, F ( E ) Representative immunoblots quantitative of ( F ) plasma GLUT4 translocation ( P = 0 . 0139 ) ± 16 - hour BAM15 treatment ( N = 3 per condition ) in C2C12 myotubes .

O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE B-CELL_TYPE O

, F (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation (P=0.0139) ± 16-hour BAM15 treatment (N=3 per condition) in C2C12 myotubes.


G ) [ 3 - 3H ] glucose uptake ± 16 hour BAM15 treatment 30 minute insulin stimulation ( N = 4 per condition ; vehicle vs . vehicle + insulin : P = 0 . 0016 , vehicle vs . BAM15 : P < 0 . 0001 , vehicle vs . BAM15 + insulin : P < 0 . 0001 , vehicle + insulin vs . BAM15 + insulin : P = 0 . 0018 , BAM15 vs . BAM15 + insulin : P = 0 . 0024 )

O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

G) [3-3H]glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation (N=4 per condition; vehicle vs. vehicle + insulin: P=0.0016, vehicle vs. BAM15: P<0.0001, vehicle vs. BAM15 + insulin: P<0.0001, vehicle + insulin vs. BAM15 + insulin: P=0.0018, BAM15 vs. BAM15 + insulin: P=0.0024)


H ) [ 1 - 14C ] palmitate oxidation ± 16 hour BAM15 treatment 5 mM glucose 1 mM pyruvate ( N = 6 per condition ; no glucose / pyruvate : P < 0 . 0001 ) in C2C12 myotubes .

O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE B-CELL_TYPE O

H) [1-14C]palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate (N=6 per condition; no glucose/pyruvate: P<0.0001) in C2C12 myotubes.


I ) Representative immunoblot quantitative ( N = 5 per condition ; P < 0 . 0001 ) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE B-CELL_TYPE O

I) Representative immunoblot and quantitative analysis (N=5 per condition; P<0.0001) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes.


J ) Representative respirometry plot of intact ± shAMPK 16 hour BAM15 treatment in C2C12 myotubes sequentially injected with oligomycin , FCCP , a cocktail of rotenone antimycin a ( N = 5 per condition )

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

J) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)


K ) basal ATP - linked respiration , proton leak , maximal uncoupling , spare respiratory capacity ( N = 5 per condition ) . Basal : shEV vs . shEV + BAM15 ( P = 0 . 0317 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) . ATP : shEV vs . shAMPK + BAM15 ( P = 0 . 0026 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P = 0 . 0003 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 040 ) . Proton leak : shEV vs . shEV + BAM15 ( P = 0 . 0052 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 0394 ) . Maximal : shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) . Spare Capacity : shEV vs . shAMPK ( P = 0 . 0028 ) , shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

K) basal and ATP-linked respiration, proton leak, maximal uncoupling, and spare respiratory capacity (N=5 per condition). Basal: shEV vs. shEV + BAM15 (P=0.0317), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001). ATP: shEV vs. shAMPK + BAM15 (P=0.0026), shEV + BAM15 vs. shAMPK + BAM15 (P=0.0003), shAMPK vs. shAMPK + BAM15 (P=0.040). Proton leak: shEV vs. shEV + BAM15 (P=0.0052), shAMPK vs. shAMPK + BAM15 (P=0.0394). Maximal: shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001). Spare Capacity: shEV vs. shAMPK (P=0.0028), shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001).


L ) [ 3 - 3H ] glucose uptake ± shAMPK , 16 hour BAM15 treatment , 30 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) . No insulin : shEV vs . shEV + BAM15 ( P = 0 . 0001 ) . Insulin : shEV vs . shEV + BAM15 ( P = 0 . 0142 ) .

O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-CELL_LINE B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O

L) [3-3H]glucose uptake ± shAMPK, 16 hour BAM15 treatment, and 30 minute insulin stimulation in C2C12 myotubes (N=3 per condition). No insulin: shEV vs. shEV + BAM15 (P=0.0001). Insulin: shEV vs. shEV + BAM15 (P=0.0142).


- O ( M ) Representative immunoblots quantitative of ( N ) AKT ( shEV + insulin : P = 0 . 0128 , shAMPK - insulin : P = 0 . 0018 , shAMPK + insulin , P = 0 . 0124 ) , ( O ) AS160 ( shEV - insulin : P = 0 . 0283 , shEV + insulin : P = 0 . 0137 ) phosphorylation ± shAMPK , 16 hour BAM15 treatment , 15 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) .

O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-CELL_LINE B-CELL_TYPE O O O O O O O O

-O (M) Representative immunoblots and quantitative analysis of (N) AKT (shEV + insulin: P=0.0128, shAMPK - insulin: P=0.0018, shAMPK + insulin, P=0.0124), and (O) AS160 (shEV - insulin: P=0.0283, shEV + insulin: P=0.0137) phosphorylation ± shAMPK, 16 hour BAM15 treatment, and 15 minute insulin stimulation in C2C12 myotubes (N=3 per condition).


