F , Western Blot of METneg METhigh subpopulations sorted from BT308NS , showing constitutive ( ctrl ) / or IR - induced phosphorylation of ATM ( pATM ) , Chk2 ( pChk2 ) H2AX ( H2AX ) , accumulation of RAD51 , 24 h after IR ( 5 Gy ) . Total ATM is also shown . β - actin was used as loading control .

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

F, Western Blot of METneg and METhigh subpopulations sorted from BT308NS, showing constitutive (ctrl) and/or IR-induced phosphorylation of ATM (pATM), Chk2 (pChk2) and H2AX (H2AX), and accumulation of RAD51, 24 h after IR (5 Gy). Total ATM protein is also shown. β-actin was used as loading control.


G , qPCR of RAD51 expression in METhigh METneg subpopulations sorted from BT308NS 24 h after IR ( 5 Gy , fold vs . non - irradiated control , ctrl ) . * : t - , P = 0 . 02 . Data are represented as mean ± .

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

G, qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR (5 Gy, fold vs. non-irradiated control cells, ctrl). *: t-test, P=0.02. Data are represented as mean ± SEM.


A , viability of BT308NS , measured 48 h after IR ( 2 or 5 Gy ) in absence ( mock ) or in presence of 10 ng / ml HGF ( fold vs . non - irradiated mock treated , mock ) . Ctrl : non - irradiated . * : t - ( 5 Gy + HGF ) vs . ( 5 Gy ) , p < 0 . 0001 .

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

A, Cell viability of BT308NS, measured 48 h after IR (2 or 5 Gy) in the absence (mock) or in the presence of 10 ng/ml HGF (fold vs. non-irradiated mock treated cells, mock). Ctrl: non-irradiated cells. *: t-test (5 Gy + HGF) vs. (5 Gy), p<0.0001.


B , Flow - cytometry of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in absence ( mock ) or in presence of HGF . Histograms show of Annexin V - positive 48 h after IR . Ctrl : non - irradiated .

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

B, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (mock) or in the presence of HGF. Histograms show the percentage of Annexin V-positive cells 48 h after IR. Ctrl: non-irradiated cells.


C , viability of BT308NS , irradiated in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of MET inhibitor JNJ38877605 ( 500 nM ) , analyzed at indicated time points . Vehicle : non - irradiated ( fold vs . vehicle - treated at time 0 , mock ) . * : t - , p < 0 . 01 .

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

C, Cell viability of BT308NS, irradiated in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM), and analyzed at the indicated time points. Vehicle: non-irradiated cells (fold vs. vehicle-treated cells at time 0, mock). *: t-test, p<0.01.


D , viability as in C , measured 5 days after IR ( 1 - 10 Gy , fold vs . vehicle - treated , mock ) . * : t - , p < 0 . 03 .

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

D, Cell viability as in C, measured 5 days after IR (1-10 Gy, fold vs. vehicle-treated cells, mock). *: t-test, p<0.03.


E , Flow - cytometry of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in absence ( vehicle ) or in presence ( JNJ ) of JNJ38877605 . Histograms show of Annexin V - positive 36 h after IR . Ctrl : non - irradiated . * : t - ( 5 Gy + JNJ ) vs . ( 5 Gy ) , P = 0 . 01 .

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

E, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Histograms show the percentage of Annexin V-positive cells 36 h after IR. Ctrl: non-irradiated cells. *: t-test (5 Gy + JNJ) vs. (5 Gy), P=0.01.


F , Western blot of BT308NS showing caspase 3 activation ( casp - 3 ) , at indicated time points after IR in absence ( - ) or in presence ( + ) of JNJ38877605 . Vinculin was used as loading control .

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

F, Western blot of BT308NS showing caspase 3 activation (cleaved casp-3), at the indicated time points after IR in the absence (-) or in the presence (+) of JNJ38877605. Vinculin was used as loading control.


G , Clonogenic with METneg METhigh subpopulations sorted from BT308NS , irradiated ( 5 Gy ) in absence ( vehicle ) or in presence of MET inhibitors ( JNJ38877605 Crizotinib , 500 nM ) . Ctrl : non - irradiated . * : t - , p < 0 . 0004 .

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

G, Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS, irradiated (5 Gy) in the absence (vehicle) or in the presence of MET inhibitors (JNJ38877605 and Crizotinib, 500 nM). Ctrl: non-irradiated cells. *: t-test, p<0.0004.


H , LDA ( sphere forming ) with METneg METhigh subpopulations sorted from BT308NS , measuring GSC frequency after irradiation in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated . * : 2 , P = 0 . 0002 .

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

H, LDA (sphere forming assay) with METneg and METhigh subpopulations sorted from BT308NS, measuring GSC frequency after irradiation in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. *: 2 test, P=0.0002.


I , Flow cytometry of phosphorylated histone H2AX ( H2AX ) in whole BT308NS at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 ( fold vs . non - irradiated at time 0 , mock ) . * : t - , p < 0 . 05 .

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

I, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) in the whole BT308NS at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605 (fold vs. non-irradiated cells at time 0, mock). *: t-test, p<0.05.


J , Neutral comet with BT308NS performed 24 h after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated . Comet tail ' s is proportional to DSB extent . Data are represented as mean ± or ± CI in H .

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

J, Neutral comet assay with BT308NS performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. Comet tail's length is proportional to DSB extent. Data are represented as mean ± SEM or ± CI in H.


A , Western blot of BT463NS showing phosphorylation of ATM ( pATM ) Chk2 ( pChk2 ) , accumulation of RAD51 at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) . Total ATM is also shown . H3 was used as loading control .

