T cells expressing GFP - CAR were plated on CD19 and ICAM - 1 coated SLBs . TIRF microscopy was implemented to visualize CAR localization . A . CAR microclusters underwent centripetal movement to form cSMAC ( upper panel ) or remained separated ( lower panel ) . B . Quantification of the categories regarding the formation of CAR cSMAC . Unstable cSMACs are defined by a cSMAC duration below 12 min . Data are shown as mean ± SD . n = 3 independent experiments . 37 , 38 , and 47 cells were quantified in individual experiments . C . Quantification of time from cell landing to the start of retrograde flow . Data are shown as mean ± SD . n = 33 ( stable ) , 31 ( unstable ) , 10 ( kinapse ) , and 41 ( no cSMAC ) cells . Results are pooled from three independent experiments .

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T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization. A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel). B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments. C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments.


D . Localization of CAR and CD45 in the immunological synapse . CAR T cells were fixed 20 min after being plated on the SLB , followed by staining with an anti - CD45 antibody . E . Line scan of CAR and CD45 across the long axis of the immunological synapse . Shown are the normalized fluorescence intensity .

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D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody. E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.


A . WT or LAT - deficient Jurkat cells ( Jcam2 . 5 ) expressing CAR - GFP were stimulated on SLB - coated with OKT3 ( anti - TCR antibody ) or CD19 ( CAR antigen ) ; in both conditions , the lipid bilayers were also coated with ICAM - 1 to facilitate cell attachment . TIRF microscopy revealed clustering of Ax647 - labeled streptavidin - Biotin - OKT3 or CD19 , which serves as a probe for TCR or CAR respectively . Scale bar , 2 µm . B . Quantification of clustering as normalized variance . n = 100 cells . Shown are the mean ± SD . Statistical test : unpaired two - tailed t - test . * * * * p < 0 . 0001 . * * 0 . 005 < p < 0 . 05 .

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A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.


B . WT or LAT - deficient ( Jcam2 . 5 ) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM - 1 . TIRF microscopy revealed clustering of CAR and Ax647 - labeled streptavidin - Biotin - CD19 . Scale bar , 2 µm . C . Quantification of clustering level of Ax647 - labeled streptavidin - Biotin - CD19 . Shown are means ± SD . Clustering is quantified as normalized variance . N = 50 cells for CAR 3rd WT and 1st WT , 63 cells for CAR 3rd lat - , and 51 cells for CAR 1st lat - . * * : 0 . 0005 < p < 0 . 005 . Statistical test : unpaired two - tailed t - test .

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B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm. C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . A . Gads was recruited to CAR microclusters in both the wild - type and LAT - deficient ( Jcam2 . 5 ) cells . n = 33 or 34 cells . Shown are the means ± SE . n . s . , p = 0 . 58 by unpaired two - tailed t - test . As a control , Gads is not recruited to LAT - deficient cells activated by OKT3 .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . B . Phosphorylated SLP76 , recognized by an anti - SLP76 pY145 antibody , was enriched in CAR microclusters in both the wild - type and LAT - deficient cells . In contrast , phosphorylated SLP76 was not enriched in TCR microclusters in LAT - deficient cells . n = 50 cells . Shown are the means ± SE . n . s . , p = 0 . 95 by unpaired two - tailed t - test .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test.


CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . C . Actin remodeling was induced in LAT - deficient cells by CD19 - , but not OKT3 - coated SLB . No ICAM - 1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA - 1 / ICAM - 1 pathway . Actin network was revealed by F - tractin - mScarlet . n = 6 cells . Shown are the means ± SD . n . s . , p = 0 . 68 , and 0 . 98 by unpaired two - tailed t - test .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.


A . Cell - cell conjugation assay . Wild - type or Lat - null ( J . LAT ) Jurkat T cells expressing CAR - GFP were mixed with Raji cells expressing mCherry - CAAX at a 1 : 1 ratio for 1 hr before being fixed . Three independent experiments were performed and over 90 cells were scored in each experiment . Scale bar : 5 µm .

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A. Cell-cell conjugation assay. Wild-type or Lat-null (J.LAT) Jurkat T cells expressing CAR-GFP were mixed with Raji cells expressing mCherry-CAAX at a 1:1 ratio for 1 hr before being fixed. Three independent experiments were performed and over 90 cells were scored in each experiment. Scale bar: 5 µm.


B . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio for 24 hrs and IL - 2 from the supernatant were measured by ELISA . C . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio and fixed at 24hr , stained with an anti - CD69 antibody , and analyzed by FACS . CD69 positive cells were scored within the CAR - GFP + population .

