DU145 V5 - SLC1A3 - DU145 were treated under indicated conditions with ASNase ( 0 . 2 U / ml ) or TFB - TBOA ( 20μM ) or both , subjected to IncuCyte for apoptotic .

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

DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.


PC3 were treated under ASNase ( 0 . 3 U / ml ) , or TFB - TBOA ( 5 μM ) conditions for 3 days lysates were extracted intracellular contents of aspartate , asparagine , glutamate glutamine were determined by liquid - chromatography mass spectrometry ( LC - MS ) . Data information : Median peak was used for raw data normalization Results were calculated on three replicates presented as mean ± SD ( unless otherwise stated ) . p - value was calculated by two - unpaired t from Prism7 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

PC3 cells were treated under ASNase (0.3 U/ml), or TFB-TBOA (5 μM) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate, asparagine, glutamate and glutamine were determined by liquid-chromatography mass spectrometry (LC-MS). Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.


key metabolites involved in urea cycle , pyrimidine synthesis , TCA cycle , oxidation , glycolysis carnitines metabolism were determined . NAD + / NADH of indicated conditions was calculated normalized to control ( mean ± ) . Dash indicates indirect effect . TCA cycle : tricarboxylic acid cycle ; OAA : oxaloacetic acid ; UMP : uridine monophosphate ; CMP : cytidine monophosphate ; PEP : phosphoenolpyruvate ; NADH : nicotinamide adenine dinucleotide ( reduced form ) ; NAD + : nicotinamide adenine dinucleotide ( oxidized form ) ; NADPH : nicotinamide adenine dinucleotide phosphate ( reduced form ) ; NADP + : nicotinamide adenine dinucleotide phosphate ( oxidized form ) ; FAD : flavin adenine dinucleotide ; GSSG : glutathione disulfide ; HMG - CoA : 3 - hydroxy - 3 - methylglutaryl - CoA . Data information : Median peak was used for raw data normalization Results were calculated on three replicates presented as mean ± SD ( unless otherwise stated ) . p - value was calculated by two - unpaired t from Prism7 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

key metabolites involved in urea cycle, pyrimidine synthesis, TCA cycle, oxidation, glycolysis and carnitines metabolism were determined. The NAD+/NADH ratio of the indicated conditions was calculated and normalized to control (mean± SEM). Dash line indicates indirect effect. TCA cycle: tricarboxylic acid cycle; OAA: oxaloacetic acid; UMP: uridine monophosphate; CMP: cytidine monophosphate; PEP: phosphoenolpyruvate; NADH: nicotinamide adenine dinucleotide (reduced form); NAD+: nicotinamide adenine dinucleotide (oxidized form); NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form); FAD: flavin adenine dinucleotide; GSSG: glutathione disulfide; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.


A - C . PC3 ( A ) , DU145 ( B ) DU145 - V5 - SLC1A3 ( C ) were treated with ASNase ( 0 . 3 U / ml in A ; 0 . 2 U / ml in B C ) , TFB - TBOA ( 5 μM ) for 3 days as indicated subjected to transcriptome . Bioinformatics pathway or gene ontology ( GO ) biological process was performed on sets of genes that were upregulated or downregulated when PC3 were treated with ASNase TFB - TBOA compared to mock . Transcriptome was on one biological replicate for each validated by real - time PCR experiments in Figures EV4A - C . presents row scaled normalized read biological signaling pathway enrichment was performed by ToppGene online

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

A-C. PC3 (A), DU145 (B) and DU145-V5-SLC1A3 (C) cells were treated with ASNase (0.3 U/ml in A; 0.2 U/ml in B and C), TFB-TBOA (5 μM) for 3 days as indicated and subjected to transcriptome analysis. Bioinformatics pathway or gene ontology (GO) biological process analysis was performed on the sets of genes that were upregulated or downregulated when PC3 cells were treated with ASNase and TFB-TBOA compared to mock. Transcriptome analysis was based on one biological replicate for each cell line and validated by real-time PCR experiments in Figures EV4A-C. Heatmap presents row scaled normalized read counts and the biological signaling pathway enrichment analysis was performed by ToppGene online program


SUM159PT human breast cancer were orthotopically injected into mammary of NSG mice . Once SUM159PT tumors reached 250mm3 volume , mice were treated with mock or ASNase ( 60U per day ) for 5 consecutive days ( n = 3 ) . Following treatment , mice were sacrificed , blood , mammary tumors were collected subjected to mass spectrometry to determine asparagine level . Essential were used for raw data normalization . Data are presented as mean ± SD . Data information : pretreatment started 2 days before injection of cancer . mice were either injected with 60 U ASNase or saline per day . p - value was calculated by two - unpaired t in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


mouse breast cancer lines 4T1 4T1 - V5 - SLC1A3 were orthotopically implanted into mammary of pretreated NSG mice . Presented is volume measurements of arising tumors at day 9 . ( n = 13 mice for each group , except for 4T1 + ASNase , n = 12 ) . Data are presented as mean ± . Data information : pretreatment started 2 days before injection of cancer . mice were either injected with 60 U ASNase or saline per day . p - value was calculated by two - unpaired t in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

The mouse breast cancer cell lines 4T1 and 4T1-V5-SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice. Presented is the volume measurements of arising tumors at day 9. (n=13 mice for each group, except for 4T1+ASNase, n=12). Data are presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


tumors were surgically removed once reached a volume of ~ 500mm3 collected subjected to LC - MS to determine of asparagine , aspartate , glutamine glutamate . Leucine level was used as a control . Results are on five samples presented as mean ± . Data information : pretreatment started 2 days before injection of cancer . mice were either injected with 60 U ASNase or saline per day . p - value was calculated by two - unpaired t in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


human breast cancer lines MDA - MB - 231 MDA - MB - 231 - V5 - SLC1A3 were intravenously injected into pretreated NSG mice . Once mice showed breathing problems , they were sacrificed , lung liver were collected blindly scored for metastasis lesions . p - value was calculated by one - unpaired t in Prism7 . Data are presented as mean ± ( n = 8 ) . Data information : pretreatment started 2 days before injection of cancer . mice were either injected with 60 U ASNase or saline per day . p - value was calculated by two - unpaired t in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

The human breast cancer cell lines MDA-MB-231 and MDA-MB-231-V5-SLC1A3 cells were intravenously injected into pretreated NSG mice. Once mice showed breathing problems, they were sacrificed, and lung and liver were collected and blindly scored for metastasis lesions. The p-value was calculated by one-tailed unpaired t test in Prism7. Data are presented as mean ± SEM (n=8). Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


( a ) Kaplan - Meier survival curves of combined WT Atg5Tg mice no . 25 ( P0 . 001 ; n = 65 for WT mice n = 70 for Atg5Tg mice ) . ( b , c ) Lifespan extension in Atg5Tg mice no . 25 ( P0 . 001 ; n = 32 for WT mice n = 36 for Atg5Tg mice no . 25 ) ( b ) Atg5Tg mice no . 25 ( P0 . 001 ; n = 33 for WT mice n = 34 for Atg5Tg mice no . 25 ) ( c ) . ( d - f ) Kaplan - Meier survival curves of combined WT Atg5Tg mice no . 43 ( d ) , no . 47 ( e ) no . 471 ( f ) ( P0 . 001 ; n = 25 for WT mice n = 30 for Atg5Tg mice ) . P - values were calculated using log - rank .