( A ) Serum concentrations of BAM15 over a 24 - hour period ( N = 3 per time point )

O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(A) Serum concentrations of BAM15 over a 24-hour period (N=3 animals per time point)


( B ) peak tissue concentrations of BAM15 ( N = 6 per tissue ) .

O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O

(B) peak tissue concentrations of BAM15 (N=6 per tissue).


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

C, D (C) Daily body weight (day 8: P=0.0454, day 9: P=0.0353, day 10: P=0.0251, day 11: P=0.0131, day 12: 0.0038, day 13: P=0.0008, day 14: P=0.0005, day 15: P=0.0003, day 16-21: P<0.0001) and (D) cumulative food intake over the 3-week treatment in CTRL and BAM15 treated animals (N=16 per group).


( E ) Whole before ( pre ) , one hour ( 1 hr Post ) , 24 hours ( 24 hrs Post ) after intraperitoneal injection of corn oil , vehicle ( corn oil saline ) , 0 . 1 , 0 . 5 , , 1 mg BAM15 per kg of ( N = 11 per group ) ,

O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group),


Whole ( F ) daily during chronic BAM15 treatment ( N = 8 per group ) .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

Whole body temperature (F) daily during chronic BAM15 treatment (N=8 per group).


G , H ( G ) Change in fat mass ( % ) ( P < 0 . 0001 ) ( H ) lean mass ( % ) ( P < 0 . 0001 ) from baseline to 3 - in CTRL BAM15 treated ( N = 8 per group ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group).


( I ) Fasting plasma glucose ( N = 8 per group ; P = 0 . 007 ) ( J ) insulin ( N = 7 per group ; P = 0 . 0024 ) after 3 of treatment in CTRL BAM15 treated .

O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

(I) Fasting plasma glucose (N=8 per group; P=0.007) and (J) insulin (N=7 per group; P=0.0024) after 3 weeks of treatment in CTRL and BAM15 treated animals.


( K ) Plasma glucose concentrations at 0 , 15 , 30 , 60 , 90 , 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of ; N = 8 per group )

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O

(K) Plasma glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( L ) total under curve glucose after 3 - in CTRL BAM15 treated ( N = 8 per group ; P = 0 . 0106 ) .

O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(L) total area under the curve glucose after 3-weeks in CTRL and BAM15 treated animals (N=8 per group; P=0.0106).


M - O ( M ) Oxygen consumption rates ( N ) energy expenditure ( daily : P = 0 . 0357 , phase : P = 0 . 030 ) , ( O ) respiratory exchange ( daily : P = 0 . 0424 , phase : P = 0 . 0016 ) measured by continuous high - resolution indirect calorimetry after 3 - of CTRL or BAM15 treatment ( N = 8 per group ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

M-O (M) Oxygen consumption rates (N) energy expenditure (daily: P=0.0357, dark phase: P=0.030), and (O) respiratory exchange ratio (daily: P=0.0424, dark phase: P=0.0016) measured by continuous high-resolution indirect calorimetry after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( P ) Fecal energy density ( % recovered in feces ) after 3 - of CTRL or BAM15 treatment ( N = 8 per group ) .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( A ) Change in ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P < 0 . 0001 ) , ( B ) fat mass ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0041 , BAM15 vs . CR : P = 0 . 043 ) , ( C ) lean mass ( CTRL vs . BAM15 : P = 0 . 0014 , CTRL vs . CR : P < 0 . 0001 , BAM15 vs . CR : P = 0 . 0047 ) from baseline to 2 - in CTRL , BAM15 , CR treated

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

(A) Change in body weight (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P<0.0001), (B) fat mass (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0041, BAM15 vs. CR: P=0.043), and (C) lean mass (CTRL vs. BAM15: P=0.0014, CTRL vs. CR: P<0.0001, BAM15 vs. CR: P=0.0047) from baseline to 2-weeks in CTRL, BAM15, and CR treated animals


( D ) daily food intake ( CTRL vs . CR : P = 0 . 0092 , BAM15 vs . CR : P = 0 . 0175 ) in CTRL , BAM15 , CR treated ( CTRL N = 7 , BAM15 CR N = 8 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(D) daily food intake (CTRL vs. CR: P=0.0092, BAM15 vs. CR: P=0.0175) in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( E ) Blood glucose concentrations at 0 , 15 , 30 , 60 , 90 , 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of ; N = 8 per group )

(E) Blood glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( F ) total under curve glucose ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P = 0 . 003 , BAM15 vs . CR : P = 0 . 0261 ) after 2 - of treatment in CTRL , BAM15 , CR treated ( CTRL N = 7 , BAM15 CR N = 8 ) .