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B. CAR T cells were incubated with Raji B cells at a 1:1 ratio for 24 hrs and IL-2 from the supernatant were measured by ELISA. C. CAR T cells were incubated with Raji B cells at a 1:1 ratio and fixed at 24hr, stained with an anti-CD69 antibody, and analyzed by FACS. CD69 positive cells were scored within the CAR-GFP+ population.


A . Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR . Shown are the means ± SD . n = 4 independent experiments .

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A. Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR. Shown are the means ± SD. n = 4 independent experiments.


B . CAR T cells were stimulated on SLBs coated with biotin - OKT3 ( left ) or biotin - CD19 ( right ) supplemented with ICAM - 1 . TIRF microscopy revealed clustering of Alexa647 - labeled streptavidin - Biotin - OKT3 or CD19 . Scale bar , 5 µm . C . Quantification of clustering of OKT3 or CD19 as normalized variance . n > 200 cells from two independent donors were scored for each condition . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 48 .

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B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm. C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48.


D . Immunofluorescence analysis revealed membrane - localized phosphorylated SLP76 . Cells were plated on supported lipid bilayer coated with ICAM - 1 only ( control ) or ICAM - 1 supplemented with CD19 or OKT3 as indicated . The white dashed circle indicated cell boundary . Scale bar , 5 µm . E . Quantification of pSLP76 level on the membrane . n > 200 cells were scored , and the cells were isolated from two independent donors . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 11 .

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D. Immunofluorescence analysis revealed membrane-localized phosphorylated SLP76. Cells were plated on supported lipid bilayer coated with ICAM-1 only (control) or ICAM-1 supplemented with CD19 or OKT3 as indicated. The white dashed circle indicated cell boundary. Scale bar, 5 µm. E. Quantification of pSLP76 level on the membrane. n > 200 cells were scored, and the cells were isolated from two independent donors. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.11.


F . T cells were incubated with Raji B cells at a 4 : 1 ratio and IL - 2 production was measured by ELISA at 24 hr . Data were pooled from three donors . * , 0 . 005 < p < 0 . 05 ; * * , 0 . 0005 < p < 0 . 005 . Unpaired two - tailed t - test was applied .

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F. T cells were incubated with Raji B cells at a 4:1 ratio and IL-2 production was measured by ELISA at 24 hr. Data were pooled from three donors. *, 0.005 < p < 0.05; **, 0.0005 < p < 0.005. Unpaired two-tailed t-test was applied.


Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of Ca2 + monitored at 280 nm absorption wavelength . Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength .

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Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.


MICU2 _ WT ( A ) , MICU2 _ K172A ( B ) were pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + . MICU2 _ EF1mut was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + ( C ) , while MICU2 _ EF2mut was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( D ) . MICU2 _ R352A was not pulled - down by GST - MICU1 in the absence of 2 mM Ca2 + ( E ) , while MICU2 _ E329A was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( F ) . MICU2 _ D185A was partially pulled - down by GST - MICU1 in the presence of Ca2 + , while they showed a strong interaction in the absence of 2 mM Ca2 + . MICU2 _ E196K was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + , while a strong interaction was observed in the absence of 2 mM Ca2 + .

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MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.


GST - MICU1 _ EF1mut failed to pull down MICU2 _ WT in the absence of 2 mM Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A & H385A failed to pull - down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . C GST - MICU1 _ E242K failed to pull - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ D231A partly pulled - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A failed to pull down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . GST - MICU1 _ H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2 + .

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GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.


ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA ( G ) and CaCl2 ( H ) .

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ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).


A . Chemical structure of Nec - 1 . The schematic diagram was drawn by S . Lee using ChemDraw Professional 15 . 0 . software .

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A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.


B , C . MTT cytotoxicity assay . ( B ) HT22 cell line was treated with various concentrations of Nec - 1 ( 0 - 200 μM ) . Aggregated Aβ ( 1 - 42 ) was used as the positive control for the induction of neural cell death . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


B , C . MTT cytotoxicity assay . ( C ) HT22 , BV2cell lines and primary astrocytes were pre - treated with Nec - 1 ( 50 μM ) 15 minutes prior to application of 10 μM of pre - aggregated Aβ ( 1 - 42 ) for indicated times . Cell proliferation was then measured by MTT assay . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


D , E . Neural cell death levels measured by staining with the Live / Dead Viability / Cytotoxicity Assay Kit ( Molecular Probes ) . Pre - treatment of cells with 50 μM Nec - 1 preceded the application of 10 μM pre - aggregated Aβ ( 1 - 42 ) by 15 minutes . The assay was performed 24 hours after Aβ ( 1 - 42 ) application . ( D ) Representation of the stained cells . Scale bar = 500 μm ( E ) Quantification of dead cells stained by EthD - 1 .

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D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.