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

(a) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 25 (P0.001; n=65 for WT mice and n=70 for Atg5Tg mice). (b,c) Lifespan extension in male Atg5Tg mice no. 25 (P0.001; n=32 for WT mice and n=36 for Atg5Tg mice no. 25) (b) and female Atg5Tg mice no. 25 (P0.001; n=33 for WT mice and n=34 for Atg5Tg mice no. 25) (c). (d-f) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P0.001; n=25 for WT mice and n=30 for Atg5Tg mice). P-values were calculated using the log-rank test.


( g ) Expression of Atg5 Atg12 - Atg5 conjugate , conversion of LC3 I into LC3 II , p62 level in hearts of Atg5 Tg mice . Whole - tissue extracts were prepared from 3 - month - old Atg5 Tg mice WT , analysed by western blotting using indicated antibodies . β - Actin served as a control . ( h ) Densitometric of signals on blots shown in ( g ) . bars represent mean ± s . d . ( n = 3 ) .

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

(g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).


( a ) Western blot showing enhanced of Atg5 , Atg12 - Atg5 conjugate LC3 conversion , reduced p62 in indicated tissues of 18 - month - old Atg5 Tg mice no . 25 ( T ) WT mice ( W ) . ( b ) Densitometric of signals on blots shown in ( a ) .

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

(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a).


( c ) Electron microscopy of autophagosomes ( arrows ) late - autophagic compartments ( arrowheads ) in liver tissues of 18 - month - old Atg5 Tg mice no . 25 WT mice . Scale bars are indicated . ( d ) numbers of autophagosomes autolysosomes per ( n = 50 ) in electron microscopic shown in ( c ) were counted . bars represent mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( e ) Fluorogenic comparison of basal autophagy activity between GFP - LC3 Tg mice Atg5 / GFP - LC3 double Tg mice . Whole bodies of 8 - week - old ( left ) ( right ) GFP - LC3 Tg ( LC3 ) , LC3 / LC3 - LC3Tg mice ( Atg5 / LC3 ) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC ( Kodak Molecular Imaging Software , Version 4 . 0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and LC3/LC3-LC3Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0).


( f ) Western blot showing autophagy markers in liver of GFP - LC3 mice . GFP - LC3 mice were injected for 5 days with siRNA - Atg5 or Atg5 - HA cDNA through tail vein . Whole - liver tissue lysates were prepared subjected to immunoblotting using indicated antibodies . β - Actin served as a control .

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

(f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.


( a ) Representative physical pictures of 18 - month - old WT Atg5 Tg mice fed with a regular diet . ( b ) chart of ageing - induced obesity in WT Atg5 Tg mice . mice were fed with a regular diet for 24 months ( n = 180 - - 250 ) their weights were monitored monthly . bars represent mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - .

(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


. ( c ) Average daily oral food intake normalized to , measured on regular diet . food intake of mice ( n = 18 - 24 ) was measured for 2 at 3 - , 6 - , 12 - 18 - month - old .

O O O O O O O O O O O O O O O O O O O O O O O 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) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age.


d ) of gonadal fat pads from 18 - month - old mice ( n = 11 ) . * * * P0 . 0001 versus control ; Student ' s t - . ( e , f ) of visceral ( e ) subcutaneous ( f ) fat pads of 18 - month - old mice ( n = 6 ) . * * * P0 . 0001 versus control ; Student ' s t - .

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

d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test.


( g ) Enhanced glucose tolerance of Atg5 Tg mice . WT Atg5 Tg mice were starved overnight then given with an i . p injection of glucose ( 1 mg g − 1 ) . results are mean ± s . d . of 12 - week - old WT Atg5 Tg mice ( n = 12 ) . * P0 . 01 , * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - .

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

(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.


( h ) Enhanced insulin sensitivity of Atg5 Tg mice . WT Atg5 Tg mice were starved for 6 h then i . p injected with 0 . 75 IU soluble insulin . * P0 . 01 , * * P0 . 001 versus control ; Student ' s t - .

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

(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.


( i ) Plasma of triglycerol ( TG ) in WT Atg5 Tg mice . mice were fasted for 3 h before measurement . Young mice ( 4 - week old , n = 8 ) ; old mice ( 18 - month old , n = 8 ) . * * * P0 . 0001 versus control ; Student ' s t -

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

(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test


( j ) Plasma leptin in WT Atg5 Tg mice ( n = 8 - 12 ) . * * * P0 . 0001 versus control ; Student ' s t - . (

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

(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. (


( k ) GSH / GSSG in liver ( n = 4 , three times trial ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - .

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

(k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.


( l ) Respiration rates of mitochondria in WT Atg5 Tg MEFs ( n = 2 ) .

(l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2).


( m ) Wire - hang endurance of Atg5 Tg WT mice ( n = 10 ) . bars represent mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - .

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

(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( a ) Comparison of Atg12 - Atg5 conjugate in WT Atg5 Tg MEFs by western blot . MEFs from WT Atg5 Tg mice were cultured at embryonic day 13 , after which extracts were examined by western blot using anti - Atg5 anti - β - actin antibodies .

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

(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies.


( b ) Increased conversion of LC3 reduced expression of p62 in Atg5 Tg MEFs no . 6 by rapamycin / or Baf . A1 . WT MEFs no . 1 Atg5 Tg MEFs no . 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in absence or presence of Baf . A1 , extracts were then analysed by western blotting . signals on blot were quantified by densitometric represented as of p62 to β - actin .

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

(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.


( c , d ) Increased resistance of Atg5 Tg MEFs to oxidative stress . WT MEFs no . 1 Atg5 Tg MEFs no . 6 were treated for 24 h with 50 , 100 , 200 300 μM H2O2 ( c ) or 300 μM H2O2 in absence or presence of 5 mM 3 - MA or 20 nM Baf . A1 ( Baf . A1 ) ( d ) . viability was assessed by propidium iodide . values are mean ± s . e . m . ( n = 3 ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - .

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

(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.


( e ) Representative showing resistance of Atg5 Tg MEFs to oxidative stress . cultured WT Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h then observed under a microscope . Scale bars , 50 μm .

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

(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.