O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(F) total area under the curve glucose (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P=0.003, BAM15 vs. CR: P=0.0261) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( G ) Plasma insulin concentrations at baseline ( PRE ) , 0 ( CTRL vs . BAM15 : P = 0 . 0154 , CTRL vs . CR : P = 0 . 0425 ) 120 minutes ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0211 , BAM15 vs . CR : P = 0 . 0264 ) after injection of glucose after 2 - of treatment in CTRL , BAM15 , CR treated ( CTRL N = 7 , BAM15 CR N = 8 )

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O

(G) Plasma insulin concentrations at baseline (PRE), and 0 (CTRL vs. BAM15: P=0.0154, CTRL vs. CR: P=0.0425) and 120 minutes (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0211, BAM15 vs. CR: P=0.0264) after injection of glucose after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8)and


( H ) Homeostatic Model Assessment of Insulin Resistance ( HOMA - IR ) ( CTRL vs . BAM15 : P = 0 . 0259 , CTRL vs . CR : P = 0 . 040 ) after 2 - of treatment in CTRL , BAM15 , CR treated ( CTRL N = 7 , BAM15 CR N = 8 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(H) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (CTRL vs. BAM15: P=0.0259, CTRL vs. CR: P=0.040) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( A ) Representative H & E stained liver , adipose tissue , skeletal muscle sections from CTRL BAM15 treated . Scale bars ( black ) = 100 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O

(A) Representative H&E stained liver, adipose tissue, and skeletal muscle sections from CTRL and BAM15 treated animals. Scale bars (black) = 100 μm.


( B ) Representative immunohistochemical of pancreatic insulin ( red ) content counterstained with diaminobenzidine ( blue ) . Scale bars ( black ) = 100 μm .

O O O O B-EXP_ASSAY O B-TISSUE B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(B) Representative immunohistochemical analysis of pancreatic insulin (red) content counterstained with diaminobenzidine (blue). Scale bars (black) = 100 μm.


C - F ( C ) Quantitative of liver steatosis ( μm2 ) ( N = 6 per group ; P < 0 . 0001 ) , ( D ) gastrocnemius muscle cross sectional ( μm2 ) ( CTRL N = 7 , BAM15 N = 8 ) , ( E ) pancreatic insulin positive ( % of total ) ( N = 6 per group ; P = 0 . 0420 ) , ( F ) β - mass ( N = 4 per group ; P = 0 . 0005 ) .

O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F (C) Quantitative analysis of liver steatosis (μm2) (N=6 per group; P<0.0001), (D) gastrocnemius muscle cross sectional area (μm2)(CTRL N=7, BAM15 N=8), (E) pancreatic insulin positive area (% of total area) (N=6 per group; P=0.0420), (F) and β-cell mass (N=4 per group; P=0.0005).


( G - I ) ( ; μm2 ) distributional ( number ) of ( G ) iWAT , ( H ) gWAT , ( I ) BAT from CTRL BAM15 treated ( N = 8 per group ) .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O

(G-I) Size (area; μm2) and distributional (number) analysis of (G) iWAT, (H) gWAT, and (I) BAT from CTRL and BAM15 treated animals (N=8 per group).


A - D ( A ) Plasma concentrations of non - esterified fatty ( NEFA ) ( CTRL N = 7 , BAM15 N = 8 , P = 0 . 0073 ) , ( B ) leptin ( N = 8 per group , P < 0 . 0001 ) , ( C ) GDF15 ( N = 7 per group , P = 0 . 035 ) , ( D ) FGF21 ( N = 7 per group , P = 0 . 0026 ) after 3 - of CTRL or BAM15 treatment .

O O O O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

A-D (A) Plasma concentrations of non-esterified fatty acids (NEFA) (CTRL N=7, BAM15 N=8, P=0.0073), (B) leptin (N=8 per group, P<0.0001), (C) GDF15 (N=7 per group, P=0.035), and (D) FGF21 (N=7 per group, P=0.0026) after 3-weeks of CTRL or BAM15 treatment.


E , F ( E ) Total ( basal : P = 0 . 015 ) ( F ) complete ( basal : P = 0 . 0137 ) oxidation of 200 μM [ 1 - 14C ] palmitate to CO2 from mixed gastrocnemius muscle homogenates ( N = 7 per group ) .

O O O O O 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-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O O O

E, F (E) Total (basal: P=0.015) and (F) complete (basal: P=0.0137) oxidation of 200 μM [1-14C]palmitate to CO2 from mixed gastrocnemius muscle homogenates (N=7 per group).


( G ) Routine ( R ) oxygen flux , leak ( L ) , OXPHOS ( P ) ETC ( E ) capacity in permeabilized mixed gastrocnemius ( N = 6 per group ) . PM : pyruvate malate , ADP : adenosine diphosphate , GS : glutamate succinate , Tm : tetramethyl - p - phenylenediamine , PalM : palmitoylcarnitine malate , Oct : octanoylcarnitine , Dur : duroquinol .

(G) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol.