F . Internalization of Aβ ( 1 - 42 ) into HT22 and BV2 cell lines in the presence or the absence of Nec - 1 pre - treatment for indicated periods .

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F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.


G . Cellular expression levels of indicated proteins in HT22 , BV2 cell lines , and primary astrocyte stimulated with Aβ ( 1 - 42 ) aggregates in the presence or the absence of Nec - 1 pre - treatment for 24 hours .

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G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.


A - E . Behavioural tests of APP / PS1mice . ( B - D ) Y - maze and passive avoidance tests on 7 - month - old APP / PS1mice after Nec - 1 administration for 12 weeks . Average alternation ( % ) for each test group ( B ) and total entry number into each arm ( C ) on Y - maze . ( D ) Average latency time in seconds for each test group on passive avoidance test .

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A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.


A - E . Behavioural tests of APP / PS1mice . ( E ) Survival of Wt and APP / PS1mice after injection of Nec - 1 ( 6 . 25 mg / kg ) .

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A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).


F - I . Behavrioural tests of Aβ ( 1 - 42 ) infusion mice . ( H , I ) Y - maze tests on Aβ ( 1 - 42 ) infusion mice model injected with Nec - 1 . Average alternation ( % ) for each test group ( H ) and total entry number into each arm ( I ) on Y - maze .

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F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.


A . Co - immunoprecipitation of RIPK3 with Aβ ( 1 - 42 ) in HT22 , BV2 cell lines , and primary astrocytes in response to Aβ ( 1 - 42 ) aggregates . IP , immunoprecipitation ; IB , immunoblot .

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A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.


B . A close - up view of the interactions between Nec - 1 and surrounding residues in RIPK1 . Nec - 1 is shown in green . The hydrophobic residues around Nec - 1 in RIPK1 kinase domain are shown in sticks . Hydrogen bonds in this and all other figures are represented by yellow dashed lines . Docking score of Nec - 1 in RIPK1 was - 9 . 550 .

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B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.


C . Surface treated view of 12 - mer Aβ fibrils ( 2LMO , VDW radius = 0 . 55 Å ) having hydrophobic pockets plausible for Nec - 1 binding .

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C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.


D . Docked structure of 12 - mer Aβ fibrils ( 2LMO ) with 4 molecules of Nec - 1 . Docking scores for four top - ranked Glide docking poses of Nec - 1 in Aβ plaques ; - 8 . 338 , - 4 . 755 , - 8 . 493 , and - 6 . 754 in clockwise order starting from the top - left corner .

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D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.


E . Surface plasmon resonance sensorgrams of Nec - 1 targeting Aβ ( 1 - 42 ) aggregates ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


4 - month - old APP / PS1 ( n = 13 , male ) and age - matched wild - type ( Wt , n = 14 , male ) mice were intravenously injected with Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for 12 weeks . After the completion of behavioural tests , the brains of each group of mice were subjected to analysis . A . ThS - stained insoluble Aβplaques in whole brain and the hippocampal region of each group . Scale bars = 1 mM ( upper ) , 200 μM ( lower ) . B - G . Statistics of ThS - positive Aβplaques in the brains of APP / PS1mice . Total numbers and areas of ThS - positive Aβplaques in the whole brains ( B and C ) , cortex ( D and E ) and hippocampus ( F and G ) .

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4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).


A . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . Diffuse plaques in the brain sections were stained by anti - Aβ antibody ( 6E10 clone , green colour ) and anti - GFAP antibody ( red colour ) . Hoechst 33342 ( blue colour ) was applied for nuclear counterstaining . Scale bars = 200 μM .

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A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.


B - D . Dot blot analysis of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) and protein oligomer ( anti - amyloidogenic protein oligomer A11 ) in the cortical ( B ) and hippocampal ( C ) region of indicated groups . ( D ) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups . Relative intensity of each band was determined by densitometry of dot blots ( B and C ) using ImageJ software and normalized to vehicle - administered APP / PS1mouse group .

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B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.


E . Sandwich ELISA of Aβ - soluble and - insoluble fractions from cortical and hippocampal lysates of APP / PS1mice .

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E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice.


F . Western blot analyses of cortical and hippocampal lysates from APP / PS1mice for APP and sAPP expressions .

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F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions.


G . ThT assays for the inhibition of Aβ aggregation . Fluorescence intensity was normalized to Aβaggregates ( 100 % , day 5 ) .

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G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5).


H . SDS - PAGE analysis of PICUP cross - linked Aβaggregates visualized by silver staining .

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H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.


A . Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . The membranes analysed in ( A ) are identical to those in Fig 5F .

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A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.


B . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for phosphorylated tau expression levels . Anti - tau ( phospho Ser199 ) antibody ( red colour ) was applied for staining tau phosphorylation on Serine - 199 and Hoechst 33342 ( blue colour ) for nuclear counterstaining in the brain sections . Scale bars = 200 μM .

