Figure 1A . RT - PCR analysis of the expression of β - actin , Trpv1 , Trpv2 , Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) and 35 ( lower ) thermal cycles with ( RT ( + ) ) and without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate the results with each plasmid DNA as a template . B . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes . Expression levels of mRNA were normalized to that of the ribosomal protein gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± SEM , n = 6 . C . Results of real - time PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression levels were normalized to that of 36B4 . Data are presented as mean ± SEM , n = 5 . D . Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes . Upper bands in the TRPV2 blots likely indicate glycosylated forms . E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) and TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm cell viability . Ratio values correspond to the real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± SEM , n = 149 of WT cells , and n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - test . F . A representative trace of whole - cell current activated by 3 mmol / L of 2APB in the presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . The left inset indicates a voltage ramp - pulse protocol . The right inset indicates the current - voltage curves of basal current ( a , black ) , current in the presence of 2APB + SKF ( b , blue ) and current in the presence of 2APB alone ( c , red ) at the time points of a , b and c , respectively . G . Comparison of the mean densities of basal currents ( Base , black ) , currents in the presence of 2APB alone ( red ) and currents in the presence of 2APB + SKF ( blue ) at - 60 mV and + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± SEM , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


Figure 2A . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression in mouse pre - adipocytes and differentiated brown adipocytes . Expression levels of mRNA were normalized to those of 36B4 . Data are presented as mean ± SEM , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - test . B . The number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± SEM , n = 6 , * P < 0 . 05 and * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . C and D . Comparison of the numbers of 6 day - differentiated mouse brown adipocytes ( C ) and triglyceride levels ( D ) in the cells from WT and TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± SEM , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


Figure 3A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) and β3 - adrenergic receptor 3 ( Adrb3 ) in the differentiated brown adipocytes from WT and TRPV2KO mice . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - test . B and C . Changes in Ucp1 ( B ) and Pgc1α ( C ) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . D and E . Changes in Ucp1 mRNA expression ( D ) and non - esterified fatty acid ( NEFA ) release ( E ) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . F and G . Changes in Ucp1 mRNA ( F ) and Pgc1α mRNA ( G ) in the differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


Figure 4A . Body weight changes from 3 - weeks to 8 - weeks of age between the two genotypes ( n = 11 ) . Data are presented as mean ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test . B and C . Changes in food intake ( B ) and water intake ( C ) from 3 - weeks to 8 - weeks of ages between WT and TRPV2KO mice ( n = 6 ) . D . Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice ( n = 15 ) . E . The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice . mRNA expression levels of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) and cluster of differentiation 36 ( Cd36 ) were examined and normalized to the levels of 36B4 . Data are presented as mean ± SEM , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


Figure 5A . Representative morphological results of iBAT by hematoxylin and eosin staining from 8 - week WT and TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , and 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± SEM , n = 4 . C . Comparison of adipocyte size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . 800 to 1000 cells were counted . Data are presented as mean ± SEM , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) and TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , and 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± SEM , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.


Figure 6A , B , C and D . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) , Pparγ ( C ) and Trpv2 ( D ) in iBAT of WT and TRPV2KO mice before ( 25oC ) , after 1 day and 3 days cold ( 4oC ) exposure . Mean ± SEM , n = 5 - 8 ; * P < 0 . 05 ; * * P < 0 . 01 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . E and F . Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before ( 25oC ) and after 3 days cold ( 4oC ) exposure ( E ) . Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice ( F ) . Mean ± SEM , n = 6 ; * P < 0 . 05 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . G and H . Averages of core body temperatures ( G ) and activities ( H ) of WT and TRPV2KO mice with or without cold ( 4oC ) exposure . Data are presented as mean ± SEM , n = 5 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.


Figure 7A , B and C . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) and Pparγ ( C ) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration ( i . p . , arrows ) of saline or a selective β3 - adrenergic receptor agonist , BRL37344 ( 600 µg / kg body weight ) . Mean ± SEM , n = 5 - 7 ; * P < 0 . 05 vs . saline group ; # P < 0 . 01 vs . WT BRL37344 administration group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction . D and E . Average traces of changes in iBAT temperature ( D ) or rectal body temperature ( E ) from WT and TRPV2KO mice after BRL37344 ( 600 µg / kg body weight ) administration ( i . p . , arrows ) . Mean ± SEM , n = 5 . * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test . F and G . iBAT NE turnover rates ( F ) and rate constants k ( G ) in WT and TRPV2KO mice upon cold exposure ( 4oC ) for 4 h . Data are presented as mean ± SEM , n = 8 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.


