sgPI4K3B : n = 12 ; sgPI4K2A + sgPI4K3B + U18 : n = 12 ; sgACBD3 : n = 15 ; sgACBD3 + U18 : n = 16 . For right graph ; control : n = 149 ; U18 : n = 251 ; sgACBD3 : n = 138 ; sgACBD3 + U18 : n = 190 . Please note that ' control ' and ' U18 ' datasets in Fig 5J are identical to those in Fig . 4C . Data for all CRISPR experiments were collected on the same days .

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I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.


A . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts expressing P4M - YFP . Black and orange squares show expanded views of PtdIns4P in Golgi and endo / lysosomal compartments respectively .

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A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.


C . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody . Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images . White dashed lines represent the outline of each cell . D . Quantification of mTORC1 mean integrated density normalized bycell area in control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts . Control : n = 15 ; NPC1I1061T : n = 14 ; NPC1I1061T + sgACBD3 : n = 11 .

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C. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell. D. Quantification of mTORC1 mean integrated density normalized bycell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.


B . Confocal images of tsA201 cells expressing PI4KIIα - GFP . Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . C . Quantification of PI4KIIα - GFP mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . D , E . Same as ( B , C ) but in tsA - 201 cells transfected with PI4KIIIβ - GFP . Control : n = 18 , U18 : n = 22 , U18 + PF : n = 23 .

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B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.


Confocal images of tsA201 cells Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . F , G . Same as ( B , C ) but in tsA - 201 cells transfected with P4M - YFP . Control : n = 39 , U18 : n = 42 , U18 + PF : n = 32 . H , I . Same as ( F , G ) but in wild - type and SCAP - / - CHO cells . Control : n = 12 , U18 : n = 11 , SCAP - / - + U18 : n = 10 .

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Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.


A . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI - M6PR . Insets show expanded views of each immunolabeling in the Golgi region ( upper insets ) or centered on endomembranes ( bottom inset , merged signals ) . Dashed grey and white lines represent the outline of each cell . B . Analysis of the mean area of M6P and Giantin colocalizing puncta ( top graph ; Control : n = 23 ; NPC1I1061T : n = 18 ) and of their mean fluorescence intensity ( bottom graph ; Control : n = 18 ; NPC1I1061T : n = 18 ) in control and NPC1I1061T patient fibroblasts .

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A. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell. B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts.


C . Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1 - GFP . Insets show enlarged views of ARF1 in the Golgi area . Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows . D . Quantification of ΑRF - 1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts . Control : n = 10 ; NPC1I1061T : n = 11 .

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C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows. D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.


B . Results of the sequential digestion workflow applied to a synthetic 7 - protein mix , compared to using trypsin alone in four replicates and parallel digestion with trypsin , AspN , chymotrypsin and GluC . A trypsin four replicate experiment shows a large overlap of the four datasets with little gain . Parallel digestions with trypsin , AspN , chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin . Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs . C . Gains of repeated analysis ( trypsin only ) , parallel digestion and sequential digestion for the same data as shown in panel B .

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B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs. C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B.


D . Crosslinked peptides obtained by sequential digestion of a synthetic 7 - protein mix are smaller than their corresponding tryptic peptides .

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D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.


B . The in vitro helicase loading assay demonstrates that an Mcm2 C - terminal deletion mutant supports complex assembly ( lanes 6 and 7 ) and blocks formation of the final helicase loading product ( lanes 8 and 9 ) .

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B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).


C . Overexpression analysis of Mcm2 - 7ΔC2 shows that this mutant causes dominant lethality , indicating that the C - terminus of Mcm2 is essential in cell survival .

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C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.


Plot showing the average Z - scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one - sample t - test of whether average Z - score ≠ 0 . Green circles represent significantly upregulated genes ( Z > 0 and P < 0 . 05 ) . Magenta circles represent significantly downregulated genes ( Z < 0 and P < 0 . 05 ) . Grey circles represent genes whose expression is not significantly changed ( P > 0 . 05 ) . All comparisons were between cancers and normal cells from the same tissue . Dashed line shows position on Y - axis that corresponds to P = 0 . 05 . Positions of MCOLN1 ( red circle ) , VAC14 ( blue circle ) , and MTM1 ( yellow circle ) are indicated . Inset , schematic showing that Mtm1 and Vac14 regulate the levels of PI ( 3 , 5 ) P2 , and thereby , influence TRPML1 activity .

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Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.


Unsupervised hierarchical clustering of Pearson ' s coefficients of pairwise correlation of gene expression reveal 4 indicated clusters . Violin plots of average Z - scores of genes that belong to the four clusters derived from hierarchical clustering . The thick horizontal dashed lines in each sample set represent median ; thin dashed lines represent the quartiles ; polygons represent density estimates of data and extend to extreme values . Statistical tests employed were 1 - sample t - test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0 .