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

A, Western blot of BT463NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). Total ATM is also shown. H3 was used as loading control.


B , Western blot of BT308NS showing phosphorylation of ATM ( pATM ) Chk2 ( pChk2 ) , accumulation of RAD51 at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of MET inhibitor JNJ38877605 ( 500 nM ) . Total ATM is also shown . β - actin was used as loading control . Veh : non - irradiated vehicle - treated . C , Densitometric of ATM phosphorylation shown in ( B ) . * : t - , p < 0 . 01 .

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

B, Western blot of BT308NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). Total ATM is also shown. β-actin was used as loading control. Veh: non-irradiated vehicle-treated cells. C, Densitometric analysis of ATM phosphorylation shown in (B). *: t-test, p<0.01.


D , Western blot of BT308NS showing phosphorylation of ATM , Chk2 , AKT ( pAKT ) , accumulation of RAD51 24 h after IR ( 5 Gy ) in absence ( vehicle , veh ) or in presence of MAPK ( PD98059 , 20 M ) , AKT ( Ly294002 , 20 M ) , MET ( JNJ38877605 ) , or ATM ( CGK733 , 10 M ) inhibitors . Total ATM AKT are also shown . β - actin was used as loading control . Ctrl : non - irradiated .

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

D, Western blot of BT308NS showing phosphorylation of ATM, Chk2, and AKT (pAKT), and accumulation of RAD51 24 h after IR (5 Gy) in the absence (vehicle, veh) or in the presence of MAPK (PD98059, 20 M), AKT (Ly294002, 20 M), MET (JNJ38877605), or ATM (CGK733, 10 M) inhibitors. Total ATM and AKT are also shown. β-actin was used as loading control. Ctrl: non-irradiated cells.


E , Western blot of BT463NS showing phosphorylation of ATM , Chk2 , Aurora Kinase A ( pAurora A ) at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + Ly ) of AKT inhibitor Ly294002 . Total ATM Aurora Kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated .

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

E, Western blot of BT463NS showing phosphorylation of ATM, Chk2, and Aurora Kinase A (pAurora A) at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + Ly) of the AKT inhibitor Ly294002. Total ATM and Aurora Kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


F , Western blot of BT308NS showing phosphorylation of Aurora Kinase A at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 . Total Aurora kinase A is also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated .

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

F, Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Total Aurora kinase A is also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


G , Western blot of BT463NS showing phosphorylation of Aurora Kinase A at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + HGF ) of HGF . Total Aurora kinase A is also shown . H3 was used as loading control ( same control applies to both panel A G ) .

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

G, Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF. Total Aurora kinase A is also shown. H3 was used as loading control (the same control applies to both panel A and G).


H , Western blot of BT308NS showing phosphorylation of ATM Aurora Kinase A at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + MLN ) of Aurora Kinase A inhibitor MLN8237 ( 20 M ) . Total ATM Aurora kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated .

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

H, Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + MLN) of the Aurora Kinase A inhibitor MLN8237 (20 M). Total ATM and Aurora kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


I , viability of BT308NS , measured 72 h after IR ( 5 Gy ) in absence ( vehicle ) or in presence of MET ( JNJ38877605 ) , Aurora Kinase A ( MLN8237 ) , or AKT ( Ly294002 ) inhibitors ( fold vs . non - irradiated vehicle - treated , mock ) . * : t - ( 5 Gy + inhibitor ) vs . ( 5 Gy + vehicle ) , p < 0 . 01 .

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

I, Cell viability of BT308NS, measured 72 h after IR (5 Gy) in the absence (vehicle) or in the presence of MET (JNJ38877605), Aurora Kinase A (MLN8237), or AKT (Ly294002) inhibitors (fold vs. non-irradiated vehicle-treated cells, mock). *: t-test (5 Gy + inhibitor) vs. (5 Gy + vehicle), p<0.01.


A , Western blot of BT371NS showing p21 phosphorylation ( pp21Thr145 ) in lysates immunoprecipitated with p21 antibodies at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) .

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

A, Western blot of BT371NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml).


B , Western blot of BT463NS showing p21 phosphorylation ( pp21Thr145 ) in lysates immunoprecipitated with p21 antibodies at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of MET inhibitor JNJ38877605 ( 500 nM ) .

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

B, Western blot of BT463NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM).


C , Western blot of BT308NS showing p21 localization in / extracts at indicated time points after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 . Vinculin lamin B were used as loading controls for fractions , respectively .

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

C, Western blot of BT308NS showing p21 localization in cytoplasmic/nuclear extracts at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions, respectively.


D , Representative immunofluorescence of p21 ( red ) on BT308NS 3 h after IR ( 5 Gy ) in absence ( vehicle ) or in presence ( JNJ ) of JNJ38877605 . Ctrl : non - irradiated . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 µm ( 63 × magnification ) . E , of of showing p21 or localization in BT308NS represented in ( D ) ( n = 10 HPF / group ) . HPF : high - power field . * : t - ( 5 Gy + JNJ ) vs . ( ctrl or 5 Gy ) , p < 0 . 0001 .

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

D, Representative immunofluorescence staining of p21 (red) on BT308NS cells 3 h after IR (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Ctrl: non-irradiated cells. Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm (63× magnification). E, Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in (D) (n = 10 HPF/group). HPF: high-power field. *: t-test (5 Gy + JNJ) vs. (ctrl or 5 Gy), p<0.0001.


F , cycle of BT314NS , performed 24 h after IR in absence ( 5 Gy ) or in presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated . * : one - way ANOVA , P = 0 . 008 .