Figure 8A . Body weight changes between WT and TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 weeks from 13 - week - old . B and C . Blood glucose level ( B ) and plasma insulin level ( C ) of WT and TRPV2KO mice after 8 - week HFD treatment . D . Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8 - week HFD treatment . E . mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8 - week HFD treatment . All data are presented as mean ± SEM , WT mice ( n = 6 ) , TRPV2KO mice ( n = 8 ) ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O B-PANEL_START O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


Figure 1FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade . Immunohistochemistry analysis of FOXA1 ( top ) and FOXA2 ( bottom ) in human PDAC . Representative G1 , G2 and G3 areas from one patient are shown . Red dotted lines indicate the borders of poorly differentiated ( G3 ) tumor areas . Percent of FOXA1 - and FOXA2 - positive nuclei in PDAC cells from areas of different grade . Tissue microarrays were automatically acquired and quantified using QuPath . The number ( n ) of analyzed areas of each grade is indicated . Expression of FOXA1 and FOXA2 in low - grade and high - grade PDAC cell lines . Vinculin is shown as loading control .

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Figure 1FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade. Immunohistochemistry analysis of FOXA1 (top) and FOXA2 (bottom) in human PDAC. Representative G1, G2 and G3 areas from one patient are shown. Red dotted lines indicate the borders of poorly differentiated (G3) tumor areas.Percent of FOXA1- and FOXA2-positive nuclei in PDAC cells from areas of different grade. Tissue microarrays were automatically acquired and quantified using QuPath. The number (n) of analyzed areas of each grade is indicated.Expression of FOXA1 and FOXA2 in low-grade and high-grade PDAC cell lines. Vinculin is shown as loading control.


Figure 2Western blot showing siRNA - mediated depletion of FOXA1 and / or FOXA2 in the low - grade CFPAC1 cell line . The three biological replicates used in the subsequent experiments are shown . Heatmap showing row - normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1 / 2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively . FOXA2 and FOXA1 expression in wild type and genome - edited high - grade PANC1 cells . Three different bulk populations were used for RNA - seq experiments . Heatmap showing row - normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively . Representative Gene Ontology ( GO ) terms enriched in genes differentially expressed upon FOXA1 / 2 depletion in CFPAC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA1 / 2 depletion on cell morphology and adhesion to multiple substrates . Three replicates per condition were used : circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition . Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates . Three different bulk populations were used as biological replicates : circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition .

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Figure 2Western blot showing siRNA-mediated depletion of FOXA1 and/or FOXA2 in the low-grade CFPAC1 cell line. The three biological replicates used in the subsequent experiments are shown.Heatmap showing row-normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1/2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.FOXA2 and FOXA1 expression in wild type and genome-edited high-grade PANC1 cells. Three different bulk populations were used for RNA-seq experiments.Heatmap showing row-normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.Representative Gene Ontology (GO) terms enriched in genes differentially expressed upon FOXA1/2 depletion in CFPAC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA1/2 depletion on cell morphology and adhesion to multiple substrates. Three replicates per condition were used: circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition.Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition.


Figure 3Scatter plots showing the correlation between FOXA2 ChIP - seq biological duplicates in CFPAC1 ( top panel ) and PANC1 cells ( bottom panel ) . Pearson ' s correlation ( r ) between samples is indicated . Scatter plot showing the correlation between FOXA2 ChIP - seq peaks in CFPAC1 and PANC1 cells . Green and blue dots indicate FOXA2 peaks that are significantly ( FDR < 0 . 001 ) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind . All other peaks are indicated as grey dots . The boxplots on the right show the intensity of CFPAC1 - specific , PANC1 - specific and other FOXA2 peaks . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Representative FOXA2 ChIP - seq snapshot showing FOXA2 peaks classified as CFPAC1 - specific , shared or PANC1 - specific .

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Figure 3Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated.Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific.