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Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters. Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0.


GSEA conducted on endolysosomal genes ranked on the basis of increasing Z - scores . Positions of the CLEAR target genes are indicated .

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GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated.


Bar graph showing the relative expression of the indicated genes in the indicated cell types . Values were normalized to respective ' HN31 + HRAS shRNA ' average and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin . Molecular weights are shown on the left . Bar graph shows relative Tfeb levels in the indicated samples . Values were normalized to ' HN31 + HRAS shRNA ' and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin. Molecular weights are shown on the left. Bar graph shows relative Tfeb levels in the indicated samples. Values were normalized to 'HN31+HRAS shRNA' and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Bar graph showing the relative expression of MCOLN1 in the indicated cell types . Values were normalized to HT1197 average and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing the relative expression of MCOLN1 in the indicated cell types. Values were normalized to HT1197 average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to ML - SA1 and GPN . Arrows serve as event markers that denote the points of time at which the drugs were added . Grey lines on top represent the time at which the bath Ca2 + was removed . All values represent mean ± SEM from 3 biological replicates .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 and GPN. Arrows serve as event markers that denote the points of time at which the drugs were added. Grey lines on top represent the time at which the bath Ca2+ was removed. All values represent mean±SEM from 3 biological replicates.


Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to ML - SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment . Lines on the right indicate peak amplitudes of Ca2 + release in response to the application of ML - SA1 . All values represent mean ± SEM from 3 biological replicates .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release in response to the application of ML-SA1. All values represent mean±SEM from 3 biological replicates.


Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to GPN . the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment . Lines on the right indicate peak amplitudes of Ca2 + release Ca2 + release was evoked using GPN .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to GPN. the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release Ca2+ release was evoked using GPN.


Peak amplitudes of Ca2 + release in cells of the indicated genotypes in response to ML - SA1 ( E ) All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 (E) All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Peak amplitudes of Ca2 + release in cells of the indicated genotypes in response to GPN ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Ratio of the amplitudes of Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Total Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments . Cell numbers were assessed after a 48 - hour period of growth in cultures seeded at equal densities . All values represent mean ± SEM . Data points represent values from biological replicates .

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Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments. Cell numbers were assessed after a 48-hour period of growth in cultures seeded at equal densities. All values represent mean±SEM. Data points represent values from biological replicates.


Representative images showing Ki67 - labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA ( left ) or MCOLN1 shRNA ( right ) . Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei . Scale bars shown ( 0 . 5 mm ) apply to all panels .

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Representative images showing Ki67-labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA (left) or MCOLN1 shRNA (right). Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei. Scale bars shown (0.5 mm) apply to all panels.


Bar graphs showing fraction ( left ) and total number ( right ) of Ki67 labeled nuclei in the indicated cell types . All values represent mean ± SEM . Values in the bar graph on the right were normalized to control mean . Data points represent values from independent eggs . Statistical test employed was Student ' s t - test .

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Bar graphs showing fraction (left) and total number (right) of Ki67 labeled nuclei in the indicated cell types. All values represent mean±SEM. Values in the bar graph on the right were normalized to control mean. Data points represent values from independent eggs. Statistical test employed was Student's t-test.


Bar graphs showing the relative cell numbers in UMSCC - 22A - HRASWT cells following the indicated perturbations . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

Bar graphs showing the relative cell numbers in UMSCC-22A-HRASWT cells following the indicated perturbations. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Kaplan - Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild - type for HRAS ( right ) or carried oncogenic HRAS mutations ( left ) . Statistical test employed was the Mantel - Cox log - rank test .

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Kaplan-Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild-type for HRAS (right) or carried oncogenic HRAS mutations (left). Statistical test employed was the Mantel-Cox log-rank test.


Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML - SI1 as indicated . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( A ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Primary antibodies used are indicated on the right .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Bar graph showing quantification of the Western blots in ( C ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (C). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Bar graph showing quantification of the Western blots in ( D ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells . repo - GAL4 was used to drive the expression of UAS - dRasG12V in glia . Images shown are volumetric reconstructions of confocal stacks . The various anatomical structures of the larval brain are annotated . VNC refers to the ' ventral nerve chord ' , which is distinct from dorsal hemispheres of the brain . GFP staining represents the total glial membranes in the larval brains . Scale bar , 100μm .

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Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells. repo-GAL4 was used to drive the expression of UAS-dRasG12V in glia. Images shown are volumetric reconstructions of confocal stacks. The various anatomical structures of the larval brain are annotated. VNC refers to the 'ventral nerve chord', which is distinct from dorsal hemispheres of the brain. GFP staining represents the total glial membranes in the larval brains. Scale bar, 100μm.