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

F, Cell cycle analysis of BT314NS, performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated cells. *: one-way ANOVA, P=0.008.


B right , of epi - GFP signal from tumors represented in ( B , left ) ( n = 4 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 5 . 5 × 10 - 5 .

O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

B right, Quantification of epi-fluorescent GFP signal from tumors represented in (B, left) (n = 4/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=5.5×10-5.


C , Growth curves of tumors , generated by subcutaneous injection of BT308NS , irradiated in absence ( 2 Gy × 3 days ) or in presence ( combo ) of JNJ38877605 , which was administered for 15 days as indicated ( n = 7 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 0 . 0006 .

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

C, Growth curves of tumors, generated by subcutaneous injection of BT308NS, irradiated in the absence (2 Gy × 3 days) or in the presence (combo) of JNJ38877605, which was administered for 15 days as indicated (n = 7/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=0.0006.


D , Survival of mice bearing tumors generated treated as in C . Black dot : censored mouse . Log - rank ( Mantel - Cox ) ( combo ) vs . ( 2 Gy × 3 days ) , P = 0 . 0019 .

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

D, Survival analysis of mice bearing tumors generated and treated as in C. Black dot: censored mouse. Log-rank (Mantel-Cox) test (combo) vs. (2 Gy × 3 days), P=0.0019.


F , In vitro LDA ( sphere - forming ) measuring GSC frequencies as described in E ( n = 3 / condition ) . * : 2 , p < 10 - 5 .

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

F, In vitro LDA (sphere-forming) measuring GSC frequencies as described in E (n = 3/condition). *: 2 test, p<10-5.


G , incidence in in vivo LDA performed on tumors generated treated as described in E .

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

G, Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E.


H , Histogram showing GSC frequencies measured by in vivo LDA , as described in E . * : 2 , P = 5 . 47 × 10 - 6 . I , Table showing GSC frequencies measured by in vivo LDA , represented in H . Data are represented as mean ± or ± CI in F , H , I .

O O O O B-CELL_TYPE 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 B-CELL_TYPE 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

H, Histogram showing GSC frequencies measured by in vivo LDA, as described in E. *: 2 test, P=5.47×10-6. I, Table showing GSC frequencies measured by in vivo LDA, represented in H. Data are represented as mean ± SEM or ± CI in F,H,I.


( A , B ) Poly ( I : C ) treatment inhibits axonal growth at 3 DIV ( A ) dendritic growth at 6 DIV ( B ) in WT neurons . Neurons were transfected with GFP at 1 or 4 DIV . One day later , neurons were treated with poly ( I : C ) for 24 h before harvest . ( C ) Axon dendrite of Tlr3 - / - neurons after poly ( I : C ) treatment . Data in A , B , C were analyzed by unpaired t - . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 20μm in A B .

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

(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B.


( D , E ) Neuronal in saline - poly ( I : C ) - treated Tlr3 + / + ; Thy1 - Yfp ( D ) Tlr3 - / - ; Thy1 - Yfp ( E ) mouse brains . Total dendrite , dendritic tip number Sholl of dendritic processes were used to examine dendrite phenotype . Data of dendrite tip number were analyzed by unpaired t - . Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . numbers of analyzed neurons mice are presented in each column . Mean values are shown . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 50 μm in D E .

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

(D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3-/-;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.


( A , B ) Quantitation of dendrite of Trif mutant neurons ( A ) Myd88 - / - neurons ( B ) after poly ( I : C ) stimulation . Trif mutant or Myd88 - / - neurons were transfected with a GFP construct at 4 DIV treated with poly ( I : C ) one day later . Neuron was analyzed at 6 DIV according to GFP signal . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by unpaired t - .

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

(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88-/- neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88-/- neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.


( C ) MYD88 but not TRIF expression reduces dendrite outgrowth . HA - tagged MYD88 TRIF were co - transfected with a GFP construct into cultured wild - type ( WT ) neurons at 4 DIV , was analyzed at 6 DIV . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by one - way ANOVA with Bonferroni ' s multiple comparison .

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

(C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni's multiple comparison test.


( D ) MYD88 knockdown , but not TRIF knockdown , robustly increases dendritic arbors loses response to poly ( I : C ) . Three DIV neurons were transfected with control shRNA ( shCtrl ) , MYD88 shRNA ( shMYD88 ) or TRIF shRNA ( shTRIF ) poly ( I : C ) was applied into culture at 5 DIV for 24 h before harvest . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . Scale bar , 20 μm . * * * P < 0 . 0001 .

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

(D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.


( A ) Coimmunoprecipitation of TLR3 MYD88 . HEK293T were transfected with indicated constructs . Twenty - four h later , immunoprecipitation ( IP ) was performed using HA antibody . precipitates were immunoblotted ( IB ) with Myc HA antibodies as indicated . EC , TLR3 ectodomain .

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

(A) Coimmunoprecipitation of TLR3 and MYD88. HEK293T cells were transfected with the indicated constructs. Twenty-four h later, immunoprecipitation (IP) was performed using HA antibody. The precipitates were immunoblotted (IB) with Myc and HA antibodies as indicated. EC, TLR3 ectodomain.


( B ) Poly ( I : C ) treatment increases interaction between TLR3 MYD88 . One day after transfection , were treated with saline or poly ( I : C ) for 30 min before harvesting .

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

(B) Poly(I:C) treatment increases the interaction between TLR3 and MYD88. One day after transfection, cells were treated with saline or poly(I:C) for 30 min before harvesting.


( C ) Upper , schematic of MYD88 constructs . DD , domain ; ID , intermediate domain , TIR , TIR domain . Lower , coimmunoprecipitation of TLR3 N - terminal region of MYD88 .