( f ) Effect of oxidative stress on autophagy activity in Atg5 Tg MEFs . WT MEFs no . 1 Atg5 Tg MEFs no . 6 were treated with 100 μM H2O2 for 24 h in presence or absence of 20 nM Baf . A1 , extracts were analysed by western blotting . About 10 30 μg of were used for LC3 ( upper panel ) p62 ( bottom panel ) detection , respectively . β - Actin served as a control .

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

(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.


B - C . Wound healing assays were performed using IBIDI ® chambers to evaluate role of AhR on migration . of wound were captured using an Axio Vert . A1 inverted microscope ( Carl Zeiss ® ) at 5x magnification . histogram represents mean ± s . d . Wound closure was determined by measuring between edges of wound at time 0 15 h ( n = 3 independent technical experiments for each lines or conditions ) compared using unpaired t - with Sidak - Bonferroni method . B . Results obtained with BRAFi - sensitive or resistant SK28 KO for AhR in absence of treatment . ( n = 3 independent technical experiments for each lines ) . C . Results obtained for migration ( 0 to 15 h ) for SK28 R KO or not for AhR after treatment or not with 10 μM CH - 223191 . ( n = 3 independent technical experiments for each lines or conditions , mean ± s . d . ) . Statistical using unpaired t - with Sidak - Bonferroni method has been performed between mean of 3 independent experiments .

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

B-C. Wound healing assays were performed using IBIDI® chambers to evaluate the role of the AhR on cell migration. Images of the wound were captured using an Axio Vert.A1 inverted microscope (Carl Zeiss®) at 5x magnification. The histogram represents the mean ± s.d. Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h (n = 3 independent technical experiments for each cell lines or conditions) and compared using unpaired t-tests with the Sidak-Bonferroni method. B. Results obtained with BRAFi-sensitive or resistant SK28 cells KO for the AhR in the absence of treatment. (n = 3 independent technical experiments for each cell lines). C. Results obtained for the migration assay (0 to 15 h) for SK28 R cells KO or not for the AhR after treatment or not with 10 μM CH-223191. (n = 3 independent technical experiments for each cell lines or conditions, mean ± s.d.). Statistical analysis using unpaired t-tests with the Sidak-Bonferroni method has been performed between the mean of the 3 independent experiments.


D . Three - dimensional spheroid growth of BRAFi - sensitive or resistant SK28 KO before or after knockout of AhR by CRISPR / Cas9 in absence of treatment . were captured four days after implantation of spheroids into collagen gel . ( n = 4 independent technical experiments , mean ± s . d . E . Three - dimensional spheroid growth of BRAFi - resistant SK28 KO before or after knockout of AhR by CRISPR / Cas9 after daily treatment with specific AhR inhibitor CH - 223191 ( 5 μM ) for one week or in absence of treatment . were captured four days after implantation of spheroids into collagen gel . ( n = 3 independent technical experiments , mean ± s . d . ) . Statistical using unpaired t - method has been performed with Sidak - Bonferroni method , ( p < 0 . 01 # # , * * ; p < 0 . 001 # # # , * * * ) .

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

D. Three-dimensional spheroid growth of BRAFi-sensitive or resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 4 independent technical experiments, mean ± s.d. E. Three-dimensional spheroid growth of BRAFi-resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 after daily treatment with the specific AhR inhibitor CH-223191 (5 μM) for one week or in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 3 independent technical experiments, mean ± s.d.). Statistical analysis using unpaired t-tests method has been performed with the Sidak-Bonferroni method, (p<0.01 ##, **; p<0.001 ###,***).


A . AhR in 501Mel CTR those transduced with sgRNA targeting AhR ( # 1 , # 2 ) were analyzed by western blotting .

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

A. AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR (#1, #2) were analyzed by western blotting.


C . Three - dimensional spheroid growth of 501Mel . were captured seven days after spheroid implantation ( n = 3 independent technical experiments , mean ± s . d . ) . Each histogram represents mean ± s . d . Comparisons with control were performed using unpaired t - with Sidak - Bonferroni method , p < 0 . 001 * * * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Three-dimensional spheroid growth of 501Mel cells. Images were captured seven days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Each histogram represents the mean ± s.d. Comparisons with control were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.


D . AhR in SK28R WT , KO , KO CA - AhR were analyzed by western blotting .

D. AhR Protein levels in SK28R WT, KO, and KO CA-AhR cells were analyzed by western blotting.


F . Three - dimensional spheroid growth of SK28 KO or not for AhR by CRISPR / Cas9 rescued by constitutive active form of AhR ( KO CA - AhR ) . were captured four days after spheroid implantation ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons were performed using unpaired t - with Sidak - Bonferroni method .

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

F. Three-dimensional spheroid growth of SK28 cells KO or not for AhR by CRISPR/Cas9 and rescued by the constitutive active form of AhR (KO CA-AhR). Images were captured four days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.


A . At basal level , AhR is located at layer in a that includes HSP90 , p23 , XAP , SRC . Immunoprecipitation of SRC was performed with specific antibodies SRC / AhR interaction analyzed by western blotting .

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

A. At the basal level, AhR is located at the membrane layer in a protein complex that includes HSP90, p23, XAP, and SRC. Immunoprecipitation of SRC was performed with specific antibodies and SRC/AhR interaction analyzed by western blotting.


B . of AhR , p - SRC ( Y416 ) , SRC , p - FAK ( Y576 / 577 ) , FAK in four different melanoma lines ( n = 3 ) . level of BRAFi resistance corresponds to our measure of IC50 ( Vemurafenib ) for different lines 501Mel ( 0 , 23uM ) , SKMel28 ( 0 , 29uM ) , M229 ( 0 , 89uM ) M238 ( 2 , 16uM ) . These lines correspond to sensitive parental .

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

B. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the four different melanoma cell lines (n=3). The level of BRAFi resistance corresponds to our measure of IC50 (Vemurafenib) for the different cell lines 501Mel (0,23uM), SKMel28 (0,29uM), M229 (0,89uM) and M238 (2,16uM). These cell lines correspond to the sensitive parental cells.


C . of AhR , p - SRC ( Y416 ) , SRC , p - FAK ( Y576 / 577 ) , FAK in SK28R KO or not for AhR by CRISPR / Cas9 or after rescue with activated - form of AhR ( CA - AhR ) .

C. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the SK28R cell line KO or not for AhR by CRISPR/Cas9 or after rescue with the activated-form of the AhR (CA-AhR).


D . of AhR , p - SRC ( Y416 ) , SRC , in SK28R after treatment with different AhR ligands for 24h ( 5 μM BaP , 5 μM indirubin , 5 μM ITE , 10 nM TCDD , 5 μM FICZ ) .

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

D. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after treatment with different AhR ligands for 24h (5 μM BaP, 5 μM indirubin, 5 μM ITE, 10 nM TCDD, and 5 μM FICZ).