Figure 4The five top transcription factor motifs over - represented in TSS - distal CFPAC1 - specific FOXA2 peaks . TFs were grouped into families or subfamilies and ranked based on the best PWM q - value . The POU domain TF family includes HNF1β . Top scoring motifs are shown on the right . Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP - seq peaks . Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS ( left ) and JUNB ( right ) ChIP - seq peaks . Two representative ChIP - seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Figure 4The five top transcription factor motifs over-represented in TSS-distal CFPAC1-specific FOXA2 peaks. TFs were grouped into families or subfamilies and ranked based on the best PWM q-value. The POU domain TF family includes HNF1β. Top scoring motifs are shown on the right.Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP-seq peaks.Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS (left) and JUNB (right) ChIP-seq peaks.Two representative ChIP-seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Figure 5Representative areas from human PDAC tissue showing expression of HNF1β in G1 , G2 and G3 areas . The red dotted line indicates the borders of a G3 area . The percentage of HNF1β - positive cells in areas of different grade from a PDAC tissue microarray is shown . Colocalization of HNF1β and FOXA2 in human PDAC areas . PDAC sections from three different patients were stained with the anti - HNF1β antibody , stripped and re - stained with the anti - FOXA2 antibody . The red dotted areas on the left represent poorly differentiated ( G3 ) tumor regions .

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Figure 5Representative areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown.Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.


Figure 6FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility ( determined by ATAC - seq ) . Clusters 1 - 4 were HNF1β - positive while cluster 5 was HNF1β - negative . The heat - maps on the left show FOXA2 , HNF1β , ATAC - seq and H3K27Ac profiles at FOXA2 - bound genomic regions . The three heat - maps on the right show JunB ChIP - seq data and ATAC - seq data in wild type or Jun / JunB double knockout ( DKO ) CFPAC1 cells . Data are shown in + / - 3 kb regions centered on the summit of FOXA2 peaks . Characterization of clusters 1 - 3 . Left : quantification of the indicated signals at FOXA2 bound regions in wild - type ( WT ) and HNF1β knock - out ( KO ) cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Middle : distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks . Right : over - represented motifs in clusters 1 - 3 and inferred binding models . Quantification of JunB ChIP - seq signals in clusters 1 to 5 . ( Right ) Quantification of ATAC - seq signals in clusters 1 to 5 in wild type and Jun / JunB double KO ( DKO ) CFPAC1 cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Representative snapshot showing changes in FOXA2 occupancy , chromatin accessibility and histone acetylation in HNF1β knock - out CFPAC1 cells . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Figure 6FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks.Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models.Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Figure 7Western blot showing HNF1β over - expression after lentiviral transduction of PANC1 cells . Expression of FOXA2 in wild type and over - expressing ( O . E . ) cells is shown . Vinculin : loading control . Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β - expressing lentivirus ( HNF1B O . E . ) and in their matched mock - infected control . The fraction of gained , unchanged and lost FOXA2 peaks overlapping HNF1β ChIP - seq peaks in transduced PANC1 cells is shown in the pie - charts on the right . TF motif over - represented in gained or lost FOXA2 - bound genomic regions . Representative snapshot from PANC1 cells transduced with the HNF1β - expressing lentivirus . HNF1B O . E . : HNF1B over - expression .

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Figure 7Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control.Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right.TF motif over-represented in gained or lost FOXA2-bound genomic regions.Representative snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.


Figure 8Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP - seq peaks . Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP - seq peaks in PANC1 cells . The FOXA2 ChIP - seq track in CFPAC1 cells is also shown for comparison . FOXA2 ChIP - seq in CFPAC1 cells transduced with a HOXB8 - expressing lentivirus and in their matched mock - infected control . Gained ( brown ) and lost ( yellow ) FOXA2 peaks are shown . Pie - charts on the right show the fraction of gained , unchanged and lost peaks overlapping HOXB8 ChIP - seq peaks in transduced CFPAC1 cells . TF motif over - represented in gained or lost FOXA2 - bound genomic regions . Expression of HNF1β protein in wild - type and HOXB8 - expressing PANC1 cells . Representative snapshots from CFPAC1 cells transduced with the HOXB8 - expressing lentivirus .

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Figure 8Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks.Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison.FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells.TF motif over-represented in gained or lost FOXA2-bound genomic regions.Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells.Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.