( H ) Representative immunoblots of phosphorylated total AMPK ACC from iWAT , mixed gastrocnemius

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(H) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT, and mixed gastrocnemius muscle


( I - K ) Densitometric ( N = 4 per group ; iWAT p - AMPKT172 : P = 0 . 0186 , iWAT p - ACCS79 : P = 0 . 0083 , mixed gastrocnemius p - AMPKT172 : P = 0 . 0286 ) .

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(I-K) Densitometric quantification (N=4 per group; iWAT p-AMPKT172: P=0.0186, iWAT p-ACCS79: P=0.0083, mixed gastrocnemius p-AMPKT172: P=0.0286).


( L - N ) mRNA expression of transcriptional regulators of lipogenesis lipolysis in iWAT ( N = 7 per group ) . Srebf1 ( P < 0 . 0001 ) , Mlxipl ( P = 0 . 0018 ) , Scd1 ( P < 0 . 0001 ) , FASN ( P = 0 . 0009 ) , LIPE ( P = 0 . 0015 ) , Pnpla2 ( P < 0 . 0001 ) , PPARg ( P < 0 . 0001 ) .

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(L-N) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT (N=7 per group). Srebf1 (P<0.0001), Mlxipl (P=0.0018), Scd1 (P<0.0001), FASN (P=0.0009), LIPE (P=0.0015), Pnpla2 (P<0.0001), PPARg (P<0.0001).


Bone marrow were polarized in presence of 40 ng / ml GM - CSF treated with 0 . 2 mM of indicated compounds for 7 days . Gr1 - CD11b + were sorted lysed for microarray . hierarchical clustering was on different expression genes between BSA ( Con ) oleate group .

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Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group.


Signature genes involved in lipid metabolism , dendritic maturation , macrophage maturation , associated macrophages ( TAMs ) phenotype , MHCII innate immune response are listed validated via flow cytometry or catalytic activity . Data are expressed as mean ± SD from two to four independent experiments . Unpaired Student ' s two - t were performed to compare expression level of indicated in control oleate groups . * p < 0 . 05 ; * * p ≤ 0 .

O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O 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-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01


Gr1 - CD11b + , polarized by indicated treatment , were purified for mitochondrial respiration detection . oxygen consumption rate ( OCR ) of these was monitored after addition of oligomycin ( OA ) ( 1 µM ) , Carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , electron transport inhibitor rotenone antimycin A ( R / AA ) ( 0 . 5 µM ) at indicated time points . basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , proton leak , ATP production maximal respiration on OCR value were quantified .

Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


Forty µM etomoxir were added starting from day 0 of bone marrow polarization , followed by mitochondrial respiration by using same amount of polarized Gr1 - CD11b + myeloid on day 7 . mitochondrial respiration was monitored analyzed via XFe96 Analyzer .

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Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer.


Exemplary plots of proportion of CD206 + , mean ( MFI ) of CD38 CD73 on polarized myeloid was determined via flow cytometry .

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Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry.


T proliferation was performed via co - culture with purified CD4 + T in ratios of M ( myeloid ) : T ( T ) = 1 : 30 .

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T cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30.


NO production from co - culture supernatant was quantified by Griess reaction .

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NO production from the co-culture supernatant was quantified by Griess reaction.


oxygen consumption rate ( OCR ) of 1 × 105 purified Gr1 - CD11b + was monitored as described in Figure 2 . basal OCR , basal ECAR ( extracellular acidification rate ) , spare respiratory capacity , proton leak , ATP production maximal respiration on OCR value were quantified .

B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O O O O

The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


polarization state was evaluated via expression of CD206 .

The polarization state was evaluated via the expression of CD206.


T proliferation was performed employing co - culture with purified CD4 + T in variant ratios .

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T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios.


lipid droplets accumulation was determined by BODIPY . of divided proliferation index were calculated .

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The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated.


Nitric oxide ( NO ) in co - culture supernatant was quantified by Griess reaction .

B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O

Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.


Bone marrow - derived myeloid were treated with BSA - control ( Con ) or oleate in presence or absence of 10 nM rapamycin ( Rapa ) . polarization of myeloid was detected via flow cytometry as indicated by CD11b CD206 expression .

B-TISSUE I-TISSUE O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O

Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.


Purified Gr1 - CD11b + myeloid were co - cultured with CD4 + T for proliferation . supernatant was collected for nitric oxide ( NO ) detection .

O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.


oxygen consumption rate ( OCR ) of 1 × 105 differentiated myeloid were monitored after addition of oligomycin ( OA ) ( 1 µM ) , uncoupler carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , electron transport inhibitor rotenone antimycin A ( R / AA , 5 µM ) at indicated time points . basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , ATP production maximal respiration on OCR value were quantified .

The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.


Mean ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid as determined by flow

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Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometry


Mean ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid lysed for western blot .

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Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.


infiltrating immune were isolated from either CT26 or MCA205 bearing model analyzed for expression of BODIPY in CD206 + ( black ) CD206 - ( grey ) .