E . of AhR , p - SRC ( Y416 ) , SRC , in SK28R after 24 h of treatment with increasing doses of ITE .

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

E. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after 24 h of treatment with increasing doses of ITE.


B . Three - dimensional spheroid growth of a 50 % / 50 % mix of SK28R WT ( in red ) KO ( in green ) over five days . were treated or not with SRC inhibitors : bosutinib ( 1 µM ) or dasatinib ( 1 µM ) every two days ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons with control ( DMSO ) were performed using unpaired t - with Sidak - Bonferroni method , p < 0 . 001 * * * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Three-dimensional spheroid growth of a 50%/50% mix of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with SRC inhibitors: bosutinib (1 µM) or dasatinib (1 µM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.


C . Three - dimensional spheroid growth of SK28R WT ( in red ) KO ( in green ) over five days . were treated or not with dasatinib ( 1 µM ) in combination with ITE ( 5 μM ) or TCDD ( 20 nM ) every two days ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons with control ( DMSO ) were performed using unpaired t - with Sidak - Bonferroni method , p < 0 . 05 * , p < 0 . 01 * * . Comparisons were performed using unpaired t - with Sidak - Bonferroni method .

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

C. Three-dimensional spheroid growth of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with dasatinib (1 µM) in combination with ITE (5 μM) or TCDD (20 nM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.05 *, p<0.01 **. Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.


D . of AhR , p - SRC ( Y416 ) , SRC in SK28R after 24 h of treatment with ITE ( 5 μM ) or SRC inhibitor ( dasatinib ) ( 1 μM ) .

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

D. Protein levels of AhR, p-SRC (Y416), and SRC in the SK28R cell line after 24 h of treatment with ITE (5 μM) or SRC inhibitor (dasatinib) (1 μM).


F . Expression of median of gene expression for various signatures ( left ) AhR target genes ( right ) from RNAseq datasets for SK28R treated or not for 24 h with SRC inhibitors : bosutinib ( Bos , 1 μM ) or dasatinib ( Das , 1 μM ) . scale corresponds to Z scores .

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

F. Expression heatmap of the median of gene expression for the various signatures (left) and AhR target genes (right) from RNAseq datasets for SK28R cells treated or not for 24 h with SRC inhibitors: bosutinib (Bos, 1 μM) or dasatinib (Das, 1 μM). The scale corresponds to the Z scores.


B . PDX volumes were measured every 2 days until reaching 1500mm3 . Values correspond to mean + / - . Statistical ( 2way ANOVA ) has been performed between different experiments ( Das or Das before combo vs Dab ) at different times . Stars represent time from when is significantly lower than group treated with BRAFi alone ( Dabrafenib ) p < 0 . 05 * , p < 0 . 01 * * .

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

B. PDX tumor volumes were measured every 2 days until reaching 1500mm3. Values correspond to the mean +/- sem. Statistical analysis (2way ANOVA) has been performed between the different experiments (Das or Das before combo vs Dab) at different times. Stars represent time from when tumor size is significantly lower than group treated with BRAFi alone (Dabrafenib) p<0.05 *, p<0.01 **.


C . Kaplan - Meier survival curve for MEL006R mice treated with different drugs . Comparison of Survival Curves have been performed using nonparametric Log - rank ( Mantel - Cox ) .

O O O O O O O O O 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. Kaplan-Meier survival curve for MEL006R mice treated with the different drugs. Comparison of Survival Curves have been performed using the nonparametric Log-rank (Mantel-Cox) test.


GSC neurospheres were dissociated to single suspension transfected with either miR - 10b inhibitor ( labeled " miR - 10b - i " ) or non - targeting control oligonucleotide , or treated with Lipofectamine 2000 only ( Mock ) . Statistical significance of difference was determined by Student ' s t , with p - values < 0 . 001 indicated by three asterisks . Numbers of replicates exact P - values are included in Appendix Table S4 . A . viability was monitored at day 5 after transfection as described in Materials Methods .

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

GSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled "miR-10b-i") or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Statistical significance of the difference was determined by Student's t test, with p-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.A. Cell viability was monitored at day 5 after transfection as described in Materials and Methods.


B . number of GSC colonies were monitored at day 5 after transfection .

B. The number and size of GSC colonies were monitored at day 5 after transfection.


C . MiR - 10b inhibition induces cleavage of caspases 3 7 in GSC , as determined by Western blot at day 5 after transfection with inhibitor . signals were quantified using ImageJ normalized to beta - actin .

O O O O O O O O O O O O O O O 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. MiR-10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta-actin.


D . Flow Cytometry of Annexin V 7 - AAD of GSC GBM8 at day 5 after miR - 10b inhibition .

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

D. Flow Cytometry analysis of Annexin V and 7-AAD staining of GSC GBM8 at day 5 after miR-10b inhibition.


Three types of GSC ( GBM4 , GBM6 , GBM8 ) were transfected with miR - 10bASO , gene expression was analyzed 24 hours later by Affymetrix microarrays . heatmaps colors demonstrate altered expression of genes ( up - or down - regulated relative to mock treated samples ) with fold change > 1 . 2 p < 0 . 05 in at least two out of three GSC cultures . A . genes associated with " cycle " bioterm have been selected using Ingenuity Pathway . treatment with miR - 10b inhibitor is indicated as " miR - 10b - i " .

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

Three types of GSC (GBM4, GBM6, and GBM8) were transfected with miR-10bASO, and gene expression was analyzed 24 hours later by the Affymetrix microarrays. The heatmaps colors intensity demonstrate altered expression of the genes (up- or down-regulated relative to the mock treated samples) with the fold change > 1.2 and p < 0.05 in at least two out of the three GSC cultures.A. The genes associated with "cell cycle" bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR-10b inhibitor is indicated as "miR-10b-i".


B . genes associated with " splicing " bioterm have been selected using Gene Ontology ( GO ) . treatment with miR - 10b inhibitor is indicated as " miR - 10b - i " . Arrows depict genes selected as candidate direct targets for further study .

O O O O O O O O O O O O O O O O O O O O O O O O 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. The genes associated with "RNA splicing" bioterm have been selected using Gene Ontology (GO). The treatment with the miR-10b inhibitor is indicated as "miR-10b-i". Arrows depict the genes selected as candidate direct targets for further study.


C . miR - 10b binding motifs are enriched in 5 ' UTRs of genes up - regulated by miR - 10b ASO . graph shows probability that enrichment of miR - 10b motifs in mRNAs up - regulated vs . unchanged ( p < 0 . 05 ) by anti - miR - 10b does not occur by chance .

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

C. miR-10b binding motifs are enriched in 5'UTRs of the genes up-regulated by miR-10b ASO. The graph shows the probability that enrichment of the miR-10b motifs in mRNAs up-regulated vs. unchanged (p<0.05) by anti-miR-10b does not occur by chance.


