( H ) SMAD3 depletion decreases expression of SMAD3 - regulated genes . SMAD3 knock - down by siRNA decreased mRNA expression of BRAFi - resistance genes in SKMel28R and Me1402 . NRP1 mRNA was not detected in our experimental conditions .

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

(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions.


IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


IncuCyte cell proliferation curves for ASNS knockout ( sgASNS ) and control ( sgNon - targeting ) PC3 cells in the absence and presence of ASNase . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen . Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate ( FDR ) < 0 . 003 .

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

Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003.


IncuCyte cell proliferation curves of SLC1A3 knockout ( sgSLC1A3 ) and control ( sgNon - targeting ) PC3 cells in the absence and presence of ASNase treatment . # 3 and # 4 represent 2 different sgRNAs targeting SLC1A3 . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Radioactive labeled aspartate and glutamate uptake measurement in control ( sgNon - targeting ) and SLC1A3 knockout ( sgSLC1A3 ) PC3 cells . # 3 and # 4 represent two different sgRNAs targeting SLC1A3 . Radioactive labeled leucine uptake was used as a control . Data was normalized to the reads of control PC3 cells .

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

Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.


Endogenous levels of aspartate , asparagine , glutamate and glutamine in control ( sgNon - targeting ) and SLC1A3 knockout ( sgSLC1A3 ) PC3 cells with or without ASNase for 3 days . Median peak intensity was used for the read normalization .

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

Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.


IncuCyte cell proliferation curves of SLC1A3 knockout ( sgSLC1A3 # 3 ) PC3 cells treated with ASNase and supplemented with either esterified - aspartate ( 6mM ) or esterified - glutamate ( 6mM ) , and esterified - leucine ( 6mM ) as a control . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


RT - qPCR analysis was used to determine the relative SLC1A3 mRNA expression ( to GAPDH ) in different prostate and breast cancer cell lines , as indicated . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-DISEASE O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR analysis was used to determine the relative SLC1A3 mRNA expression (to GAPDH) in different prostate and breast cancer cell lines, as indicated. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


cell lines were transduced with either control ( sgNon - targeting ) or sgSLC1A3 . Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines . Leucine uptake level was used for normalization . The numbers above the control column denote the basal aspartate uptake capacity . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

cell lines were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


RT - qPCR was used to determine the relative mRNA levels ( to GAPDH ) of aspartate / glutamate transporter genes ( SLC1A1 , SLC1A2 , SLC1A3 , SLC1A6 and SLC1A7 ) in LNCaP , BT549 and SUM159PT cells . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

RT-qPCR was used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations . ' ns ' indicates no significant difference . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations. 'ns' indicates no significant difference. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


MCF7 and DU145 cells were transduced with either lentiviral empty vector ( control ) or lentiviral vector containing a V5 - tagged SLC1A3 coding sequence ( V5 - SLC1A3 ) . Relative SLC1A3 mRNA levels ( to GAPDH ) were determined by RT - qPCR .

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MCF7 and DU145 cells were transduced with either lentiviral empty vector (control) or lentiviral vector containing a V5-tagged SLC1A3 coding sequence (V5-SLC1A3). Relative SLC1A3 mRNA levels (to GAPDH) were determined by RT-qPCR.


Immunofluorescence staining of the V5 - tagged SLC1A3 in MCF7 and DU145 cells using anti - V5 antibody . Green staining indicates the plasma membrane localization of V5 - SLC1A3 and blue DAPI staining marks the nuclei . Scale bar stands for 5 μm .

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Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.


Relative aspartate uptake levels in control and V5 - SLC1A3 - expressed MCF7 and DU145 cells . Leucine uptake level was used for normalization .

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Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.


Control and V5 - SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations .

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

Control and V5-SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations.


DU145 cells were supplemented with cell - permeable aspartate ( 6mM , esterified ) or glutamate ( 3mM , esterified ) following ASNase treatment , with esterified leucine ( 6mM or 3mM ) as control .

B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O

DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.


PC3 , DU145 and V5 - SLC1A3 - DU145 cells were subjected to ASNase and TFB - TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay .

PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.


PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions . ASNase ( 0 . 3 U / ml ) , TFB - TBOA ( 5 μM ) .

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PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).


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 .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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 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 .

B-CELL_LINE B-ORGANISM B-DISEASE I-DISEASE O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O B-ORGANISM B-DISEASE I-DISEASE O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O B-ORGANISM B-DISEASE I-DISEASE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD B-ORGANISM 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 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 ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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 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 ) .

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

(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 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 .

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

(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 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 ) .

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

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

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

(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 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 .

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

. (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 ) 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 .

O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 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 .

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

(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 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 .

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

(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 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 -

O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-ORGANISM O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-TISSUE B-SMALL_MOLECULE O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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(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 and Atg5 Tg MEFs ( n = 2 ) .

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


( 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 .

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

(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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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 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 # # # , * * * ) .

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

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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 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 * * * .

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

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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 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 .

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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 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 .

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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 . 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 .

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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 . 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 ) .

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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 . 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 ) .

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

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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 ) 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 * * * .

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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 ) 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 .

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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 . 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 ) .

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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 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 .

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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 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 * * .

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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 the different drugs . Comparison of Survival Curves have been performed using the nonparametric Log - rank ( Mantel - Cox ) test .

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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 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 .

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

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


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 .

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

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 , 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 " .

O O O B-CELL_TYPE O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 . 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 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-DISEASE O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O B-DISEASE O O O O O O O O O O O O O O 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-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O B-TISSUE 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 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 .

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

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

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 analysis demonstrates that miR - 10b inhibition in orthotopic GBM8 leads do de - repression of its mRNA targets . mRNA expression levels were normalized to GAPDH .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD 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 levels and expression of its mRNA targets in resected GBM8 tumors .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_LINE O O

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


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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 individual tumors , based on the ratios of bioluminescence signals to the baseline signals at day 5 .

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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 the tumor - generated signal reached 1 . 5x107 photons / sec , and Kaplan - Meier survival plots were built retrospectively .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 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 .

O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O B-ORGANISM 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 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

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 levels and 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 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 .

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

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 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 .

O O O O O O O B-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 ) .

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

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 .

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B. Representative immunostaining of tumors for PCNA proliferation marker.


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

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


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

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


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

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

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


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 .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE B-CELL_LINE 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 photographs of GL261 cultures at day 6 post - transfection . B . Growth curves of cultured GL261 cells , based on the viability assay .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 cell implantation . The uptake of ASOs was confirmed by IHC for PS - containing oligonucleotides ( green ) . GL261 tumor cells expressing M - Cherry are red .

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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 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 .

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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.


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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 .

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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 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 .

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(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 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 ) .

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(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 and Trem2 knockout ( ko ) animals using antibody 5F4 .

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