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B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.


C . Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP / PS1mice injected with Nec - 1 ( upper ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


C . Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec - 1 ( lower ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


D , E . ThT assays for the inhibition of tau aggregation ( D ) and for disaggregation of tau ( E ) . Fluorescence intensity was normalized to tauaggregates ( 100 % , day 5 ) .

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D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).


F . Surface plasmon resonance sensorgrams of Nec - 1 targeting tau ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h and stained as in ( A ) . aspect ratio ( O ) , branch length ( P ) , and roundness ( Q ) of mitochondria in the cells in ( N ) .

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MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


TMRE ( 100 nM ) and MitoTracker green ( 200 nM ) staining in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE intensity in ( A ) on a per cell basis . MitoSOX ( 5 μM ) and MitoTracker Green ( 200 nM ) staining in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX intensity for ( C ) on a per cell basis .

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TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed and imaged in pairs ( SD and HFD ) .

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NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).


Citrate synthase staining in the arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 .

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Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.


imaging was performed in the areas of the cortex


NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from the mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H intensity from ( B ) calculated on a per cell basis ; 16 - 25 cells from each of the 5 mice per group were quantified ( 87 - 109 total cells per treatment group ) .

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NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).


TMRE ( 100 nM ) and MitoTracker Green staining ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) intensity for ( E ) calculated on a per cell basis ; 15 - 24 cells from each of the 5 mice in each group were quantified ( 99 - 104 total cells per treatment group ) .

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TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).


MitoSOX ( 5 μM ) and MitoTracker Green staining in eWAT resected from the mice in ( D ) treated as in ( B and E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) intensity calculated as in ( F and G ) ; 10 - 13 cells from each of the 5 mice in each group were quantified ( 57 - 58 total cells per treatment group ) .

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MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).


Chop , Xbp1s , Atf6 , and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , and Friday for the final 3 weeks ; n = 7 - 9 .

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Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 weeks then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with the amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

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Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Average daily distance run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , and Fridays over 4 weeks when housed with a running wheel .

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Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.


Body weight change in the mice in ( O , P ) over the 24 h after the first treatment ( Q ) or after the 3 week course of treatment ( R ) ; n = 9 .

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Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Representative H & E stained liver sections from the mice shown in Fig . 6F after 4 weeks of treatment with vehicle or 120 mg / kg SH - BC - 893 . Scale bar , 10 μm .

Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.


Blood glucose levels ( F ) or area under the curve ( AUC , ( G ) ) during an oral glucose tolerance test performed in the same mice as in ( E ) .

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Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).


Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH - BC - 893 from the mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 weeks on HFD ) ; n = 8 . Insulin levels in the plasma collected 4 h after treatment with vehicle or SH - BC - 893 from the same mice as in Fig . 5A , B ; n = 8 .

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Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


C TIMM50S levels in primary and immortalized skin fibroblast from control ( C1 - 4 ) and mutant TIMM50 ( Pt ) . SDHA and ETHE1 are shown as loading controls .

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C TIMM50S levels in primary and immortalized skin fibroblast from control (C1-4) and mutant TIMM50 (Pt). SDHA and ETHE1 are shown as loading controls.


D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts . Data are shown as mean ± SD , n = 4 .

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D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts. Data are shown as mean ± SD, n = 4.


A Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium .

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A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.


B Western - blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium .

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B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.


C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC - 1 staining in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


D TMRM live staining of control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in either glucose or galactose containing medium . Scale bar corresponds to 10 μm .

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

D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm.


A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP . The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1 . Coomassie Blue ( CB ) staining was used as loading control and the radiolabeled protein ( TNT ) as reference for quantification . The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . Western blot for TIMM50 is shown as cell line control . B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor ( empty arrowheads ) and the mature protein ( filled arrowhead ) are also shown . D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector ( EV ) or with wild - type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 .

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

C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05.


Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Two biological replicates are shown for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red .

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

Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red.


B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Two biological replicates were obtained for each cell line and condition . Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red .

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

B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red.


Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


A ROS production as measured by intracellular oxidation of the cell - permeant probe DCF - DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose . Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min . Data are shown as mean ± SD , n = 3 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

A ROS production as measured by intracellular oxidation of the cell-permeant probe DCF-DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 grouping both controls together .

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

B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01 grouping both controls together.


D Steady state levels of mitophagy related proteins VDAC1 , p62 and LC3 - II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . E Quantification of the steady state levels of VDAC1 , p62 and LC3 - II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 grouping both controls together .

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

D Steady state levels of mitophagy related proteins VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. E Quantification of the steady state levels of VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.