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Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.


Balb / c mice were inoculated with 5 × 105 CT26 subcutaneously . Since day 6 , vehicle or DGAT inhibitors ( iDGAT ) were administrated via intra - tumoral injection once daily .

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Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily.


At day 13 , infiltrated immune were isolated stained for CD206 CD11b .

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At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.


5 × 105 MCA205 were injected subcutaneously into C57BL / 6 mice . PBS , liposome control or iDGAT - liposome was injected into peritoneum every other day starting at day 7 . were measured every 7 days mice were sacrificed on day 31 for . Immune from spleen , - draining lymph nodes ( TDLN ) or were isolated stained for CD11b with BODIPY for lipid droplets in myeloid .

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5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.


infiltrating CD8 + T ( red ) were stained together with nuclei ( DAPI / blue ) after sacrificing bearing mice .

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Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.


1 × 106 CD14 + myeloid from healthy donors were polarized by 1 ng / ml GM - CSF in presence of BSA - control ( C ) , 0 . 2 mM oleate ( O ) or 0 . 2 mM stearate ( S ) over a 6 - day period . expression of CD206 , CD204 CD38 was analyzed via flow cytometry .

O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.


level of lipid droplets was determined via BODIPY .

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The level of lipid droplets was determined via BODIPY staining.


oxygen consumption rate ( OCR ) of differentiated myeloid was monitored as described in Materials Methods .

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The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.


( D ) purified CFSE - labeled autologous CD4 + T were co - cultured with polarized myeloid with indicated treatment for T proliferation .

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(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.


tissue ( ) as well as corresponding non - tumorous adjacent tissue ( control ) were collected from colorectal cancer prepared for histopathology . expression of CD68 ( red ) , CD206 ( green ) ADRP ( blue ) indicate lipid droplets in infiltrating myeloid . Nuclei ( ) were stained using DAPI . absolute number of positive was quantified in 5 high - power fields ( hpf ) ( scale bar = 20 µm ) .

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Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).


A . Immunofluorescence microscopy of control , parental fibroblasts labeled for LAMP - 1 . fibroblasts ( VPS41S285P / R662 * VPS41c . 1423 - 2A > G / R662 * ) show more LAMP - 1 , which are distributed throughout . ( A ' ) shows a significant increase in number of LAMP - 1 positive compartments for both . > 15 per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.


D . HeLaVPS41KO transfected with VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 constructs labeled for LAMP - 1 V5 immunofluorescence microscopy . VPS41 variants co - localize with LAMP - 1 , indicating that VPS41S285P VPS41R662 * are recruited to late endosomes / lysosomes . Scale bars 10µm ; zoom of squared , 1µm .

O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O

D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.


H . VPS41WT / WT , VPS41WT / R662 * VPS41S285P / R662 * fibroblasts incubated with Dextran - 568 for 0 . 5 , 2 , 5 , 8 24 hours . Colocalization of Dextran - 568 SiR - Lysosome Cathepsin D ( SiR - Lyso ) reveals a delay in lysosomal delivery in maternal ( VPS41WT / R662 * ) ( VPS41S285P / R662 * ) at 2 hours of Dextran uptake . After 5 hours , maternal show similar to control colocalization . show only after 24 hours colocalization similar to control , indicating a delay rather than a block in late endosome - lysosome fusion . > 10 per were quantified .

O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O

H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.


A . Western blot of LC3 expression in control fibroblasts . In steady state ( 0 hours ) conditions , fibroblasts have a higher of lipidated LC3 ( LC3II ) : LC3I than control . Induction of autophagy by nutrient starvation ( 2 hours incubation with minimal EBSS medium ) results in a raise in LC3II : LC3I in both control , but in VPS41S285P / R662 * fibroblasts this increase is only modest ( quantified in A ' ) ( n = 2 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).


E . Immunofluorescence of HeLaWT HeLaVPS41KO transfected with LC3GFP / RFP tandem construct . increased of GFP / RFP - positive compartments in HeLaVPS41KO indicate a block in autophagic flux ( quantified in E ' ) . > 12 per were quantified . Scale bars 10µm ; zoom , 1µm .

O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O 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. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.


A . Immunofluorescence of control , parental fibroblasts labeled for LAMP - 1 mTOR . In steady state conditions ( 0 hours ) VPS41S285P / R662 * fibroblasts show less colocalization between mTOR LAMP - 1 . VPS41WT / WT , VPS41WT / S285P VPS41WT / R662 * show an appropriate mTOR response upon nutrient deprivation ( 2 hours ) or restimulation ( 2 hours starvation followed by 15 minutes restimulation ) ( Appendix Fig S5A ) . > 10 per were quantified ( A ' ) . Quantifications are performed relative to colocalization under steady state conditions per ( n = 3 ) . Scale bars , 10µm ; zoom , 1µm .

O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.