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

(C) Upper, schematic of MYD88 constructs. DD, death domain; ID, intermediate domain, TIR, TIR domain. Lower, coimmunoprecipitation of TLR3 and the N-terminal region of MYD88.


( D ) Full - N - terminal MYD88 inhibit dendrite outgrowth . Myd88 - / - neurons were transfected with control vector ( Ctrl ) , HA - tagged MYD88 ( MYD88 ) , N - terminal MYD88 ( MYD88 - N ) , or C - terminal MYD88 ( MYD88 - C ) with a GFP construct . Poly ( I : C ) was applied into culture at 5 DIV neuronal was analyzed at 6 DIV . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . Scale bar , 20 μm .

O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-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 O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Full-length and N-terminal MYD88 inhibit dendrite outgrowth. Myd88-/- neurons were transfected with control vector (Ctrl), HA-tagged MYD88 (MYD88), N-terminal MYD88 (MYD88-N), or C-terminal MYD88 (MYD88-C) with a GFP construct. Poly(I:C) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm.


( E ) Expression patterns of full - truncated fragments of MYD88 in Myd88 - / - neurons . Representative are shown .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O

(E) Expression patterns of full-length and truncated fragments of MYD88 in Myd88-/- neurons. Representative images are shown.


( A ) qPCR of proinflammatory cytokines Il - 6 , Tnfα , Il - 1β antiviral cytokine Ifnβ in WT cultured cortical hippocampal neurons . Data were analyzed by unpaired t - . Il - 6 Tnfα N = 8 ; Ifnβ Il - 1β , N = 6 .

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

(A) qPCR analysis of proinflammatory cytokines Il-6, Tnfα, Il-1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons. Data were analyzed by unpaired t-test. Il-6 and Tnfα N=8; Ifnβ and Il-1β, N=6.


( B , C ) Poly ( I : C ) treatment inhibits dendrite outgrowth of Il - 6 - / - neurons ( B ) Tnfα - / - neurons ( C ) . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by unpaired t - .

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

(B, C) Poly(I:C) treatment inhibits dendrite outgrowth of Il-6-/- neurons (B) and Tnfα-/- neurons (C). Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.


( D ) Left , schematic of experiment using conditioned medium . Upper right , conditioned medium applied to WT neurons . Lower right , conditioned medium applied to Tlr3 - / - neurons . Dendrite was analyzed one day after adding conditioned medium . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by unpaired t - . * P < 0 . 05 , * * * P < 0 . 0001 .

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 B-GENEPROD O O O B-CELL_TYPE 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 B-CELL_TYPE O O O O O O O O O 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) Left, schematic of experiment using conditioned medium. Upper right, conditioned medium applied to WT neurons. Lower right, conditioned medium applied to Tlr3-/- neurons. Dendrite morphology was analyzed one day after adding conditioned medium. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. * P < 0.05, *** P < 0.0001.


At 4 DIV , cultured cortical neurons were transfected with indicated plasmids . Three days later , neurons were treated with 10 μg / ml poly ( I : C ) for 24 h . Neuronal was monitored by GFP signals at 8 DIV . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . Scale bar , 20 μm . * * * P < 0 . 0001 , ns : non - significant .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-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 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 O O O O O O O O O O O O O O O O

At 4 DIV, cultured cortical neurons were transfected with indicated plasmids. Three days later, neurons were treated with 10 μg/ml poly(I:C) for 24 h. Neuronal morphology was monitored by GFP signals at 8 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001, ns: non-significant.


( A ) Expression of 12 neuropsychiatric disorder - related genes in saline - poly ( I : C ) - treated WT neurons .

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

(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons.


( B ) Expression of 9 neuropsychiatric disorder - related genes in poly ( I : C ) - treated Tlr3 - / - neurons . Relative expression compared with vehicle control are shown . In ( B - D ) , dashed lines indicate level of saline control .

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

(B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control.


( C , D ) Expression of Auts2 , Disc1 , Fmr1 , Pten Ube3a in poly ( I : C ) - treated Myd88 - / - neurons ( C ) poly ( I : C ) - treated P5 WT mouse brains ( D ) . In ( B - D ) , dashed lines indicate level of saline control .

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

(C, D) Expression of Auts2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88-/- neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). In (B-D), dashed lines indicate the level of saline control.


( E ) DIV 5 WT neurons were treated with poly ( I : C ) for 24 h . total neuronal lysates were subjected to immunoblotting with indicated antibodies . VCP β - actin were used as loading controls . were normalized with β - actin . Data were analyzed by unpaired t - . Numbers of experimental repeats ( N ) are shown in each column . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 .

O O O O O O B-CELL_TYPE O O O B-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 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 O

(E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.


( A ) Overexpression of WT DISC1 , but not DISC1 L604F mutant , in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation . Mean values of representatives of three independent experiments are shown . numbers of analyzed neurons in representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . Scale bar , 20 μm . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O B-GENEPROD 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 O O O O O O O O O O O O O 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) Overexpression of WT DISC1, but not the DISC1 L604F mutant, in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. * P < 0.05, ** P < 0.001, *** P < 0.0001.


( B - C ) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly ( I : C ) - triggered reduction of dendritic arborization . Vector control ( Ctrl ) ( B ) or Myc - DISC1 ( C ) was co - expressed with GFP in cortical layer 2 / 3 neurons in mouse brain . After poly ( I : C ) injection at P4 P5 , neuronal was analyzed at P7 by tracing GFP signals . data of dendrite tip number were analyzed by unpaired t - . Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison . numbers of analyzed neurons collected from 3 - 4 mice of each group are indicated . Mean values are shown . Scale bar , 30 μm . * P < 0 . 05 , * * * P < 0 . 0001 .