Figure 1Figure 1 . CHCHD10 resides in the same complex than Mitofilin , CHCHD3 and CHCHD6 . A . Second dimension of the Blue - Native ( BN ) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin , CHCHD3 and CHCHD6 in isolated mitochondria from mouse brainB . BN - PAGE of the MICOS and OXPHOS complexes in control ( C ) and patient ( P1 , P2 ) fibroblasts . Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody ( CI ) , 70kDa subunit antibody ( CII ) , core I antibody ( CIII ) and cytochrome c oxidase subunit I antibody ( CIV ) . MICOS complex was detected with an anti - Mitofilin antibody . C . Second dimension of the BN - PAGE showing that the steady - state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts . D . Analysis of OXPHOS supercomplexes in control and patient fibroblasts . BN - PAGE from isolated mitochondria permeabilized with 6g / g ( w / v ) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies . SC , supercomplexes I + III2 + IVn . The dividing lines correspond to gels sections visible in the raw data file .

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Figure 1Figure 1. CHCHD10 resides in the same complex than Mitofilin, CHCHD3 and CHCHD6. A. Second dimension of the Blue-Native (BN) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin, CHCHD3 and CHCHD6 in isolated mitochondria from mouse brainB. BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P1, P2) fibroblasts. Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody (CI), 70kDa subunit antibody (CII), core I antibody (CIII) and cytochrome c oxidase subunit I antibody (CIV). MICOS complex was detected with an anti-Mitofilin antibody.C. Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts.D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.


Figure 2Figure 2 . Interaction of CHCHD10 with components of MICOS complex . A . Co - immunonoprecipitation ( IP ) of endogenous mitofilin , CHCHD10 and CHCHD3 in control and patient fibroblasts ( 1mg of total extracts were used for each IP ( left lower panel ) and 200µg were used for the input ( left upper panel ) ) . The same results were found in P2 fibroblasts ( not shown ) . Reverse co - IP experiment in HeLa cells with an antibody against CHCHD10 ( right lower panel ) . 1mg of total extracts were used for each IP and 200µg were used for the input ( right upper panel ) . The dividing lines correspond to gels sections visible in the raw data file . B . Duolink proximity ligation assay between Mitofilin and CHCHD10 ( upper panels ) and , CHCHD10 and CHCHD6 ( lower panels ) in control and patient fibroblasts observed by confocal microscopy . Mitochondria were stained with Mitotracker . Duolink spots per cell were quantified for 30 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) . Scale bar = 10 µm .

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Figure 2Figure 2. Interaction of CHCHD10 with components of MICOS complex. A. Co-immunonoprecipitation (IP) of endogenous mitofilin, CHCHD10 and CHCHD3 in control and patient fibroblasts (1mg of total extracts were used for each IP (left lower panel) and 200µg were used for the input (left upper panel)). The same results were found in P2 fibroblasts (not shown). Reverse co-IP experiment in HeLa cells with an antibody against CHCHD10 (right lower panel). 1mg of total extracts were used for each IP and 200µg were used for the input (right upper panel). The dividing lines correspond to gels sections visible in the raw data file.B. Duolink proximity ligation assay between Mitofilin and CHCHD10 (upper panels) and, CHCHD10 and CHCHD6 (lower panels) in control and patient fibroblasts observed by confocal microscopy. Mitochondria were stained with Mitotracker. Duolink spots per cell were quantified for 30 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001). Scale bar = 10 µm.


Figure 3Figure 3 . Decrease of nucleoid number without mtDNA depletion in patient fibroblasts . A . In control and patient fibroblasts , nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker . Image analysis was performed by confocal microscopy . Scale bar : 10 µm . B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p < 0 . 001 ) . Nucleoid number ( B ) : patient 1 versus control * * * : p = 0 . 0001 , patient 2 versus control * * * : p = 0 . 001 . Nucleoid size ( C ) : patient 1 versus control * * * : p = 0 . 0001 . D . Mitochondrial DNA content in control and patientfibroblasts was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nuclear DNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