D . Rescue experiments of HeLaVPS41KO . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on or secreted secretogranin II ( SgII ) of PC12 VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . were washed incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII was observed between VPS41WT VPS41S285P .

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B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing of with 6 normal dopaminergic neurons in anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( UA44 ) with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( UA391 ) fails to rescue neurodegeneration . At day 10 , none of heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for α - synuclein ( α - syn ) ( total of 90 worms ) n = 90 for each independent transgenic ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads missing neurons are indicated with arrows . Scale bar 20µm .

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C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


A Gene targeting strategy . Left : max genomic locus , targeting construct max targeted locus . In vitro Flipase ( Flip ) deletion of selectable markers lacZ neo results in a max targeted locus with exons 4 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 MaxF10 . Right : genomic PCR of tail from F1 mice . PCR products of max wt ( 430 bp ) max flox ( 526 bp ) alleles . M , 1kb ladder .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O 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-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O

A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.


Flow cytometry analyses of B populations ( GFP + ) in Bone Marrow ( BM ) spleen . Single - suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . For BM , were isolated from femora tibiae . Pro - B ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , mature B ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO ( GFP + ) plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


Flow cytometry analyses of B populations ( GFP + ) in Bone Marrow ( BM ) Single - suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . For BM , were isolated from femora tibiae . Pro - B ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B ( Ly - 6c - CD49b - B220 + IgM - CD43 - )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)


Flow cytometry analyses of B populations ( GFP + ) in spleen . Single - suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . spleen follicular B ( B220 + CD23 + CD21int ) , marginal zone B ( B220 + CD23 - CD21hi ) immature ( newly formed / transitional stage 1 ) B ( B220 + CD23 - CD21lo ) . To identify KO ( GFP + ) plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) control HET - mb1 ( n = 4 ) mice . Columns represent mean values error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . different B populations were identified by flow cytometry Experiment representative of at least three independent experiments .

O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O 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-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.


I cycle by flow cytometry of sorted GFP + pre - pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) mature ( B220hiIgM + ) bone marrow B from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU analyzed 4 h later . Single - suspensions were prepared , stained sorted . Sorted were subsequently stained with propidium iodide for content . Experiment representative of at least three independent experiments . Dotted is shown for EdU MFI comparison .

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I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.


J of annexin - V + GFP + in BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) control ( n = 4 ) mice . Pro - B , pre - B , immature mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment representative of at least three independent experiments .

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J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.


Flow cytometry absolute numbers of B220loCD138 + ( plasmablasts ) ( A , B ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted activated in vitro with LPS plus IL - 4 for 72 h . were stained with monoclonal antibodies against indicated markers analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


Flow cytometry absolute numbers of B220 + IgG1 + switched B ( C , D ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted activated in vitro with LPS plus IL - 4 for 72 h . were stained with monoclonal antibodies against indicated markers analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


E Spleen B220 + GFP + B lymphocytes were stained with 7AAD annexin - V to monitor apoptosis . 7 - AAD - annexin - v - ( alive ) , 7 - AAD - annexin - v + ( early apoptotic ) , 7 - AAD + annexin - v + ( late apoptotic ) , 7 - AAD + annexin - v - ( necrosis ) . F Relative numbers of live , early apoptotic , late apoptotic necrotic from E .

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E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E.


G CD69 surface expression in purified B220 + GFP + mature spleen . Sorted B were activated in vitro with LPS plus IL - 4 for 72 h , stained analyzed by flow cytometry . solid grey peak represents expression of CD69 marker in non - activated mature B lymphocytes at day 0 . H Relative numbers of CD69 + B lymphocytes in cultures from G ( n = 4 ) . Experiment representative of at least three independent experiments .

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G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments.


I of germinal center ( GC ) formation in spleen of MaxKO - cd19 , Myc - KO - cd19 , DKO - cd19 heterozygous control mice immunized with TNP - KLH . Representative of frozen spleen sections stained with IgM ( grey / blue ) , PNA ( GC marker ; red ) , GFP ( Max - , c - Myc - or c - Myc / Max - deficient B ; green ) . Scale bar , 80µm .

O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O

I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.


J Upper , Number of GCs per mouse in spleen sections of immunized MaxKO - cd19 ( n = 3 mice , n = 186 follicles analyzed ) , MycKO - cd19 ( n = 3 mice , n = 178 follicles analyzed ) , DKO - cd19 ( n = 3 mice , n = 191 follicles analyzed ) heterozygous control mice ( n = 3 mice , n = 203 follicles analyzed ) . Lower , Frequencies of GC ( PNA + ) GFP + ( deleted ) or GFP - ( non - deleted ) in immunized MaxKO - cd19 ( n = 13 GCs ) , MycKO - cd19 ( n = 21 GCs ) , DKO - cd19 ( n = 27 GCs ) heterozygous control ( n = 62 GCs ) mice . was performed at 13 days post - immunization . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-EXP_ASSAY O B-TISSUE O B-ORGANISM O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O O O B-TISSUE O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


A Number of divisions in activated mature B lymphocytes . Sorted B220 + GFP + spleen from HET - cd19 ( n = 5 ) , MaxKO - cd19 ( n = 5 ) , MycKO - cd19 ( n = 3 ) , DKO - cd19 ( n = 3 ) mice were stained with a tracer , activated in vitro with LPS plus IL - 4 for 72 h analyzed by flow cytometry . Experiment representative of at least three independent experiments B from cultures C of B cultures Scale bar , 500 µm . 4 × magnification . D Forward scatter ( FSC ) histograms of activated B lymphocytes An arbitrary dashed is shown for comparison .