D . miR - 10b octamer motifs ' composition of 5 ' UTRs was compared between transcripts up - down - regulated on microarrays . relative frequencies of various miR - 10b binding motifs are shown , indicating that mostly miR - 10b 3 ' - end binding motifs are enriched in up - regulated mRNAs .

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

D. The miR-10b octamer motifs' composition of the 5'UTRs was compared between transcripts up- and down-regulated on the microarrays. The relative frequencies of various miR-10b binding motifs are shown, indicating that mostly miR-10b 3'-end binding motifs are enriched in the up-regulated mRNAs.


Figure 3 . Expression of splicing factor mRNAs in various GBM datasets . A . genes encoding splicing factors down - regulated by miR - 10b are expressed at lower in various GBM datasets relative to their expression in normal brain tissues . B . In contrast , many splicing factors up - regulated by miR - 10b are overexpressed in GBM datasets . Six high - content GBMmicroarray datasets from Oncomine resource ( https : / / www . oncomine . org / resource / main . html ) , including TCGA _ BrainGBM ( 2 ) , Bredel Brain2 ( 31 ) , Lee Brain ( 32 ) , Liang Brain ( 33 ) , Murat Brain ( 34 ) Sun Brain ( 35 ) , that collectively contain information for 858 GBM 52 control samples , have been utilized for . data is presented as log2 fold change between GBM normal brain tissues .

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

Figure 3.Expression analysis of splicing factor mRNAs in various GBM datasets.A. The genes encoding splicing factors down-regulated by miR-10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues.B. In contrast, many splicing factors up-regulated by miR-10b are overexpressed in the GBM datasets.Six high-content GBMmicroarray datasets from the Oncomine resource (https://www.oncomine.org/resource/main.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34) and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues.


B . qRT - PCR validates that mRNA of MBNL1 - 3 , SART3 , RSRC1 other splicing factors are de - repressed by miR - 10b ASO in different GSC GBM lines . mRNA expression were normalized to GAPDH expression .

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

B. qRT-PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1 and other splicing factors are de-repressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.


C . Regulation of representative splicing - related by miR - 10b mimic in GSC , as demonstrated by Western blot . signals were quantified using ImageJ normalized to beta - actin . ratios between miR - 10b mimic expressing control samples are indicated .

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

C. Regulation of representative splicing-related proteins by miR-10b mimic in GSC, as demonstrated by Western blot analysis. The signals were quantified using ImageJ and normalized to beta-actin. The ratios between miR-10b mimic expressing and control samples are indicated.


D . miR - 10b mimic regulates 5 ' UTR Luciferase reporter containing a single miR - 10b complementary site .

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

D. miR-10b mimic regulates 5'UTR Luciferase reporter containing a single miR-10b complementary site.


E . miR - 10b mimic regulates 5 ' UTR Luciferase reporters of some splicing factors genes bearing wild type ( WT ) , but not mutated ( Mut ) miR - 10b binding sites . Statistical significance of differences was determined by Student ' s t , with p - values < 0 . 05 indicated by asterisks , p < 0 . 01 by two asterisks , p < 0 . 001 by three asterisks . Numbers of replicates exact P - values are included in Appendix Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. miR-10b mimic regulates 5'UTR Luciferase reporters of some splicing factors genes bearing wild type (WT), but not mutated (Mut) miR-10b binding sites. Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks, p < 0.01 by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.


B . 2 ' - O - MOE - POmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control ( 1 μg of each ) formulated with InVivo - jet PEI were injected intratumorally at days 20 25 after implantation . efficacy of miR - 10b inhibition was assessed by qRT - PCR of resected tumors , with miR - 10b expression normalized to miR - 125b .

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

B. 2'-O-MOE-POmiR-10b inhibitor (miR-10b-i) or non-targeting control (1 μg of each) formulated with InVivo-jet PEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR-10b inhibition was assessed by qRT-PCR analysis of the resected tumors, with miR-10b expression levels normalized to miR-125b.


C . qRT - PCR demonstrates that miR - 10b inhibition in orthotopic GBM8 leads do de - repression of its mRNA targets . mRNA expression were normalized to GAPDH .

O O O O O O O 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. qRT-PCR analysis demonstrates that miR-10b inhibition in orthotopic GBM8 leads do de-repression of its mRNA targets. mRNA expression levels were normalized to GAPDH.


D . Inverse correlation between miR - 10b expression of its mRNA targets in resected GBM8 tumors .

D. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.


E . Inhibition of miR - 10b markedly reduces burden . left panels illustrate imaging in representative at day 29 . bars represent average signal ratios for each group at day 29 ( after treatment ) to day 20 ( at beginning of treatment ) . N = 7 per group at treatment initiation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Inhibition of miR-10b markedly reduces tumor burden. The left panels illustrate tumor imaging in representative animals at day 29. The bars represent average signal ratios for each group at day 29 (after treatment) to day 20 (at the beginning of treatment). N = 7 animals per group at treatment initiation.


F . Growth curves of tumors , on ratios of bioluminescence signals to baseline signals at day 5 .

F. Growth curves of individual tumors, based on the ratios of bioluminescence signals to the baseline signals at day 5.


G . Each mouse was sacrificed when - generated signal reached 1 . 5x107 photons / sec , Kaplan - Meier survival plots were built retrospectively .

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

G. Each mouse was sacrificed when the tumor-generated signal reached 1.5x107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.


A . Intravenously injected Cy5 - labeled 2 ' - O - MOE - PSoligonucleotide is distributed to intracranialGBM8tumor . In normal brain , signal is observed in blood vessels but not within brain parenchyma . " T " - , " B " - brain tissue . Each image is representative of three mice analyzed .

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

A. Intravenously injected Cy5-labeled 2'-O-MOE-PSoligonucleotide is distributed to intracranialGBM8tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. "T" - tumor, "B" - brain tissue. Each image is representative of three mice analyzed.


B - D . Systemic inhibition of miR - 10b markedly reduces burden . Uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control of same chemistry were injected at 80 mg / kg through tail vein at days indicated by arrows . B . left panels illustrate tumorimages of representative at day 34 , average signals ( photons / sec ) are indicated below . bars represent average signal ratios for each group at day 34 , relative to day

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

B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6


B - D . Systemic inhibition of miR - 10b markedly reduces burden . Uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control of same chemistry were injected at 80 mg / kg through tail vein at days indicated by arrows . B . left panels illustrate tumorimages of representative at day 34 , average signals ( photons / sec ) are indicated below . bars represent average signal ratios for each group at day 34 , relative to day 6 . C . Each mouse was sacrificed when - generated signal reached 5X107 photons / sec , Kaplan - Meier survival plots were built retrospectively . D . Growth curves of tumors on ratios of bioluminescence signals to baseline signals at day 6 .