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Figure 3Figure 3. Decrease of nucleoid number without mtDNA depletion in patient fibroblasts. A. In control and patient fibroblasts, nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p<0.001). Nucleoid number (B): patient 1 versus control ***: p=0.0001, patient 2 versus control ***: p=0.001. Nucleoid size (C): patient 1 versus control ***: p=0.0001. D. Mitochondrial DNA content in control and patientfibroblasts was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nuclear DNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 4Figure 4 . Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles . A . Transfections were performed with empty vector ( EV ) or vectors encoding wild - type CHCHD10 - FLAG ( WT ) , CHCHD10 - FLAG ( S59L ) or CHCHD10 - FLAG ( P34S ) mutants . Mitochondria were stained with Mitotracker . Cells overexpressing wild - type and mutant CHCHD10 were labeled with FLAG antibodies . Nucleoids were visualized with an antiserum against DNA . Image analysis was performed by confocal microscopy . Scale bar : 10 µm . B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : very significant ( * * : p = 0 . 0280 ) or extremely significant ( * * * : p = 0 . 0001 ) . D . Mitochondrial DNA content was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nDNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 5Figure 5 . Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress . A . Total extracts from control ( C ) fibroblasts and intact isolated mitochondria from control and patient fibroblasts ( P1 , P2 ) were analyzed by immunoblotting using antibodies against PCNA ( nuclear protein ) , GAPDH ( cytosolic protein ) or SMAC ( mitochondrial intermembrane space protein ) ( upper panel ) . Mitochondria from patient and control fibroblasts were incubated with NP - 40 and separated into pellets ( P ) and supernatants ( S ) . The fractions of each extraction were subjected to western blot analysis . VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins , respectively ( middle panel ) . Ratio of mtDNA amplified from supernatant / mtDNA amplified from pellet , by qPCR , was quantified in control and patient fibroblasts ( lower panel ) . B . Determination of the mtDNA copy number after H2O2 treatment . mtDNA / nDNA values in control , patient 1 and patient 2 fibroblasts . mtDNA : mitochondrial DNA , nDNA : nuclear DNA . C . mtDNA repair after H2O2 treatment . A long - range PCR was used to evaluate the oxidative damage , induced by H2O2 treatment , in mtDNA . The relative PCR amplification of a 15 . 6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment . mtDNA repair activity in control , patient 1 and patient 2 relative PCR . B - C . Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times . Untreated control cultures were incubated in serum - free medium alone . Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed . Values were normalized to untreated cells and differences were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) , very significant ( * * : 0 . 01 > p > 0 . 001 ) . Patient 1 versus control : * : p = 0 . 015 ( recovery 1h ) , * : p = 0 . 022 ( recovery 2h ) , * * : p = 0 . 003 ( recovery 4h ) . Patient 2 versus control : * : p = 0 . 0230 ( recovery 2h ) , * : p = 0 . 041 ( recovery 4h ) . NT : cells not treated with

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel).B. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 relative PCR. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2


Figure 6Figure 6 . No autophagy inhibition ( A - C ) and no mitophagy defect ( D - E ) in patient fibroblasts . A . Immunoblot of LC3B - I / - II expression in fibroblasts of patient 2 ( 1 , 2 ) , patient 1 ( 3 , 4 ) and control individual ( 5 , 6 ) in absence ( - ) or presence ( + ) of chloroquine ( CLQ ) . B . Quantitative analysis of LC3B - II / β - tubulin ratio from immunoblots ( n = 3 ) . C . Quantitative analysis of LC3B - II + CLQ / LC3B - II - CLQ ratio from immunoblots ( n = 3 ) . D . Confocal images of fibroblasts derived from a control , patient 1 and patient 2 . Boxed regions are enlarged . Scale bar : 20µm . E . Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol ( FL lyso / FL mito ) . Statistical analysis were performed on mean ± SEM using Student ' s t - test ( two - sided ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Figure 6. No autophagy inhibition (A-C) and no mitophagy defect (D-E) in patient fibroblasts. A. Immunoblot of LC3B-I/-II expression in fibroblasts of patient 2 (1,2), patient 1 (3,4) and control individual (5,6) in absence (-) or presence (+) of chloroquine (CLQ). B. Quantitative analysis of LC3B-II/β-tubulin ratio from immunoblots (n=3). C. Quantitative analysis of LC3B-II + CLQ/ LC3B-II - CLQ ratio from immunoblots (n=3).D. Confocal images of fibroblasts derived from a control, patient 1 and patient 2. Boxed regions are enlarged. Scale bar: 20µm. E. Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol (FL lyso/FL mito ). Statistical analysis were performed on mean ± SEM using Student's t-test (two-sided).