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

(B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.


( A ) Cortical hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV treated with poly ( I : C ) ( pIC ) at 17 DIV for 24 h . Spine was examined at 18 DIV . Three secondary dendrites of each neuron were selected to analyze spine density , width of spine head spine . Mean values of representatives of three independent experiments are shown . For saline group , 15 neurons / 45 dendrites were examined ; for poly ( I : C ) group , 16 neurons / 48 dendrites were examined . Data were analyzed by unpaired t - . Scale bar , 20 μm in right upper panel 5 µm in lower left panel . * * * P < 0 . 0001 .

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

(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001.


( C , D ) Spine of P21 Thy1 - Yfp ( C ) Tlr3 - / - ; Thy1 - Yfp mice ( D ) after poly ( I : C ) stimulation at P4 P5 . One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine spine . Three mice were analyzed for each group . Saline - treated Thy1 - Yfp , 44 neurons ; poly ( I : C ) - treated Thy1 - Yfp , 43 neurons ; saline - treated Tlr3 - / - ; Thy1 - Yfp , 46 neurons ; poly ( I : C ) - treated Tlr3 - / - ; Thy1 - Yfp , 50 neurons . Mean values are shown . Data were analyzed by unpaired t - . Scale bar , 5 μm . * * * P < 0 . 0001 .

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

(C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3-/-; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3-/-;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3-/-;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.


reversal potential of currents was positively shifted when a long voltage ramp followed depolarizing steps . ( top panel ) voltage protocol used for experiment . ( bottom panel ) Representative traces of currents elicited by voltage ramp pulses are shown . ( top panel ) . red represents arithmetical difference between currents elicited by 500 - ms ( 2 ) 2 - ms pulses ( 1 ) . B . summary bar graph represents mean values of reversal potentials of currents . ( 9 from 3 mice , paired Student t - ; data are presented as mean values ± s . e . m ) .

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

The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps. (top panel) The voltage protocol used for the experiment. (bottom panel) Representative traces of currents elicited by voltage ramp pulses are shown. (top panel). The red line represents the arithmetical difference between currents elicited by 500-ms (2) and 2-ms pulses (1). B. The summary bar graph represents the mean values of the reversal potentials of currents. (9 cells from 3 mice, paired Student t-test; data are presented as mean values ± s.e.m).


Tail currents elicited by hyperpolarizing voltage steps following depolarizing steps of variable durations . Representative traces of currents elicited by voltage protocol with a pipette solution containing 150 mM Cl - 0 . 2 mM EGTA ( top panel ) , 150 mM gluconate ( middle panel ) , or 2 mM BAPTA ( bottom panel ) .

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

Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations. Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl- and 0.2 mM EGTA (top panel), 150 mM gluconate (middle panel), or 2 mM BAPTA (bottom panel).


A summary graph of averaged tail current amplitudes as a function of depolarizing step duration ( numbers on right indicate numbers of recorded , data are presented as mean values ± s . e . m . )

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

A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration (the numbers on the right indicate the numbers of recorded cells, data are presented as mean values ± s.e.m.)


A bar graph of averaged values of tail current amplitudes summarizing results of ( c ) at 500 ms . ( numbers inside each bar indicate numbers of recorded from at least 3 mice , unpaired Student t - ; data are presented as mean values ± s . e . m . ) .

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

A bar graph of averaged values of the tail current amplitudes summarizing the results of (c) at 500 ms. (The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice, unpaired Student t-test; data are presented as mean values ± s.e.m.).


A summary bar graph showing quantitative real - time PCR data for TMEM16A , TMEM16B , BEST1 with cDNA samples prepared from mHb hippocampus of adult mouse brains ( N = 3 , n = 3 each , unpaired Student t - , data are presented as mean values ± s . e . m . ) .

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

A summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.).


RNAscope ISH showed an apparent expression of TMEM16A mRNA co - localized with ChAT Tac1 signals in mHb compared to ones in lHb . Enlarged showed that signals of TMEM16A Tac1 in dorsal mHb or ChAT in ventral mHb reside together at single level .

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

RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level.


Immunohistochemical of 40 - µm coronal brain sections with anti - TMEM16A antibody . In mHb , TMEM16A signals colocalize with cholinergic marker ChAT ( arrows ) . High magnifications of indicated areas are shown in merged panel .

B-EXP_ASSAY O O O O O B-TISSUE O O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O

Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.


Immunohistochemical of 40 - µm coronal brain sections with an anti - TMEM16A antibody . TMEM16A signals in mHb of TMEM16A cKO mice were compared to ones of littermate controls . TMEM16A colocalized with cholinergic marker ChAT in mHb . specific deletion of TMEM16A in mHb is evident .

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

Immunohistochemical staining of 40-µm coronal brain sections with an anti-TMEM16A antibody. The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls. TMEM16A colocalized with the cholinergic marker ChAT in the mHb. The specific deletion of TMEM16A in the mHb is evident.


Representative traces demonstrating spontaneous activity of mHb neurons in a - attached mode from TMEM16A cKO mice compared to one from littermate controls . D . A summary bar graph showing frequencies of spontaneous firing of mHb neurons in TMEM16A cKO ( 4 mice ) littermate controls ( numbers inside each bar indicate numbers of recorded from 4 mice each , paired Student t - , data are presented as mean values ± s . e . m . ) .

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

Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell-attached mode from TMEM16A cKO mice compared to the one from littermate controls. D. A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO (4 mice) and littermate controls (The numbers inside each bar indicate the numbers of recorded cells from 4 mice each, paired Student t-test, data are presented as mean values ± s.e.m.).