O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O

A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.


cycle of sorted B220 + GFP + B lymphocytes activated in vitro . were activated EdU was added 4 h before harvesting . were stained with propidium iodide to measure content . Experiment representative of at least three independent experiments . An arbitrary dashed shows difference of EdU MFI . S phase mean ( MFI ) : 7871 HET , 6512 MaxKO , 5860 MycKO , 6420

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO


Representation of labeling with nucleotide analogues CldU ( red ) IdU ( green ) of from sorted B220 + GFP + B lymphocytes activated for 48 h . Scale bar , 10 µm .

O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.


H Fork rate of fibres from B lymphocytes sorted activated Data is pooled from three replicate experiments . Horizontal bars represent median values . 150 - 250 forks were measured per condition . * * * , p < 0 . 001 ; * * , p < 0 .

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01


I Western blotting of replication in activated B lymphocytes Sorted B220 + GFP + spleen were activated in vitro with LPS plus IL - 4 for 72 h . MEK2 , SMC1 are shown as loading controls . blots shown are representative of three independent experiments .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O

I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.


A Bar plot showing number of DEGs ( adj . p - value < 0 . 01 , FC + 1 . 5 in red , 1 . 5 < FC > - 1 . 5 in , FC < - 1 . 5 in blue ) . Sorted B220 + GFP + spleen B from MaxKO - cd19 , MycKO - cd19 , DKO - cd19 control HET - cd19 mice were activated with LPS plus IL - 4 for 72 h analyzed . Two samples for each condition were used .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-TISSUE B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.


C Hierarchical clustering heat map of DEGs in any comparison ( two samples per condition ) . blue in left panel indicates significance for that contrast column . Colour code : red represents upregulation , , unchanged , blue downregulation of expression . Gender : colour code for genetic condition . Pink are blue colour mice .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O

C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.


D Venn diagrams of up - ( left ) down - regulated genes ( right ) for three conditions ( + / - 1 . 5 - FC p < 0 . 01 ) . Heatmaps of genes only deregulated in MaxKO - cd19 , MycKO - cd19 , or DKO - cd19 mice are shown .

O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-ORGANISM O O O

D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.


B Heatmaps of genes involved in relevant pathways Colour code : red represents upregulation , , unchanged , blue downregulation of expression .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.


C qPCR of up - down - regulated genes Pooled was prepared from sorted B220 + GFP + spleen B of MaxKO - cd19 ( n = 4 ) , MycKO - cd19 ( n = 4 ) , or DKO - cd19 ( n = 4 ) control HET - cd19 ( n = 4 ) mice activated Genes were randomly selected qPCR was performed using specific primers for cd72 , med13l , card11 , ssh2 , cdc7 , mrpl12 , rpl39 , cysc . max c - myc were included as controls actin was used for normalization .

O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O

C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.


growth assessed by color change due to medium acidification for both undefined ATCC 1161 semi - defined CSY media with or without sucrose , 4 % 1 % respectively . ( + ) , inoculated wells ; ( - ) , non - inoculated control . picture was taken after a 24 - hour incubation period .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.


Impact of horse serum ( HS ) yeast extract ( YE ) dilution on M . florum doubling time . green red dotted lines indicate HS YE concentrations found in ATCC 1161 medium ( 20 % HS , 1 . 35 % YE ) CSY medium ( 0 . 313 % HS , 0 . 02 % YE ) , respectively . Doubling times were measured in a CMRL - 1066 base medium using colorimetric assays . Dots error bars indicate mean standard deviation calculated from three biological replicates .

O O B-ORGANISM B-TISSUE O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates.


maximal biomass observed for M . florum cultures growing in CSY medium with varying initial concentrations of sucrose . Biomass was measured using colony forming units ( CFU / ml ; left axis ) converted to grams of dry ( gDW / L ; right axis ) . A rectangular hyperbola fit is shown ( yellow ) , dotted represents maximal biomass value predicted by fit ( 1e8 x 5 . 95 CFU / ml ; 0 . 013 gDW / L ) .

O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O

The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).