Figure 7Figure 7 . CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts . A . Control ( C1 , C2 ) and patient ( P1 , P2 ) fibroblasts were treated with 1 μM of staurosporine ( STS ) for 16 h . Cell death was determined by flow cytometry using Annexin V / DAPI staining . Three independent experiments were performed per condition with two points analyzed per experiment . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : p = 0 . 0116 ) or extremely significant ( * * * : p = 0 . 0001 ) . B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control ) B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control ) or by DEVDase activity measurement ( C ) . Two ( B ) or three ( C ) independent experiments were performed per condition . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) . D - E . Mitochondrial membrane potential ( ΔΨM ) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h ( E ) and quantified in D from two independent experiments . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) . Patient 1 ( P1 ) versus control ( C2 ) : * : p = 0 . 0414 ( 24h ) . Patient 2 ( P2 ) versus control ( C2 ) : * : p = 0 . 0461 ( 24h ) . D - E . Mitochondrial membrane potential ( ΔΨM ) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h ( E ) and quantified in D from two independent experiments . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) . Patient 1 ( P1 ) versus control ( C2 ) : * : p = 0 . 0414 ( 24h ) . Patient 2 ( P2 ) versus control ( C2 ) : * : p = 0 . 0461 ( 24h ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001).B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control)B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001).D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).


Figure 8Figure 8 . Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death . Transfections were performed with empty vector ( EV ) or vectors encoding either wild - type CHCHD10 - FLAG ( WT ) or mutant CHCHD10 - FLAG ( P34S and S59L ) . A . Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90 . B - D . HeLa cells transiently expressing the WT or mutated forms of CHCHD10 - FLAG ( P34S or S59L ) were treated with 1 μM Actinomycine D ( ActD ) for 4 , 6 or 8 h with measurement of Annexin V / DAPI staining ( B ) , DEVD - ase activity ( C ) from three independent experiments . Differences between the mutated and non mutated alleles were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) or very significant ( * * : 0 . 01 > p > 0 . 001 ) . P34S versus WT : * * : p = 0 . 0055 ( 4h ) , * : p = 0 . 0103 ( 8h ) . S59L versus WT : * : p = 0 . 0304 ( 4h ) , * : p = 0 . 0129 ( 8h ) . D . Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac . Hsp90 was used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Figure 8. Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death. Transfections were performed with empty vector (EV) or vectors encoding either wild-type CHCHD10-FLAG (WT) or mutant CHCHD10-FLAG (P34S and S59L). A. Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90.B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h).D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.


Figure 1Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with ( A ) a murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) . Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with a human macrophage cell line ( U937 ) , treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) . Heat map represents cytokine secretion by in vitro expanded ( C ) murine splenic iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates . Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates . Wild type C57BL / 6 mice were injected intravenously with thapsigargin - loaded PLGA nanoparticles or vehicle , and 12 h later , hepatic iNKT cells were analysed by flow cytometry . Histograms represent expression of intracellular IL - 4 and IFN - γ levels in hepatic iNKT cells from mice injected with thapsigargin - loaded PLGA nanoparticles ( red histogram ) or vehicle - treated ( grey histogram ) mice . Dot plots represents MFI for intracellular IL - 4 and IFN - γ expression in hepatic iNKT cells from thapsigargin - loaded PLGA nanoparticles ( red dots ) or vehicle - treated ( black dots ) mice . Data plots show mean ± SEM from n = 5 mice per group . * * P ≤ 0 . 001 ( Mann - Whitney U test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).


Figure 2Thapsigargin or αGalCer treated J774 . 2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d - specific antibody ( black circles ) or isotype ( red squares ) for 16 hours . The addition of an anti - CD1d antibody blocked the activation of 2C12 demonstrating that the NKT - cell activation was CD1d - specific . Graphs show mean ± SEM from n = 2 biological replicates . Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin - or tunicamycin - treated CD1d knockdown ( white bars ) or untransfected ( black bars ) . Knockdown of CD1d blocked IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate . Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured either with thapsigargin - or tunicamycin - treated primary BMDM isolated from CD1d - / - ( white bars ) or wild type ( black bars ) mice . 2C12 cocultured with CD1d - / - BMDMs resulted in reduced IL - 2 secretion . Graphs show mean ± SEM from n = 3 biological replicates . * * * P < 0 . 001 ( Unpaired t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).