Representative averaged traces of sAPs of mHb neurons with pipette solution ( 40 mM Cl - 0 . 5 mM EGTA ) from TMEM16A cKO mice littermate controls .

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

Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls.


Summary bar graphs showing frequency , half - width , AHP slope of sAPs of mHb neurons from TMEM16A cKO mice littermate controls ( numbers inside each bar indicate numbers of recorded , unpaired Student t - , data are presented as mean values ± s . e . m . ) .

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

Summary bar graphs showing the frequency, half-width, and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls (The numbers inside each bar indicate the numbers of recorded cells, unpaired Student t-test, data are presented as mean values ± s.e.m.).


Immunohistochemical of IPN region from control TMEM16A cKO mice . c - Fos signal was greatly reduced in TMEM16A cKO mice . Scale bar = 200 μm .

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

Immunohistochemical images of the IPN region from control and TMEM16A cKO mice. The c-Fos signal was greatly reduced in TMEM16A cKO mice. Scale bar = 200 μm.


A summary bar graph showing reduced c - Fos expression in IPN in TMEM16A cKO mice . ( NCTL = 7 ( n = 42 ) , NcKO = 6 ( n = 36 ) , unpaired Student t - , data are presented as mean values ± s . e . m . ) .

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

A summary bar graph showing reduced c-Fos expression in the IPN in TMEM16A cKO mice. (NCTL=7 (n=42), NcKO=6 (n=36), unpaired Student t-test, data are presented as mean values ± s.e.m.).


Representative traces of sEPSCs of neurons in medial part of IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls .

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

Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls.


Summary bar graphs of frequency amplitude of sEPSCs of IPN neurons in TMEM16A cKO ( cKO ) mice littermate controls ( CTL ) . Data are presented as mean values ± s . e . m . ( paired Student t - ) . numbers inside each bar indicate numbers of recorded .

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

Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO (cKO) mice and littermate controls (CTL). Data are presented as mean values ± s.e.m. (paired Student t-test). The numbers inside each bar indicate the numbers of recorded cells.


In EPM task , time spent in open arms was significantly decreased in cKO mice compared to that in control ( CTL ) group ( nCTL = 20 , ncKO = 12 , unpaired Student t - ) . B . In LDB task , time spent in zone by cKO mice was significantly decreased compared to that spent by CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - ) . C . In PA task , entry latency of cKO mice was significantly decreased compared to that of CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - ) . . D . Nestlet shredding behaviors of cKO group were also decreased compared to those of CTL group . of shredded nestlets of cKO group was significantly lower than that of CTL group ( nCTL = 20 , ncKO = 12 , unpaired Student t - ) .

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

In the EPM task, the time spent in the open arms was significantly decreased in cKO mice compared to that in the control (CTL) group (nCTL=20, ncKO=12, unpaired Student t-test). B. In the LDB task, the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test). C. In the PA task, the entry latency of cKO mice was significantly decreased compared to that of the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test)..D. Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group. The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group (nCTL=20, ncKO=12, unpaired Student t-test).


In 3 - chamber , cKO mice exhibited a decreased interest for stranger mouse ( S1 ) against objects ( O ) compared to CTL group ( nCTL = 12 , ncKO = 14 , unpaired Student t - ) . F . When cKO mice were exposed to a new stranger mouse ( S2 ) against familiar S1 mouse , they also showed a decreased interest for S2 mouse compared to that of CTL group . preference index showed decreased sociability of cKO group in both sessions ( nCTL = 12 , ncKO = 14 , unpaired Student t - ) . Data information : data are presented as mean values ± s . e . m .

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

In the 3-chamber test, the cKO mice exhibited a decreased interest for the stranger mouse (S1) against objects (O) compared to the CTL group (nCTL=12, ncKO=14, unpaired Student t-test). F. When cKO mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to that of the CTL group. The preference index showed the decreased sociability of the cKO group in both sessions (nCTL=12, ncKO=14, unpaired Student t-test). Data information: All data are presented as mean values ± s.e.m.


Representative traces showing that spontaneous firing of mHb neurons from ChAT ( + ) - cre : : hM4D ( Gi ) mice were reduced when CNO ( 10 μM ) was applied in ACSF .

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

Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF.


A summary bar graph showing reduction of normalized frequencies of spontaneous firing of mHb neurons transfected with AAV - hM4Di - mCherry before during CNO treatment ( numbers inside each bar indicate numbers of recorded ) .

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

A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells).


In EPM task , time spent in open arms of CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that of PBS - treated controls ( CTL ) . ( nCTL = 6 , ncKO = 9 , unpaired Student t - ) . E . In LDB task , time spent in zone by CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that spent by PBS - treated control mice . ( nCTL = 6 , ncKO = 9 , unpaired Student t - ) . F . In PA task , entry latency of CNO - treated mice was significantly decreased compared to that of CTL group . ( nCTL = 6 , ncKO = 9 , unpaired Student t - ) . G . Nestlet shredding behaviors of CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were also decreased . ( nCTL = 6 , ncKO = 9 , unpaired Student t - ; ) .

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

In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;).


In 3 - chamber , subject mouse of PBS - treated mice CNO - treated mice explored freely three chambers . time spent in each chamber during two sessions was measured . CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice exhibited a decreased interest for a stranger mouse ( S1 ) against objects ( O ) compared to PBS - treated group ( left two panels ) ( nCTL = 6 , ncKO = 9 , unpaired Student t - ) . I . When CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were exposed to a new stranger mouse ( S2 ) against familiar S1 mouse , they also showed a decreased interest for S2 mouse compared to PBS - treated control group ( right two panels ) . preference index showed decreased sociability of CNO - treated group in both sessions ( nCTL = 6 , ncKO = 9 , unpaired Student t - ; ) .