Sucrose specific uptake rate ( e ) combined lactate / acetate specific secretion rates ( f ) at varying initial sucrose concentrations in CSY medium . Boxplots represent median interquartile range of uptake or secretion rate values calculated at different time intervals during exponential growth phase of M . florum cultures . Whiskers indicate minimal maximal values . Dotted lines indicate selected uptake ( - 5 . 26 mmol ∙ gDW - 1 ∙ hr - 1 ) secretion rate ( 8 . 69 mmol ∙ gDW - 1 ∙ hr - 1 ) values used for modeling . Sucrose quantifications were performed in technical duplicate , whereas lactate acetate quantifications were performed in single replicates . See Appendix Figure S5 for further details .

B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.


Week 10 COs stained for ventricular radial glia ( vRGs ) marker CRYAB , together with WWOX pan - radial glia marker SOX2 . on right are enlargements of boxed . dashed defines ventricular space . Scale = 50µm ( left ) , 25µm ( right ) ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches ) .

O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).


Immunofluorescent ( IF ) for glutamatergic neurons marker VGLUT1 GABAergic neurons marker GAD67 ( GAD1 ) in week 10 COs ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 100µm . of represented in figure 1E . VGLUT1 GAD67 were quantified as surface covered by were normalized to number of nuclei in each image . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . As samples were not normally distributed , statistical significance was determined using Kruskal - Wallis with Dunn ' s multiple comparisons . Outliers were removed using ROUT ( Q = 1 % ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . Sample traces show visible differences in local field potential , with WWOX - KO COs ( Red ) showing increased activity compared to WT ( Blue ) in baseline condition ( Left ) in presence of 100µM 4AP ( Right ) .

O O O O O O O O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O 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-SMALL_MOLECULE O O O O

Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . WWOX ' s coding was re - introduced into week 6 WWOX - KO COs using lentiviral transduction ( lenti - WWOX ) ( F ) Immunofluorescent showing WWOX expression in different populations in WWOX - KO organoids following infection with lentivirus . NT = non - treated . Scale = 50µm .

O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O

Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .


Week 15 week 24 COs stained for astrocytic radial glia marker GFAP , astrocyte - specific marker S100β ( WT W15 : n = 9 from 3 batches , KO W15 : n = 16 from 3 batches , W - AAV W15 : n = 4 organoids from 1 batch . WT W24 : n = 10 from 4 batches , KO W24 : n = 9 from 3 batches , W - AAV W24 : n = 4 from 1 batch ) . Scale =

O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O 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-ORGANISM O O O O O O O O O O O O

Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µm


IF of week 6 COs for astrocytic markers in surrounding of VZs ( WT : n = 6 from 2 batches , KO : n = 6 from 2 batches ) . Scale = 100µm ( left ) , 50µm ( right ) . for damage markers γH2AX 53BP1 in nuclei of in VZ of week 6 COs at physiological conditions , together with pan - radial glia marker SOX2 ( WT : n = 8 from 3 batches , KO : n = 12 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 50µm ( left ) , 25µm ( right ) . of γH2AX ( top ) 53BP1 foci ( bottom ) in nuclei of composing innermost layer of VZ , normalized to total number of nuclei in this layer . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 8 organoids from 3 batches , KO : n = 12 organoids from 3 batches , W - AAV : n = 4 from 1 batch ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O 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-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-TISSUE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.


sequencing ( - seq ) of week 15 COs transcriptome ( WT : n = 2 , KO : n = 4 ) . qPCR for selected Wnt target genes validating results of - seq . Y - axis indicates relative expression fold change . Data are represented as mean ± . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 4 from 1 batch , KO : n = 4 from 1 batch , W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 16 COs were sub - fractionated into a ( C ) ( N ) fractions . experiment was run twice with a total of 2 WT organoids 4 KOs organoids ( 2 for each KO ) . KAP - 1 marks nucleus HSP90 marks cytoplasm . numbers at bottom are a of fraction of β - catenin band intensities normalized to fraction [ β - C ( N / C ) ] .

O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].


IF in week 15 COs validating decreased of deep - layer cortical markers CTIP2 ( BCL11B ) TBR1 , superficial - layer marker SATB2 ( WT : n = 3 , KO : n = 4 , W - AAV : n = 4 ) . Scale = 50µm . of cortical markers seen in G , normalized to total number of nuclei . Y - axis indicates fold change compared to average of WT COs . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 9 from 3 batches , KO : n = 16 from 3 batches , W - AAV : n = 4 organoids from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.   Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . Week 10 WSM COs stained for progenitor marker SOX2 , neuronal marker β3 - Tubulin WWOX ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-DISEASE O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.   Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . Average firing rate over 24 neurons from WSM P COs ( 4 organoids ) , 41 neurons from WSM S COs ( 3 organoids ) 40 neurons from WSM S W - AAV organoids ( 3 organoids ) . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons . Bars represents mean ± . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-DISEASE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM. Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . GAD67 VGLUT1 immunostaining in week 10 WSM COs ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of data shown in fig 5D . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-DISEASE O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.