Figure 3qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) . qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) . Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor ( 4μ8c ) ( black bar ) or PERK inhibitor ( GSK ) ( grey bar ) or ATF6α inhibitor ( Ceapin - a7 ) ( red bar ) respectively . Inhibition of IRE1α or PERK resulted in significant reduction in IL - 2 secretion by iNKT hybridoma compared to controls . In total , n = 2 biological replicates were performed ( two and four technical replicates per biological replicate respectively ) . Graphs show mean ± SEM of each biological replicate . Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774 . 2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene . Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with thapsigargin - treated murine IRE1α or PERK knockdown macrophage cell line ( J774 . 2 ) . Knockdown of IRE1α ( black bar ) or PERK ( grey bar ) resulted in significant reduction in IL - 2 secretion by iNKT hybridoma . In total n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene.Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Figure 4Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 6 ) were isolated from J774 . 2 macrophages stimulated with thapsigargin , tunicamycin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or tunicamycin - treated ( red bars ) J774 . 2 cells . Polar lipid fractions induce very little IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate . Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin - treated J774 . 2 at acidic pH and reduced mCD1d loading at neutral pH . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) . Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) , isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges , were co - cultured with purified NKT1 subsets . Bar graphs represent IFN - γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) primary murine macrophages . Polar lipid fractions induce a minimal amount of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate . Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents IFN - γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) human primary macrophages . Polar lipid fractions induce only minimal amounts of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Figure 5Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL - 2 secretion by NKT cells . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate . qPCR analyses showing the expression ( fold induction ) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin or DMSO for 8 hours . In total n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate . Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells . IL - 2 secretion in UPR - induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin . Graphs show mean ± SEM from n = 3 biological replicates . n . s ( non - significant ) ( One - way ANOVA ) biological replicates . L363 antibody does not impact UPR - induced NKT cell activation . 2C12 NKT cells were co - cultured with ER - stressed macrophages J774 . 2 cells and treated with L363 antibody or isotype control . α - GalCer induced responses served as positive control . In total , n = 2 biological replicates were performed ( Four technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Figure 1Meth Induces Rapid Alkalization of Acidic Organelles , and Changes in Endosomal Structure in Dendritic Cells ( A ) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment . Similar results were obtained in three independent experiments . ( Statistics n = 25 - 35 , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . ) ( B ) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence ( ) or presence ( ▴ ) of 50 μM Meth . p < 0 . 001 for all time points versus control ( n = 25 - 35 , mean ± SEM , Student ' s two - tailed t - Test ) . ( C ) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement . ( Statistics n = 30 - 90 cells , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . ) ( D ) Representative electron micrographs showing multivesicular endosomal compartments ( arrows ) in control , Clq - and Meth - treated cells at 4 h . Scale bar , 200 nm . ( E ) Ultra - thin cryosections of control , Clq - and Meth - treated dendritic cells as in ( D ) , immunolabeled for LAMP - 1 ( 15 nm gold particles ) and MHC class II ( 10 nm gold particles ) . Scale bar , 200 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test).(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm.(E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.


Figure 3 ( A ) Accumulation of GFP - LC3 labeled autophagosomes in Meth - or Clq - treated dendritic cells at 2 h and 24 h . Fisher ' s exact test P values versus control ( n = 300 - 400 cells , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 0001 ) . ( B ) GFP - LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment . Similar results were obtained in three independent experiments . Fisher ' s exact test versus control ( n = 300 - 400 cells , mean ± SEM , * * * p < 0 . 0001 ) . ( C ) Representative fluorescence micrographs of GFP - LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h . Arrow denotes examples of autophagosomes . Scale bar , 10 μm .

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Figure 3(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001).(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.


Figure 4 ( A ) Cells were incubated in indicated concentrations of Meth or Clq for 2 h , and then IgG coated erythrocytes were added . Extracellular uningested EIgG were lysed and removed . Phagocytic index ( PI ) was quantified and inhibition is indicated as percent of control . Data were collected from four to seven experiments ( n = 300 cells , mean ± SEM , * * * p < 0 . 0001 , two - tailed ANOVA ) . ( B ) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment . Arrows denote phagocytosed erythrocytes . Scale bar , 10 μm . ( C , D ) J774 . 16 cells were exposed to PBS , chloroquine , or methamphetamine for 2 h followed by incubation with Cn or Ca . The phagocytic indices ( ratio of intracellular yeast to the number of macrophages counted ) were determined after 1 h or 30 min for Cn and Ca , respectively ( C ) . ( Statistics n = 300 , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) CFU determinations were preformed after 24 h incubations ( D ) . The values are the mean and standard deviation from determinations made in triplicate wells ( n = 300 cells , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) . Experiments were repeated three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.