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

In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).


surface H2 - Kb of wild type TAP1 - / - BMDC transduced with control vector or a vector expressing hβ2m were analyzed .

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

Cell surface H2-Kb levels of wild type and TAP1-/- BMDC transduced with control vector or a vector expressing hβ2m were analyzed.


Cross - presentation of OVA by wild type TAP1 - / - BMDC expressing hβ2m were compared to control BMDC , by measuring IL - 2 production by a Kb - SIINFEKL specific T - hybridoma ( B3Z ) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA - coated latex beads for 6hr , prior to fixation .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O

Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.


effect of hβ2m on presentation of peptides derived from endogenous antigen was assessed by measuring IL - 2 in culture supernatant of B3Z hybridoma incubated with fixed VV - OVA infected WT TAP1 - / - BMDC transduced with control vector or hβ2m .

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

The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA infected WT and TAP1-/- BMDC transduced with control vector or hβ2m.


Surface H2 - Kb of wild - type TAP1 - / - BMDC co - expressing Rab mutants hβ2m were analyzed by flow cytometry .

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

Surface H2-Kb levels of wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry.


Cross - presentation of OVA by wild - type TAP1 - / - BMDC co - expressing Rab mutants hβ2m were analyzed IL - 2 release is

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

Cross-presentation of OVA by wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed and IL-2 release is shown


TAP1 - / - BMDC transduced with vector control ( A ) or hβ2m ( C ) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb - binding SIINFEKL peptide analyzed for surface expression of Kb by flow cytometry . Representative plots are shown ( A , C ) . impact of Epoxomicin on surface Kb was analyzed by plotting Kb surface expression on incubated with Epoxomicin , normalized to that on incubated without Epoxomicin ( n = 3 ) ( B , D ) .

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

TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).


effect of Epoxomicin on cross - presentation of OVA - coated beads by TAP1 - / - BMDC expressing hβ2m was determined by incubating BMDC with OVA coated latex beads in presence of varying doses of Epoxomicin 10μM gB peptide . After 6hrs were fixed , incubated with B3Z IL - 2 production was

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

The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured


293T - FcR - Kb co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or US6 were analyzed for cross - presentation of OVA using B3Z . effects of US6 on cross - presentation were analyzed by plotting mean of IL - 2 release by co - expressing US6 ( A ) with control vector or Rab mutants compared to co - expressing LacZ with control vector or Rab mutants , respectively .

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

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or ICP47 ( B ) , were analyzed for cross - presentation of OVA using B3Z . effects of ICP47 on cross - presentation were analyzed by plotting mean of IL - 2 release by co - expressing ICP47 ( B ) with control vector or Rab mutants compared to co - expressing LacZ with control vector or Rab mutants , respectively .

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

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb expressing Rab5ACA were incubated with opsonized OVA coated latex beads in presence of varying doses of Epoxomicin . After 6hrs were fixed , incubated with B3Z IL - 2 production was measured .

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

293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb expressing Rab22ACA were incubated with opsonized OVA coated latex beads in presence of varying doses of Epoxomicin . After 6hrs were fixed , incubated with B3Z IL - 2 production was measured .

293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb expressing Rab7ADN ( E ) were incubated with opsonized OVA coated latex beads in presence of varying doses of Epoxomicin . After 6hrs were fixed , incubated with B3Z IL - 2 production was measured .

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

293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


EM of double immuno - gold labeling using antibodies against immunoproteasome subunit LMP2 endolysosomal marker LAMP1 in BMDC , WT MEF LMP2 KO MEF . Large gold particles ( 15 nm , black arrow head ) labels LMP2 small gold particles labels LAMP1 ( 5 nm , arrow head ) ( Scale bars = 500nm ) . insets are magnification of region of interest containing LAMP1 positive vacuole ( Scale bars = 100nm ) , marked by black rectangle .

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

EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.


distribution of LMP2 positive signals ( n = 22 ) assessed by plotting number of gold particles labeling LMP2 within LAMP1 positive compartment versus those outside LAMP1 positive organelles .

O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-SUBCELLULAR O

The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.


Immuno - gold labeling with antibodies against constitutive proteasome subunits , α5 , β5 19 S2 ( 15 nm , black arrow head ) , co - labeled with an anti - LAMP1 antibody ( 5 nm , arrow head ) in BMDC ( Scale bars = 500nm ) . Regions of interest containing LAMP1 positive vacuoles are marked by black rectangles magnified in insets ( Scale bars = 100nm ) .

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

Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5 and 19 S2 (15 nm, black arrow head), co-labeled with an anti-LAMP1 antibody (5 nm, white arrow head) in BMDC (Scale bars = 500nm). Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets (Scale bars = 100nm).


Immuno - electron microscopy ( n = 23 ) were analyzed by plotting total number of gold particles labeling proteasome subunits inside outside LAMP1 positive compartments ( D ) . These data are also presented as of total gold particles that are within LAMP1 positive organelle ( E ) .

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

Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).