A Cell size in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


B Growth curves of control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Cell growth was monitored continuously by the Incucyte live cell imager ( Essen Bioscience ) . One of the three independent experiments carried out is presented . Data are shown as mean of three technical replicates ± SD .

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

B Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD.


C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Cells in the late stage of apoptosis are boxed in the top right corner .

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

C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner.


D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts , transduced with the empty vector ( EV ) or with wild - type TIMM50 grown in glucose or galactose . Data are shown as mean ± SD , n = 5 or 4 biological replicates in the case of transduced cells ; ∗ * ∗ p < 0 . 001 .

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

D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001.


A Western - blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium .

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

A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium.


B Mitochondrial membrane potential in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC - 1 staining in untreated or FCCP treated conditions . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


C Western blot for representatives of all five respiratory complexes in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . GAPDH is from the same blot as panel A . Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red .

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

C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.


D Oxygen consumption measurements in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . Values of basal and maximal respiration along with ATP - production dependent , proton leak respiration and spare capacity are shown . Data are shown as mean ± SD , n = 4 biological replicates ; ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ * ∗ p < 0 . 001 .

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

D Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.


E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1 , p62 and LC3 in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control . GAPDH is from the same blot as panel A .

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

E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A.


F Quantification of the number of late apoptotic cells in control and TIMM50 , RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose . Data are shown as mean ± SD , n = 4 or more biological replicates ; ∗ * ∗ p < 0 . 001 .

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

F Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001.


Huh7 cells were infected with HCV ( strain Jc1 ) using 30 TCID50 / cell and 48 h later cells were fixed and processed for fluorescence microscopy . In case of samples shown in panels B - D , cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above . Samples were analyzed with a Nikon TE2000 - E inverted confocal microscope at 60 × magnification . ( A ) - ( D ) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase ( PDI ; an ER marker ) , GFP - Rab21 ( marker for early endosomes ) , GFP - Rab7 ( marker for late endosomes ) or βCOP - YFP ( marker for COP I vesicles ) . The upper panels represent a low magnification overview ; boxed areas are shown as enlargement in the corresponding panel below . The nucleus was stained with DAPI ( blue ) . Scale bars represent 10 µm ( top panels ) and 2 µm ( lower panels ) . The quantification of the degree of colocalization ( Pearson ' s correlation coefficient ) is given at the top of the enlarged pictures .

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

Huh7 cells were infected with HCV (strain Jc1) using 30 TCID50/cell and 48 h later cells were fixed and processed for fluorescence microscopy. In case of samples shown in panels B-D, cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above. Samples were analyzed with a Nikon TE2000-E inverted confocal microscope at 60× magnification. (A)-(D) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase (PDI; an ER marker), GFP-Rab21 (marker for early endosomes), GFP-Rab7 (marker for late endosomes) or βCOP-YFP (marker for COP I vesicles). The upper panels represent a low magnification overview; boxed areas are shown as enlargement in the corresponding panel below. The nucleus was stained with DAPI (blue). Scale bars represent 10 µm (top panels) and 2 µm (lower panels). The quantification of the degree of colocalization (Pearson's correlation coefficient) is given at the top of the enlarged pictures.


( A ) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP - tagged NS5A . Huh7 - Lunet cells were transfected with replicon RNA and seeded onto carbon - patterned sapphire discs . Twenty - four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM . ( a ) Fluorescence image ; ( b ) enlarged fluorescence image of the cell of interest ; ( c ) merge of bright field and fluorescence images . Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells . White squares in a and c enclose the cell shown in b . ( B ) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image . Areas marked with a green dotted line indicate regions of intense fluorescence . Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell . ( C ) Higher magnification images of the two different regions , labeled 1 and 2 in panel B , corresponding to regions with intense fluorescence ( 1 ) or a region corresponding to the intersection of high to low fluorescence ( 2 ) . Region 1 ( top panel ) corresponds to a DMV - containing area residing in close proximity of the ER ; region 2 ( bottom panel ) corresponds to a LD - enriched area containing DMVs in very close proximity . Areas marked with white squares in the left images are magnified in the corresponding right panels . LD , lipid droplet ; ER , endoplasmic reticulum ; DMV , double membrane vesicle ; m , mitochondrium ; if , intermediate filaments .

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

(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.


( A ) Images of mock - infected high - pressure - frozen and freeze - substituted Huh7 . 5 cells . ( B - E ) Micrographs of high pressure frozen freeze - substituted Huh7 . 5 cells harvested 16 , 24 , 36 and 48 h after infection with Jc1 ( 100 TCID50 / cell ) . Yellow squares indicate the areas that are shown at higher magnification on the right of each subpanel . Note the time - dependent increase of complexity of HCV - induced membrane alterations . ER , endoplasmic reticulum ; m , mitochondria ; MVB , multi - vesicular bodies ; LD , lipid droplet ; DMV , double membrane vesicle ; MMV , multi membrane vesicle ; DMT , double membrane tubule ( labeled with a black arrow ) .