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

B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6.C. Each mouse was sacrificed when the tumor-generated signal reached 5X107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.D. Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6.


E . efficacy of miR - 10b inhibition in intracranial tumors was assessed by qRT - PCR of resected tissues , with miR - 10b expression normalized to miR - 125b .

O O O O O O O O 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. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b.


F . qRT - PCR demonstrates that miR - 10b targets were de - repressed in orthotopic GBM8 upon systemic administration of miR - 10b inhibitor . Seven tumors per condition , two specimens per have been analyzed . mRNA expression were normalized to GAPDH .

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

F. qRT-PCR analysis demonstrates that miR-10b targets were de-repressed in orthotopic GBM8 upon systemic administration of the miR-10b inhibitor. Seven tumors per condition, and two specimens per tumor have been analyzed. mRNA expression levels were normalized to GAPDH.


G . Inverse correlation between miR - 10b expression of its mRNA targets in resected GBM8 tumors .

G. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.


A . Uptake of uncomplexed Cy5 - labeled 2 ' - O - MOE - PSoligonucleotide ( 80 mg / kg injected via tail vein ) by normal extracranial tissues was examined by microscopy24 h after injections .

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

A. Uptake of the uncomplexed Cy5-labeled 2'-O-MOE-PSoligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy24 h after injections.


B - D . Systemic treatment of intracranialGBM8tumors with uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control oligonucleotide ( at 80 mg / kg ) was not associated with toxic effects . B . No significant difference in average mice was observed between anti - miR - 10b control treatment groups . C . No significant difference in average organs ' was observed between anti - miR - 10b control treatment groups . D . No significant difference in tissue histology using Hematoxylin Eosin was observed between anti - miR - 10b control treatment groups .

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

B-D. Systemic treatment of intracranialGBM8tumors with uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects.B. No significant difference in average mice weight was observed between the anti-miR-10b and control treatment groups.C. No significant difference in average organs' weight was observed between the anti-miR-10b and control treatment groups.D. No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti-miR-10b and control treatment groups.


A . Continuous osmotic delivery of miR - 10b inhibitor markedly reduces burden . osmotic pumps , loaded with lipid nanoparticles formulated with 2 ' - O - MOE - POmiR - 10b inhibitor or non - targeting control infused 2 μg of oligonucleotides per day introtumorally , over 13 days . Tumors growth was monitored by WBI , left panels illustrate imaging of representative at end of treatment . bars represent average signal ratios for each group at day 13 ( end of treatment ) , relative to day 2 ( treatment initiation ) .

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

A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2'-O-MOE-POmiR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).


B . Representative immunostaining of tumors for PCNA proliferation marker .

B. Representative immunostaining of tumors for PCNA proliferation marker.


C . Representative immunostaining of tumors for KI67 proliferation marker .

C. Representative immunostaining of the tumors for KI67 proliferation marker.


D . Representative immunostaining for Caspase 3 as a marker of apoptosis .

D. Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis.


E . invasion was examined by microscopy for mCherry - positive migrating through border .

E. Tumor cell invasion was examined by fluorescence microscopy for mCherry-positive cells migrating through the tumor border.


F . Quantitative immunostainings indicates that proliferation apoptosis markers are affected by anti - miR - 10b treatment . No significant effect on invasion of intracranial GBM8 was observed .

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

F. Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti-miR-10b treatment. No significant effect on invasion of intracranial GBM8 was observed.


A , B . miR - 10b inhibition decreases GL261 cellviability . Cellviability was measured at days 3 - 7 after transfection with miR - 10b inhibitor , non - targeting control , or Lipofectamine 2000 alone ( mock ) . A . Phase contrast of GL261 cultures at day 6 post - transfection . B . Growth curves of cultured GL261 , on viability .

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

A, B. miR-10b inhibition decreases GL261 cellviability. Cellviability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.B. Growth curves of cultured GL261 cells, based on the viability assay.


C . InVivo - jet PEI - formulated 2 ' - O - MOE - PS / PO miR - 10b inhibitor or non - targeting control were infused to orthotopic GL261 tumors by osmotic pumps , starting at day 6 after implantation . uptake of ASOs was confirmed by for PS - containing oligonucleotides ( green ) . GL261 expressing M - Cherry are red .

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

C. InVivo-jet PEI - formulated 2'-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red.


D . Osmotic delivery of miR - 10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice . Micephotographs show imaging in representative at day 3 after pump implantation , average signals in photons per second are indicated . Tumorgrowth rates were calculated as ratios of signals at day 3 of treatment to day 1 prior to initiation of treatment .

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

D. Osmotic delivery of miR-10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice. Micephotographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment.


F . qRT - PCR demonstrates that miR - 10b target p21 was de - repressed in GL261 tumors upon miR - 10b inhibition . mRNA expression were normalized to GAPDH .

F. qRT-PCR analysis demonstrates that miR-10b target p21 was de-repressed in GL261 tumors upon miR-10b inhibition. mRNA expression levels were normalized to GAPDH.


( B ) Western blot of lysates media from wt mutant N9 ( N9 wt / mu ) using antibody anti - murine TREM2 ( clone 5F4 ) , which is raised against murine TREM2 extracellular domain . sTREM2 , soluble TREM2 * Indicate unspecific bands . Anti - calnexin antibody was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control.


( C ) Phagocytosis of 1 µM HiLyte ™ Fluor 488 Aβ1 - 42 ( fAβ42 ) by N9 wt N9 mu in presence or absence of antibody 2D8 or non - binding antibody 6687 . Cytochalasin D ( CytoD , 10 mM ) was used as control to verify phagocytic uptake . ( n = 4 , + / - ; 2 way ANOVA , interaction P = 0 . 61 , genotype P < 0 . 0001 , treatment P = 0 . 0001 ; post hoc wt vs . mu for following conditions : fAβ42 P = 0 . 0043 , fAβ42 - 2D8 P = 0 . 0436 ) .

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

(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).


( D ) Western blot of BMDM derived from wt Trem2 knockout ( ko ) using antibody 5F4 .

(D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4.


( E ) Phagocytosis of fAβ42 by BMDM from wt Trem2 ko in presence or absence of 2D8 , or non - binding control antibody 6687 . ( n = 3 , + / - ; 2 way ANOVA , interaction P = 0 . 0005 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc wt vs . ko for following conditions : fAβ42 P = 0 . 0021 , fAβ42 - 2D8 1 µg / ml P < 0 . 0001 , fAβ42 - 2D8 5 µg / ml P < 0 . 0001 , fAβ42 - 2D8 10 µg / ml P < 0 . 0001 , fAβ42 / 6687 10 µg / ml P = 0 . 0007 ) ( F ) of relative fAβ42uptake to lowest antibody used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used.