Bar graph showing relative glial cell volume in larvae of the indicated genotypes . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing relative glial cell volume in larvae of the indicated genotypes. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes . He - GAL4 was used to drive the expression of UAS - dRasG12V in macrophages . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( D ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes. He-GAL4 was used to drive the expression of UAS-dRasG12V in macrophages. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using fat - body extracts isolated from 3rd instar larvae of the indicated genotypes . cg - GAL4 was used to drive the expression of UAS - HRASG12V in larval fat - bodies . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Representative Western blots generated using fat-body extracts isolated from 3rd instar larvae of the indicated genotypes. cg-GAL4 was used to drive the expression of UAS-HRASG12V in larval fat-bodies. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Weighted mean univariate K - function curves of immunogold labeled GFP - HRASG12V under indicated conditions , n ≥ 15 for each condition . The gold point patterns were analyzed with univariate K - functions expressed as L ( r ) - r . The bar graph on the right shows the peak value Lmax of the L ( r ) - r curve , which represents the extent of GFP - HRASG12V nanoclustering . Values are normalized to DMSO average and represent mean ± SEM ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap tests .

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 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

Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs . Values are normalized to DMSO average and represent mean ± SEM , n ≥ 15 for each condition . Statistical test employed was Student ' s t - test with Bonferroni post hoc correction to account for multiple pairwise comparisons .

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

Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons.


The extent of GFP - HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations . Values are normalized to DMSO average and represent mean ± SEM ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap tests .

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

The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs . but in cells exposed to the indicated perturbations . Values shown are normalized to DMSO average .

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

Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average.


Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom . Primary antibodies used are indicated on the right .

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Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom . Bar graph showing quantification of the Western blots in ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was ANOVA .

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

Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA.


Bar graph showing the relative levels of total in HN31 cell extracts exposed to the indicated perturbagens Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Bar graph showing the relative levels of total in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing the relative levels of free / total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Bar graph showing the relative levels of free/total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing the relative levels of free / total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Bar graph showing the relative levels of free/total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Confocal images of MDCK cells expressing mCherry - D4H . Colocalization with the cortical actin ( phalloidin ) indicates that mCherry - D4H is predominantly localized to the plasma membrane in DMSO treated cells . Within 24 hours of ML - SI1 treatment , the mCherry - D4H redistributes to intracellular punctae . Although minimal mCherry - D4H signal remained at the plasma membrane in ML - SI1 - treated cells , the phalloidin signal ( cortical actin ) remained unchanged . Scale bar ( 10μm ) shown in the panel on the top right applies to all panels . Bar graph showing quantification of mCherry - D4H punctae in DMSO or ML - SI1 - treated cells . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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 O O B-MEASURED_VAR 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 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

Confocal images of MDCK cells expressing mCherry-D4H. Colocalization with the cortical actin (phalloidin) indicates that mCherry-D4H is predominantly localized to the plasma membrane in DMSO treated cells. Within 24 hours of ML-SI1 treatment, the mCherry-D4H redistributes to intracellular punctae. Although minimal mCherry-D4H signal remained at the plasma membrane in ML-SI1-treated cells, the phalloidin signal (cortical actin) remained unchanged. Scale bar (10μm) shown in the panel on the top right applies to all panels. Bar graph showing quantification of mCherry-D4H punctae in DMSO or ML-SI1-treated cells. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Confocal images of MDCK cells expressing mCherry - D4H and LAMP1 - GFP treated with ML - SI1 . Scale bar ( 10μm ) shown in the panel on the top right applies to all panels .

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Confocal images of MDCK cells expressing mCherry-D4H and LAMP1-GFP treated with ML-SI1. Scale bar (10μm) shown in the panel on the top right applies to all panels.


Confocal images of control and MCOLN1 shRNA - expressing HN31 cells treated with the cholesterol sensor , filipin . Scale bar ( 10μm ) shown in the panel on the top applies to both panels . Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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 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

Confocal images of control and MCOLN1 shRNA-expressing HN31 cells treated with the cholesterol sensor, filipin. Scale bar (10μm) shown in the panel on the top applies to both panels. Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Fraction of phase - separated GPMVs at the indicated temperatures . Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs . Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs . Values shown represent mean ± SEM . Lines were generated by fitting the data to sigmoidal functions .

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Fraction of phase-separated GPMVs at the indicated temperatures. Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs. Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs. Values shown represent mean±SEM. Lines were generated by fitting the data to sigmoidal functions.


Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Fluorescence polarization ( FP ) competition derived affinity assessment of consecutive truncations of the DAT C13 C - terminus as indicated by the sequences . Bars indicate the mean fold change relative to C13 ( Ki = 1 . 69 µM ) . Data is shown as mean with error - bars as SEM with n = 6 . Representative binding curves are shown in EV1A .

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Fluorescence polarization (FP) competition derived affinity assessment of consecutive truncations of the DAT C13 C-terminus as indicated by the sequences. Bars indicate the mean fold change relative to C13 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1A.