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(A) Images of mock-infected high-pressure-frozen and freeze-substituted Huh7.5 cells. (B-E) Micrographs of high pressure frozen freeze-substituted Huh7.5 cells harvested 16, 24, 36 and 48 h after infection with Jc1 (100 TCID50/cell). Yellow squares indicate the areas that are shown at higher magnification on the right of each subpanel. Note the time-dependent increase of complexity of HCV-induced membrane alterations. ER, endoplasmic reticulum; m, mitochondria; MVB, multi-vesicular bodies; LD, lipid droplet; DMV, double membrane vesicle; MMV, multi membrane vesicle; DMT, double membrane tubule (labeled with a black arrow).


Immuno - EM localization of NS5A and NS3 in HCV - infected cells . Huh7 . 5 cells were infected with 100 TCID50 / cell of Jc1 and fixed 16 h post - infection . ( A ) Cells shown in panels b , c and e to g were post - fixed with 0 . 1 % OsO4 providing best membrane preservation , especially in case of DMVs ( f and g ) and LDs ( e ) . Both NS5A and NS3 localize predominantly to the ER ( b and d ) and 50 - 70 nm diameter single - membrane vesicles ( SMVs ; subpanel a , c and d ) . NS5A also localizes to lipid droplets ( e ) .

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

Immuno-EM localization of NS5A and NS3 in HCV-infected cells.Huh7.5 cells were infected with 100 TCID50/cell of Jc1 and fixed 16 h post-infection. (A) Cells shown in panels b, c and e to g were post-fixed with 0.1% OsO4 providing best membrane preservation, especially in case of DMVs (f and g) and LDs (e). Both NS5A and NS3 localize predominantly to the ER (b and d) and 50-70 nm diameter single-membrane vesicles (SMVs; subpanel a, c and d). NS5A also localizes to lipid droplets (e).


( B ) Amount of gold particles per µm2 in Jc1 - infected versus mock - infected cells after immunolabeling with NS3 - and NS5A - specific primary antibodies . Note the higher immunolabeling with samples prepared without OsO4 , but also the lower membrane preservation under this condition .

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(B) Amount of gold particles per µm2 in Jc1-infected versus mock-infected cells after immunolabeling with NS3- and NS5A-specific primary antibodies. Note the higher immunolabeling with samples prepared without OsO4, but also the lower membrane preservation under this condition.


( C ) Relative labeling distribution of NS3 and NS5A . Thawed cryosections of cells post - fixed or not with OsO4 were labelled with NS3 - or NS5A - specific antibodies by using two different blocks and 3 different labeling experiments . Per labeling experiment two grids were considered , counting ∼ 100 - 200 gold particles per grid and attributing the particles to the indicated structures . In the case of uninfected cells only background labeling in the cytoplasm , on mitochondria and undefined structures was seen . Numbers refer to the percent of total gold particles counted per sample . ER , endoplasmic reticulum ; SV , small vesicles ; Cyto , cytosol ; Mito , mitochondria ; n . d . , non - defined structures ; NE , nuclear envelope ; EE / LE , early / late endosomes ; PM , plasma membrane ; DMV , double membrane vesicle ; LD , lipid droplet .

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

(C) Relative labeling distribution of NS3 and NS5A. Thawed cryosections of cells post-fixed or not with OsO4 were labelled with NS3- or NS5A-specific antibodies by using two different blocks and 3 different labeling experiments. Per labeling experiment two grids were considered, counting ∼100-200 gold particles per grid and attributing the particles to the indicated structures. In the case of uninfected cells only background labeling in the cytoplasm, on mitochondria and undefined structures was seen. Numbers refer to the percent of total gold particles counted per sample. ER, endoplasmic reticulum; SV, small vesicles; Cyto, cytosol; Mito, mitochondria; n.d., non-defined structures; NE, nuclear envelope; EE/LE, early/late endosomes; PM, plasma membrane; DMV, double membrane vesicle; LD, lipid droplet.


Correlation between HCV RNA replication and appearance of double membrane vesicles . ( A ) Time course of spread of HCV infection in Huh7 . 5 cells infected with 100 TCID50 / cell of Jc1 . Infected cells were detected by immunofluorescence using an NS5A - specific antiserum ( upper panels ) . The graph shown below represents the result of counting ∼ 200 cells for each time point to determine the percentage of infected cells . Scale bars represent 50 µm .

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Correlation between HCV RNA replication and appearance of double membrane vesicles.(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID50/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm.