( G ) Phagocytosis of fAβ42 by BMDM from wt Trem2 ko in presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 4 , + / - ; 2 way ANOVA , interaction P = 0 . 0223 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc wt vs . ko for following conditions : fAβ42 - mAb11 1 µg / ml P = 0 . 0391 , fAβ42 - mAb11 5 µg / ml P = 0 . 0069 , fAβ42 - mAb11 10 µg / ml P < 0 . 0001 , fAβ42 - mAb11 20 µg / ml P = 0 . 0001 , fAβ42 - mAb11 50 µg / ml P < 0 . 0001 ) . ( H ) of relative fAβ42 uptake to lowest antibody used .

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

(G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used.


( I ) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2 - deficient BMDM . Increasing amounts of sTREM2 were added to media of wt or Trem2 ko BMDM in presence or absence of mAb11 ( 10 µg / ml ) .

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

(I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml).


( J ) Western blot of microglia from wt or Trem2 ko using antibody 5F4 .

(J) Western blot of primary microglia from wt or Trem2 ko animals using antibody 5F4.


( K ) Phagocytosis of fAβ42 by microglia from wt Trem2 ko in presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 5 , + / - ; 2 way ANOVA , interaction P = 0 . 4797 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc wt vs . ko for following conditions : fAβ42 - mAb11 5 µg / ml P = 0 . 0449 , fAβ42 - mAb11 10 µg / ml P = 0 . 0370 , fAβ42 - mAb11 20 µg / ml P = 0 . 0299 , fAβ42 - mAb11 50 µg / ml P = 0 . 0120 ) . ( L ) of relative fAβ42uptake to lowest antibody used .

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

(K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.


( A ) Representative histograms for Fcγ - receptors - PE ( FcγR - PE ) expression as used for . Stacked histograms for log PE of wt Trem2 ko BMDM are shown for respective FcγR ( I , IIB / III IV ) . ( B ) Relative of surface of FcγR molecules . Absolute number of surface FcγR - PE molecules was determined by BD QuantiBRITE ® method ( see methods section for details ) normalized to expression of respective wt control . ( n = 4 , + / - , t , two - ; wt vs . ko : FcγRI - PE P = 0 . 0008 , FcγRIIB / III - PE P = 0 . 0033 , FcγRIV - PE P = 0 . 7001 ) .

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

(A) Representative histograms for Fcγ-receptors-PE (FcγR-PE) expression levels as used for quantification. Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR (I, IIB/III and IV).(B) Relative quantification of cell surface levels of FcγR molecules. Absolute number of cell surface FcγR-PE molecules was determined by the BD QuantiBRITE® method (see methods section for details) and normalized to expression levels of the respective wt control. (n=4, +/- SEM, t test, two-tailed; wt vs. ko: FcγRI-PE P=0.0008, FcγRIIB/III-PE P=0.0033, FcγRIV-PE P=0.7001).


( C ) mRNA of FcγR are increased in Trem2 ko BMDM . Fold changes of respective FcγR ( I , IIB , III , IV ) mRNA in Trem2 ko BMDM were determined by quantitative real - time PCR . ( n = 6 , + / - ; one - sample t , two - ; wt vs . ko : FcγRI P = 0 . 0118 , FcγRIIB P = 0 . 0006 , FcγRIII P = 0 . 0004 , FcγRIV P = 0 . 0284 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA levels of FcγR are increased in Trem2 ko BMDM. Fold changes of the respective FcγR (I, IIB, III, IV) mRNA levels in Trem2 ko BMDM were determined by quantitative real-time PCR. (n=6, +/- SEM; one-sample t test, two-tailed; wt vs. ko: FcγRI P=0.0118, FcγRIIB P=0.0006, FcγRIII P=0.0004, FcγRIV P=0.0284)


( D ) Phosphorylated Syk ( P - Syk ) total Syk ( T - Syk ) were determined by western blotting in lysates from wt Trem2 ko BMDM after 1 h treatment with Aβ alone , together with antibody 2D8 ( Aβ - 2D8 ) , or an isotype control ( Aβ - IC ) . Actin was used as a loading control . ( E ) of P - Syk normalized to T - Syk . ( n = 4 , + / - ; 2 way ANOVA , interaction P = 0 . 0490 , genotype P = 0 . 0898 , treatment P < 0 . 0001 . Bonferroni - corrected pair - wise post hoc , * * P = 0 . 0075 vs . wt ) .

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

(D) Phosphorylated Syk (P-Syk) and total Syk (T-Syk) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone, together with antibody 2D8 (Aβ-2D8), or an isotype control (Aβ-IC). Actin was used as a loading control.(E) Quantification of P-Syk normalized to T-Syk. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0490, genotype P=0.0898, treatment P<0.0001. Bonferroni-corrected pair-wise post hoc tests, ** P =0.0075 vs. wt).


( B ) mAb11 but not IC co - localized with methoxy - X04 .

(B) mAb11 but not the IC co-localized with methoxy-X04.


BMDM ( C ) or microglia ( D ) were cultured on cryosections pre - incubated with mAb11 ( 1 µg / ml ) . After 24 h , sections were processed for immunostaining using antibody against CD68 to identify myeloid methoxy - X04 to visualize Aβ . Note that both types internalize Aβ into intracellular vesicles ( right panels show enlargement of insets ) . Scale bar : 10µm .

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

BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.


( A ) BMDM from wt or Trem2 ko mice were cultured on APP / PS1micebrain cryosections incubated with or without mAb11 ( 1 µg / ml ) or an isotype control ( IC ; 1 µg / ml ) for 24 h . Sections were then probed with methoxy - X04 . Scale bar , 500 µm . ( B ) amyloid plaque load was quantified from entire sagittal section . Sections incubated with medium ( no ) were set as baseline . ( n = 6 , + / - ; 2 way ANOVA , interaction P < 0 . 0001 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; Tukey ' s multiple comparisons ; wt vs . ko for following conditions : no antibody P = 0 . 0304 , IC P = 0 . 0049 , mAb11 P = 0 . 0212 ; wt : IC vs . wt : mAb11 P = 0 . 0008 ; ko : IC vs . ko : mAb11 P = 0 . 0001 ) .

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

(A) BMDM from wt or Trem2 ko mice were cultured on APP/PS1micebrain cryosections incubated with or without mAb11 (1 µg/ml) or an isotype control (IC; 1 µg/ml) for 24 h. Sections were then probed with methoxy-X04. Scale bar, 500 µm.(B) The amyloid plaque load was quantified from the entire sagittal section. Sections incubated with medium (no cell) were set as baseline. (n = 6, +/- SEM; 2 way ANOVA, interaction P<0.0001, genotype P<0.0001, treatment P<0.0001; Tukey's multiple comparisons tests; wt vs. ko for the following conditions: no antibody P=0.0304, IC P=0.0049, mAb11 P=0.0212; wt : IC vs. wt : mAb11 P=0.0008; ko : IC vs. ko : mAb11 P=0.0001).


