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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O 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 , 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 .

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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 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 .

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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 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 .

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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 test , p < 10 - 5 .

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


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

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 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 .

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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 ) 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 .

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

(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 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 .

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

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

(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 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 .

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(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 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 .

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

(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 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 .

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

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 , death domain ; ID , intermediate domain , TIR , TIR domain . Lower , coimmunoprecipitation of TLR3 and the N - terminal region of MYD88 .

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(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 - 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 .

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

(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 - length and truncated fragments of MYD88 in Myd88 - / - neurons . Representative images are shown .

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


( 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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

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 - and poly ( I : C ) - treated WT neurons .

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(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 levels compared with vehicle control are shown . In ( B - D ) , dashed lines indicate the level of saline control .

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(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 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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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) 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 . 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 .

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(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 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 .

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(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 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 .

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

(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 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 .

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

(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 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 .

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

(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.


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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 the depolarizing step duration ( the numbers on the right indicate the numbers of recorded cells , data are presented as mean values ± s . e . m . )

O O O O O O O O O O O O O O O O O O O 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 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 . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 . ) .

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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 the mHb neurons with the pipette solution ( 40 mM Cl - and 0 . 5 mM EGTA ) from TMEM16A cKO mice and littermate controls .

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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 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 . ) .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls .

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

Representative 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 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 .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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 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 ) .

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

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 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 ; ) .

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

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 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 ; ) .

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

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;).


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

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

Cell 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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

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 and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed and IL - 2 release is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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).


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)


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 .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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

(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 ± 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 .

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

(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 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 .

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

(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 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 .

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

(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.


( C - D ) TPC2 - A1 - P ( 30 µM , 48h ) rescued NPC1 and MLIV cholesterol accumulation . 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 D , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TPC2-A1-P (30 µM, 48h) rescued NPC1 and MLIV cholesterol accumulation. 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 D, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images ( E - F ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 48h ) .

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Confocal images (E-F) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h).


statistical analysis ( G ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or 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 multiple comparisons test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

statistical analysis (G) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or 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 multiple comparisons test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO 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 ) two - tailed Student ' s t - test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO 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) two-tailed Student's t-test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human 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 ) ; two - tailed Student ' s t - test K ) . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Confocal images and statistical analysis of human 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); two-tailed Student's t-test K). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Statistics ( L ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells . 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 , L ) * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Statistics (L) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells. 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 , L) *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


electron microscopy images ( M ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells .

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electron microscopy images (M) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells.


( A ) Confocal images of CTR and JNCL fibroblasts . Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength , respectively , corresponding to the lipofuscin autofluorescence spectrum . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) following cell cycle arrest ( 2h mitomycin C treatment ) . ( B ) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength ( used for LAMP1 staining ) .

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(A) Confocal images of CTR and JNCL fibroblasts. Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength, respectively, corresponding to the lipofuscin autofluorescence spectrum. Cells were treated with DMSO or TPC2-A1-P (30 µM) following cell cycle arrest (2h mitomycin C treatment). (B) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength (used for LAMP1 staining).


( C ) Mean autofluorescence intensity in LAMP1 + area . 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 . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(C) Mean autofluorescence intensity in LAMP1+ area. 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. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Mean intensity of shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest . 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 . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Mean intensity of shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest. 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. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images of Gb3 accumulation stained with shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest .

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Confocal images of Gb3 accumulation stained with shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( C - D ) Western blot analysis of cathepsin B ( CtsB ) in CTR and MCOLN1IVS3 - 2A > G neurons treated with TPC2 - A1 - P ( 30 µM ) or DMSO . 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 , obtained from at least three distinct neuronal differentiations ) ; two - tailed Student ' s t - test ( C ) . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (C-D) Western blot analysis of cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons treated with TPC2-A1-P (30 µM) or DMSO. 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, obtained from at least three distinct neuronal differentiations); two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker ( LyTr ) . Endolysosomal expansion was observed in MCOLN1IVS3 - 2A > G , CLN3D416G and CLN3ΔEx4 - 7 neurons , which was ameliorated by TPC2 - A1 - P ( 30 µM ) treatment . 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 , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker (LyTr). Endolysosomal expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P (30 µM) treatment. 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, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( H - I ) Electron microscopy analysis of neuronal rosettes ( neuronal progenitor cells , NPC ) treated with DMSO or TPC2 - A1 - P . TPC2 - A1 - P treatment significantly decreased the number of inclusion bodies ( black arrow heads ) in MCOLN1IVS3 - 2A > G . CLN3D416G and CLN3ΔEx4 - 7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies . However , CLN3ΔEx4 - 7 NPC showed significantly more mitochondria with aberrant cristae numbers ( white arrow heads ) , a phenotype which was rescued by TPC2 - A1 - P ( 30 µM ) treatment . 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 , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (H-I) Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies. However, CLN3ΔEx4-7 NPC showed significantly more mitochondria with aberrant cristae numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. 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, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( A ) Confocal images of plasma membrane ( PM ) LAMP1 immunofluorescence in CTR fibroblasts . LAMP1 on the PM is expressed as fold - change relative to DMSO - treated cells . ( B ) Statistical analysis of lysosomal exocytosis data as shown in A . ( C ) Lysosomal exocytosis in CTR , MLIV , NPC1 and JNCL human fibroblasts . PM - localized LAMP1 was measured by flow cytometry , expressed as % of CTR DMSO - treated cells . Ionomycin in A - C ( 4 µM ; 10 min treatment ) was used as positive control . TPC2 - A1 - P and ML - SA1 ( 30 µM , each ; 90 min treatment in A - C ) . Statistics ( B - C ) : Shown are mean values ± SEM . n > 3 for each tested condition ( in B each dot represents an imaged frame containing several cells , in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1 * 104 events ) ; two - way ANOVA , post hoc Dunnett ' s ( B ) or Tukey ' s ( C ) multiple comparisons test , * p - value < 0 . 05 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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(A) Confocal images of plasma membrane (PM) LAMP1 immunofluorescence in CTR fibroblasts. LAMP1 on the PM is expressed as fold-change relative to DMSO-treated cells. (B) Statistical analysis of lysosomal exocytosis data as shown in A. (C) Lysosomal exocytosis in CTR, MLIV, NPC1 and JNCL human fibroblasts. PM-localized LAMP1 was measured by flow cytometry, expressed as % of CTR DMSO-treated cells. Ionomycin in A-C (4 µM; 10 min treatment) was used as positive control. TPC2-A1-P and ML-SA1 (30 µM, each; 90 min treatment in A-C). Statistics (B-C): Shown are mean values ± SEM. n > 3 for each tested condition (in B each dot represents an imaged frame containing several cells, in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1*104 events); two-way ANOVA, post hoc Dunnett's (B) or Tukey's (C) multiple comparisons test, *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001.


Immunoblot analysis of endogenous LC3 ( LC3I - II ) following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patientfibroblasts .

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Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts.


analysis of endogenous LC3 following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patientfibroblasts . Graphs show densitometry of LC3II bands normalized to actin . Statistics ( G , Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - tailed Student ' s t - test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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

analysis of endogenous LC3 following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts. Graphs show densitometry of LC3II bands normalized to actin. Statistics (G, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.