( B ) Time course of accumulation of intracellular HCV RNA in infected Huh7 . 5 cells . The graph shows the result of two independent experiments ( 3 replicas each ) . Whiskers indicate the minimum and maximum values .

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(B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values.


( C ) Colocalization of dsRNA and NS5A in cells infected with Jc1 ( 10 TCID50 / cell ) . Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy . DNA was stained with DAPI ( blue ) . Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels . The quantification of the degree of colocalization ( Pearson ' s correlation coefficient ) is given in the enlarged pictures . Scale bars represent 10 µm and 2 µm ( left and right panels , respectively ) .

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(C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID50/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively).


( D ) Time course of accumulation of DMVs and MMVs . For each time point , 10 cellular profiles were counted . The Mann - Whitney ( non - parametric ) test was applied to determine statistical significance . Error bars refer to the standard deviation . Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi .

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(D) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.


( A ) Huh7 . 5 cells were infected with 100 TCID50 / cell of Jc1 , fixed 16 h later and after HPF - FS processed for ET as described in materials and methods . Left : slice of a dual axis tomogram showing the various membrane alterations . Right : 3D reconstruction of the complete tomogram . Note the high number of DMVs . Panels B , C and D are part of the tomogram displayed in panel A and their position in panel A is highlighted by either a yellow dashed square ( B and C ) or by a star ( D ) . In the 3D models shown on the right , the ER is depicted in dark brown , the inner membrane of DMVs and DMTs in yellowish brown and their outer membrane in semi - transparent light brown . Single membrane vesicles are colored in pink , intermediate filaments in dark blue and the Golgi apparatus in green . ( B ) Left : serial single slices through the same tomogram shown in panel ( A ) displaying a connection between the outer membrane of a DMV and the ER membrane ( black arrows ) . Right : 3D surface model showing the membrane connection . ( C ) Left : serial single slices through the same tomogram illustrating a lasso - like structure of a DMV that after rendering reveals a pore - like opening that connects the interior of the DMV with the cytosol . The position of this opening in the 2D slice is marked with a black arrow . Right : 3D view of this DMV showing the ' pore ' . ( D ) Left : serial single slices through the same tomogram showing a DMV with a large inter - membrane space between its inner and outer membranes . Right : 3D view of this DMV . Scale bars represent 100 nm . This tomogram is shown in movie S1 .

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

(A) Huh7.5 cells were infected with 100 TCID50/cell of Jc1, fixed 16 h later and after HPF-FS processed for ET as described in materials and methods. Left: slice of a dual axis tomogram showing the various membrane alterations. Right: 3D reconstruction of the complete tomogram. Note the high number of DMVs. Panels B, C and D are part of the tomogram displayed in panel A and their position in panel A is highlighted by either a yellow dashed square (B and C) or by a star (D). In the 3D models shown on the right, the ER is depicted in dark brown, the inner membrane of DMVs and DMTs in yellowish brown and their outer membrane in semi-transparent light brown. Single membrane vesicles are colored in pink, intermediate filaments in dark blue and the Golgi apparatus in green. (B) Left: serial single slices through the same tomogram shown in panel (A) displaying a connection between the outer membrane of a DMV and the ER membrane (black arrows). Right: 3D surface model showing the membrane connection. (C) Left: serial single slices through the same tomogram illustrating a lasso-like structure of a DMV that after rendering reveals a pore-like opening that connects the interior of the DMV with the cytosol. The position of this opening in the 2D slice is marked with a black arrow. Right: 3D view of this DMV showing the 'pore'. (D) Left: serial single slices through the same tomogram showing a DMV with a large inter-membrane space between its inner and outer membranes. Right: 3D view of this DMV. Scale bars represent 100 nm. This tomogram is shown in movie S1.


( A ) Left : slice of a dual axis tomogram taken from Huh7 . 5 cells 36 h after infection with 100 TCID50 / cell of Jc1 . Right : 3D reconstruction of the tomogram . Note the extensive membrane reorganization and the appearance of MMVs predominating at late time points after infection . Black arrows show invaginations of DMVs . The white star indicates a large MMV ; due to its complexity only its middle part could be rendered . ( B ) Left : slices through the same tomogram highlighting a DMT enwrapping a DMV and presumably leading to the formation of a MMV . Right : 3D surface rendering of this structure . ( C ) Left : slices through the same tomogram highlighting a ' self - invagination ' event of a DMV , also leading to the formation of a MMV . Right : 3D surface rendering of this late structure , revealing an opening of this MMV towards the cytosol as a result of the self - invagination . Panels B and C are part of the tomogram displayed in panel A and their positions are highlighted by yellow dashed squares in panel A . Scale bars represent 100 nm . For further details see legend to Figure 6 . This tomogram is shown in movie S2 .