( C , D ) Equal numbers of wt Trem2 ko BMDM were added numbers were analyzed after termination of experiments by quantifying CD68 positive on top of sections . ( n = 4 , + / - ; t - ; n . s . , nonsignificant , P = 0 . 5004 ) . Scale bar , 200 µm .

(C, D) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections. (n = 4, +/-SEM; t-test; n.s., nonsignificant, P=0.5004). Scale bar, 200 µm.


( E ) Aβ was extracted by urea buffer from replicate slices of experiment shown in ( A ) total Aβ was identified by western blotting .

O O O 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) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.


( A ) Cryosections from unfixed brain of 6 - months old APP / PS1mice were pre - incubated with increasing concentrations of mAb11 ( 0 . 001 , 0 . 01 , 0 . 1 , 1 , 5 µg / ml ) . BMDM from wt or Trem2 ko mice were added for 24 h . Sections were stained with methoxy - X04 . Scale bar , 500 µm . ( B ) Methoxy - X04 signals were quantified from entire sagittal section . ( n = 5 , + / - ; 2 way ANOVA , interaction P = 0 . 0082 , genotype P < 0 . 0001 , treatment P < 0 . 0001 . Fisher ' s LSD post hoc comparisons ; * show statistics between wt ko under same experimental condition . # in black show wt compares to no antibody stimulation ; # in grey show ko compares to no antibody stimulation ; wt vs . ko for following conditions : no antibody P = 0 . 0053 , mAb11 0 . 001 µg / ml P = 0 . 0003 , mAb11 0 . 01 µg / ml P < 0 . 0001 , mAb11 0 . 1 µg / ml P = 0 . 0001 , mAb11 1 µg / ml P = 0 . 0007 , mAb11 5 µg / ml P = 0 . 0011 ; Following conditions compare to wt / no antibody : wt / mAb11 0 . 01 µg / ml P = 0 . 0166 , wt / mAb11 0 . 1 µg / ml P = 0 . 0002 , wt / mAb11 1 µg / ml P < 0 . 0001 , wt / mAb11 5 µg / ml P < 0 . 0001 ; Following conditions compare to ko / no antibody : ko / mAb11 0 . 1 µg / ml P = 0 . 0099 , ko / mAb11 1 µg / ml P < 0 . 0001 , ko / mAb11 5 µg / ml P < 0 . 0001 .

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

(A) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.


Dynamic patterns according to density in 8 × GTIIC - EmGFP 293T .

Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.


EmGFP in LATS2 knockdown , or forced expression of a constitutively active form of YAP ( YAP - S127A ) . Scale bars indicate 100 μm .

EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.


Scatter plots of mean frequency of each shRNA per gene . Average of v HOXA4 is indicated as a red dot .

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

Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.


Augmented 8 × GTIIC - luciferase reporter activity by loss of HOXA4 . ShSAV1 was used as an experimental control . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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

Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Quantitative real - time PCR of ANKRD1 expression in HEK 293T transfected with

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4


Quantitative real - time PCR of ANKRD1 expression in HEK 293T transfected with HOXA4 expression

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression vector


Quantitative real - time PCR of CTGF , CYR61 , BIRC5 in HEK 293T with knockdown of endogenous HOXA4 . ShLATS1 / 2 shYAP were used as an experimental control . * P < 0 . 05 , * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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

Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


8 × GTIIC - luciferase reporter assays in HEK 293T transfected HOXA4 / or shLATS1 / 2 . * * P < 0 . 01 vs shCtrl , by two - way ANOVA with Sidak ' s correction .

8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.


Quantitative real - time PCR of HOXA4 , YAP , CTGF , CYR61 in HEK 293T with overexpression of HOXA4 / or constitutive active form of YAP ( YAP - 5SA ) . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with LacZ , by ANOVA with Sidak ' s correction .

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

Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.


Quantitative real - time PCR of HOXA4 , YAP , CTGF , CYR61 in HEK 293T with knockdown of endogenous HOXA4 / or overexpression of YAP - 5SA . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with shCtrl , by ANOVA with Sidak ' s correction .

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

Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.


Association of HOXA4 with TEAD1 indicated by co - IP experiments . HEK 293T were co - transfected with FLAG - HOXA4 Myc -

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

Association of HOXA4 with TEAD1 indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1


Association of HOXA4 but not YAP indicated by co - IP experiments . HEK 293T were co - transfected with FLAG - HOXA4 Myc - YAP .

Association of HOXA4 but not YAP indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-YAP.


Interaction between endogenous TEAD1 HOXA4 by co - IP experiments in HEK 293T . interaction between endogenous TEAD1 YAP was used as a positive control .

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

Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T transfected with plasmids expressing HOXA4 truncation mutants TEAD1 .

Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T transfected with plasmids expressing TEAD1 truncation mutants HOXA4 . Arrowheads indicate IgG heavy chain . Arrows indicate TEAD1 ( a . a . 1 - 426 ) .

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

Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).


Requirement of TEAD1 ( a . a . 30 - 135 ) for its interaction with HOXA4 via its homeodomain ( a . a . 216 - 274 ) .

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

Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).


Failure of TEAD1 ( a . a . 30 - 102 ) , TEA domain alone , to interact with HOXA4 .

Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.


ChIP assays of CTGF CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T transfected with plasmids expressing HOXA4 . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - . Data are expressed as means ± of three independent experiments .

ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Competitive co - IP densitometry showing significantly reduced TEAD - YAP interaction by overexpression of HOXA4 in a dose - dependent manner in HEK 293T . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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

Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Expression of Hoxa4 in various mouse tissues using qRT - PCR . Expression of glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) mRNA was used as an internal control . WAT ; adipose tissue , BAT ; brown adipose tissue , Skm ; skeletal muscle .

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

Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.


Quantitative real - time PCR of differentiated VSMC marker genes in human VSMCs with PDGF - BB stimulation for 24 hours . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Quantitative real - time PCR of smooth muscle specific genes in human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 YAP . infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

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

Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of YAP / TAZ Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

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

Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Quantitative real - time PCR of YAP / TEAD target genes in human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 YAP . infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

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

Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then were collected counted at each time point as indicated . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

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

Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with indicated genes . of BrdU positive in each group is shown . Nine to twelve were randomly acquired analyzed . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

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

BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then were collected counted at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction .

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

Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with indicated genes . of BrdU positive in each group is shown . Nine to twelve were randomly acquired analyzed . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 TEAD1 / 3 / 4 . of alpha smooth muscle actin calponin is expressed as means ± of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction .

Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then viability was analyzed by Counting Kit - 8 at indicated days at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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

Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Representative immunoblotting of interaction between endogenous TEAD1 HOXA4 or YAP using co - IP experiments in human VSMCs with knockdown of HOXA4 . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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

Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.