BMDC transduced with genes encoding GFP , GFP - Rab5ACA GFP - Rab22ACA under control of an inducible promoter were fixed stained for LMP2 LAMP1 analyzed by confocal microscopy 24hr after doxycycline induction 4hr after Alexa 647 - OVA coated bead uptake . A single optical section of 10 optical sections of a representative are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by rectangles , are magnified as inset ( Scale bars = 1μm )

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

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


BMDC transduced with genes encoding GFP , GFP - Rab5ACA GFP - Rab22ACA under control of an inducible promoter were fixed stained for LMP2 LAMP1 analyzed by confocal microscopy 3D - rendering of 10 optical sections of a representative are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by rectangles , are magnified as inset ( Scale bars = 1μm )

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

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


same were analyzed for LMP2 within phagosomal lumen as defined by a region of interest positive for Alexa 647 - OVA enclosed within a limiting positive for LAMP1 . of LMP2 within phagosomal lumen normalized to Alexa647 - OVA was calculated data compiled from independent experiments ( n = 3 ) are plotted ( H ) . For each transduced type , of at least 27 acquired across three independent experiments were analyzed each is represented as a data point .

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

The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.


BMDC co - expressing LMP2 - RFP GFP - Rab5ACA were live imaged 16hr after doxycycline induction 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . were acquired every 3min taken at 12min interval are presented ( I - K ) . arrowheads in panel ( I J ) mark phagosome or vacuoles containing LMP2 , respectively .

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

BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.


BMDC co - expressing LMP2 - RFP GFP - Rab22ACA ( J , K ) were live imaged 16hr after doxycycline induction 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . were acquired every 3min taken at 12min interval are presented ( I - K ) . arrowheads in panel ( I J ) mark phagosome or vacuoles containing LMP2 , respectively . boxed region in panel ( J ) marks a phagosome that has been enlarged in panel ( K ) ( Bar 1μm ) .

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

BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).


Activity of proteasomes within phagosomes was determined by measuring of fluorogenic proteasome substrate incubated with latex beads from detergent - treated phagosomes treated or untreated with Epoxomicin . intensities were measured at 1hr intervals values plotted represent of signals in absence versus presence of

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

Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin


Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric of phagocytosed latex beads covalently conjugated with Alexa647 - OVA . Latex beads were extracted by mechanical disruption of BMDC incubated with ATP , lysosomal protease inhibitor cocktail varying dose of Epoxomicin at 37oC for 16hrs . Post incubation latex beads were treated with detergent analyzed by flow cytometry ( B ) . mean of Alexa647 associated with latex beads incubated with Epoxomicin were normalized to of untreated beads from independent experiments were plotted ( n = 4 ) ( C ) .

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

Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C).


Ubiquitination of phagosomal cargo within phagosomes was assessed by measuring acquisition of GFP by phagocytosed latex beads conjugated with OVA post - detergent extraction from BMDC expressing GFP or GFP - Ub ( D ) . mean of intensities of GFP on beads from independent experiments was plotted ( E ) .

B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post-detergent extraction from BMDC expressing GFP or GFP-Ub (D). The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted (E).


( A ) Colocalization of LacCer LysoTracker ( LyTr ) in different CTR LSD fibroblasts . Mander ´ s coefficients were calculated using Fiji JACoP plugin . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , post hoc Bonferroni ' s ( A , multiple comparisons . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of LacCer and LysoTracker (LyTr) in different CTR and LSD patient fibroblasts. Mander´s coefficients were calculated using the Fiji JACoP plugin. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's (A, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( B - C ) Confocal ( B ) statistical ( C ) showing colocalization of LacCer LyTr in human CTR MLIV fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 16h ) . ( D - E ) Confocal ( D ) statistical ( E ) showing colocalization of LacCer LyTr in human CTR NPC1 fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 48h ) . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , post hoc Bonferroni ' s C , E , multiple comparisons . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

(B-C) Confocal images (B) and statistical analysis (C) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts, treated with TPC2-A1-P (30 µM, 16h). (D-E) Confocal images (D) and statistical analysis (E) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts, treated with TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's C, E, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( F - G ) Confocal statistical showing LacCer / LyTr colocalization in MLIV fibroblasts which were mock - electroporated treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector treated with either DMSO or TPC2 - A1 - P ( 30 µM , 16h ) . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , post hoc Bonferroni ' s G , multiple comparisons . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

(F-G) Confocal images and statistical analysis showing LacCer/LyTr colocalization in MLIV patient fibroblasts which were mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector and treated with either DMSO or TPC2-A1-P (30 µM, 16h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's G, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( H - I ) Ca2 + chelation ( BAPTA - AM ) dose - dependently impairs LacCer trafficking in CTR fibroblasts . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , post hoc Bonferroni ' s I ) multiple comparisons . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

(H-I) Ca2+ chelation (BAPTA-AM) dose-dependently impairs LacCer trafficking in CTR fibroblasts. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's I) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( J - K ) Confocal ( J ) statistical ( K ) of NPC1 fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . were then treated with DMSO or TPC2 - A1 - P ( 30 µM ) . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , Tukey ' s ( K ) multiple comparisons . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

(J-K) Confocal images (J) and statistical analysis (K) of NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, Tukey's (K) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( A - B ) Confocal ( A ) statistical ( B ) of cholesterol accumulation in human CTR , MLIV , JNCL , NPC1 fibroblasts . Cholesterol accumulation was evident for NPC1 MLIV fibroblasts , but not for JNCL fibroblasts . show filipin to visualize cholesterol accumulation TO - PRO3 as . Statistics : Shown are mean values ± . n > 3 technical biological replicates for each tested condition ( each dot represents an imaged frame containing several ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons ( B , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

(A-B) Confocal images (A) and statistical analysis (B) of cholesterol accumulation in human CTR, MLIV, JNCL, and NPC1 fibroblasts. Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts, but not for JNCL fibroblasts. The images show filipin staining to visualize cholesterol accumulation and TO-PRO3 as nuclear staining. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test (B, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.