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

(A) Left: slice of a dual axis tomogram taken from Huh7.5 cells 36 h after infection with 100 TCID50/cell of Jc1. Right: 3D reconstruction of the tomogram. Note the extensive membrane reorganization and the appearance of MMVs predominating at late time points after infection. Black arrows show invaginations of DMVs. The white star indicates a large MMV; due to its complexity only its middle part could be rendered. (B) Left: slices through the same tomogram highlighting a DMT enwrapping a DMV and presumably leading to the formation of a MMV. Right: 3D surface rendering of this structure. (C) Left: slices through the same tomogram highlighting a 'self-invagination' event of a DMV, also leading to the formation of a MMV. Right: 3D surface rendering of this late structure, revealing an opening of this MMV towards the cytosol as a result of the self-invagination. Panels B and C are part of the tomogram displayed in panel A and their positions are highlighted by yellow dashed squares in panel A. Scale bars represent 100 nm. For further details see legend to Figure 6. This tomogram is shown in movie S2.


Membrane alterations induced by expression of individual HCV proteins . Huh7 . 5 cells transfected with replicon RNAs ( A , B ) or Huh7 - Lunet - T7 cells transfected with pTM - based expression constructs ( C - H ) specified in the left of each panel , respectively , were high pressure frozen 24 h after transfection , freeze substituted , embedded into epon resin and sections were analyzed by transmission EM . Representative images showing HCV - induced membrane alterations are shown .

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Membrane alterations induced by expression of individual HCV proteins.Huh7.5 cells transfected with replicon RNAs (A, B) or Huh7-Lunet-T7 cells transfected with pTM-based expression constructs (C-H) specified in the left of each panel, respectively, were high pressure frozen 24 h after transfection, freeze substituted, embedded into epon resin and sections were analyzed by transmission EM. Representative images showing HCV-induced membrane alterations are shown.


( I ) Average diameter of specific vesicular structures , either DMVs or SMVs , detected in cells that had been transfected with constructs specified in the upper panels . Whiskers represent minimum and maximum values . ( J ) Number of vesicular structures detected in profiles of 10 transfected cells . Cells transfected with the pTM expression vector without HCV insert were used as reference . The number of vesicular structures per µm2 is given ; whiskers represent minimum and maximum values . ( K ) Relative abundance of membranous structures . Note that only upon expression of the NS3 - 5A polyprotein and NS5A two different structures were observed . In all other cases , only one membranous structure was detected .

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(I) Average diameter of specific vesicular structures, either DMVs or SMVs, detected in cells that had been transfected with constructs specified in the upper panels. Whiskers represent minimum and maximum values. (J) Number of vesicular structures detected in profiles of 10 transfected cells. Cells transfected with the pTM expression vector without HCV insert were used as reference. The number of vesicular structures per µm2 is given; whiskers represent minimum and maximum values. (K) Relative abundance of membranous structures. Note that only upon expression of the NS3-5A polyprotein and NS5A two different structures were observed. In all other cases, only one membranous structure was detected.


B . Coomassie - stained SDS - PAGE ( from left to right ) of Hchim , V1ΔH , VoND , and C .

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B. Coomassie-stained SDS-PAGE (from left to right) of Hchim, V1ΔH, VoND, and C.


C . Representative ATPase activity assay ( ATP regenerating system ) . At the indicated time , 200 nM ConA is added to the assay .

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C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.


C . Growth phenotypes of wild type and c ″ - mutant yeast strains probed on ( 1 ) rich media buffered to pH 5 ( YPD pH 5 ) ; ( 2 ) synthetic dropout ( SD ) plus all amino acids ( SD + all a . a . ) ; ( 3 ) SD minus uracil ( SD - ura ) ; ( 4 , 5 ) YPD buffered to pH 7 plus 4 mM Zn2 + or 60 mM Ca2 + , respectively ; ( 6 ) YPD buffered to pH 7 ; ( 7 ) YPD plus 60 mM Ca2 + .

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C. Growth phenotypes of wild type and c″-mutant yeast strains probed on (1) rich media buffered to pH 5 (YPD pH 5); (2) synthetic dropout (SD) plus all amino acids (SD + all a.a.); (3) SD minus uracil (SD - ura); (4,5) YPD buffered to pH 7 plus 4 mM Zn2+ or 60 mM Ca2+, respectively; (6) YPD buffered to pH 7; (7) YPD plus 60 mM Ca2+.


B . Mass spectrometry analysis of V1Hchim . Protein IDs are plotted as a function of overall peptide spectrum matches ( PSM ) and the posterior error probability ( PEP ) scores .

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B. Mass spectrometry analysis of V1Hchim. Protein IDs are plotted as a function of overall peptide spectrum matches (PSM) and the posterior error probability (PEP) scores.