( C ) Representative fluorescence microscopy images of HIV - infected macrophages which were fixed , permeabilized then stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) and antibody to LC3B ( green ) , SQSTM1 ( magenta ) , HIV p24 and p17 ( red ) . Co - localization of HIV and LC3B is represented by orange pixels , and co - localization of HIV , SQSTM1 , and LC3B is represented by cream pixels . Scale bars indicate 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm.


( D ) Macrophages were transduced with shNS or ATG13 shRNA ( shATG13 ) and analyzed for ATG13 expression . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( E ) Macrophages transduced with shNS or shATG13 from ( D ) were exposed to infectious HIV , 5 μg / mL ssRNA40 , 5 μg / mL ssRNA41 , or 100 nmol / L sirolimus for 24 h , harvested , lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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

(E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( F ) Macrophages were pretreated with 100 nmol / L bafilomycin A1 then exposed to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV , LyoVec , 5 μg / mL ssRNA41 , 5 μg / mL ssRNA40 , or 100 nmol / L sirolimus for 24 h , harvested , lysed and analyzed for endogenous LC3B , CYP27B1 and VDR by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


Macrophages were infected with HIV and cells harvested at 1 , 3 , 5 , 7 , and 10 days post - infection . ( A ) Cells were analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

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

Macrophages were infected with HIV and cells harvested at 1, 3, 5, 7, and 10 days post-infection. (A) Cells were analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


( B ) Cells were harvested then subjected to flow cytometry analysis of saponin - resistant LC3B - II in macrophages . Representative histograms of cells displaying saponin - resistant LC3B - II are shown .

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 B-CONTROLLED_VAR O O O O O O O O

(B) Cells were harvested then subjected to flow cytometry analysis of saponin-resistant LC3B-II in macrophages. Representative histograms of cells displaying saponin-resistant LC3B-II are shown.


( C ) Cells were harvested , lysed then analyzed for CYP27B1 and VDR by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were harvested, lysed then analyzed for CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


( A ) Macrophages were exposed to increasing concentrations of purified HIV for 24 h , harvested , lysed , fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Top , a representative blot is shown . Bottom , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( B ) Macrophages were infected with HIV and at 1 , 3 , 5 , 7 , and 10 days post - infection cells were harvested , lysed and fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


C ) Representative fluorescence microscopy images of HIV - infected macrophages which were fixed , permeabilized then stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; green ) and antibody to TFEB ( red ) at the indicated times post - infection . Co - localization of TFEB and DAPI staining is represented in the image by orange pixels . Scale bars indicate 20 μm .

C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm.


( D ) Macrophages were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated iodixanol velocity gradient purified HIVBa - L for 24 h , harvested , lysed fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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

(D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( E ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) and analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( F ) Macrophages transduced with shNS or shTLR8 from ( E ) were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated purified HIV for 24 h . Cells were then harvested , lysed , fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Top , a representative blot is shown . Bottom , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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

(F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , or TFEB shRNA ( shTFEB ) were infected with HIV for 10 days . Cells were lysed and analyzed for TFEB and ACTB content by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages transduced with non-specific scrambled shRNA (shNS), or TFEB shRNA (shTFEB) were infected with HIV for 10 days. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) At 1 , 3 , 5 , 7 , and 10 days post - infection cells from ( A ) were harvested , lysed , and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , representative blots are shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) Macrophages transduced with non - specific cDNA ( Control ) , or TFEB cDNA ( TFEB ) then exposed to HIV . Cells were lysed and analyzed for TFEB and ACTB content by Western blotting . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(C) Macrophages transduced with non-specific cDNA (Control), or TFEB cDNA (TFEB) then exposed to HIV. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( D ) Transduced cells from ( C ) were exposed to purified HIV for the indicated time ( minutes ) harvested , lysed , and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , representative blots are shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transduced cells from (C) were exposed to purified HIV for the indicated time (minutes) harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) qRT - PCR analysis of mRNA expression of autophagy ( UVRAG and ATG9B ) and lysosomal ( MCOLN1 ) genes 24 h post - exposure to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV . Data are reported as mean ± s . e . m . , n = 4 .

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

(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) and analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(B) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) Macrophages from ( B ) were exposed to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV for 24 h . Cells were harvested and qRT - PCR for UVRAG , ATG9B , MCOLN1 was performed . Data were normalized to the shNS mock infected control for each gene . Bar charts are reported as mean ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , or BECN1 shRNA ( shBECN1 ) were infected with HIV for 10 days . Cells were lysed and analyzed for BECN1 and ACTB content by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(A) Macrophages transduced with non-specific scrambled shRNA (shNS), or BECN1 shRNA (shBECN1) were infected with HIV for 10 days. Cells were lysed and analyzed for BECN1 and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) At 1 , 3 , 5 , 7 , and 10 days post - infection cells from ( A ) were harvested , lysed and analyzed for endogenous LC3B , SQSTM1 , and ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) At 1 , 3 , 5 , 7 , and 10 days post - infection cells from ( A ) were harvested , lysed , fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) Macrophages were infected with HIVNL ( AD8 ) or HIVNL ( AD8 ) ΔNef . At 1 , 3 , 5 , 7 , and 10 days post - infection cells were harvested , lysed and analyzed for endogenous LC3B , SQSTM1 , and ACTB by Western blotting . Left , representative blots are shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) Macrophages were infected with HIVNL ( AD8 ) or HIVNL ( AD8 ) ΔNef . At 1 , 3 , 5 , 7 , and 10 days post - infection cells were harvested , lysed , fractionated for cytoplasmic and nuclear content , and analyzed for TFEB , ACTB and H3 histone by Western blotting . Left , representative blots are shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


Immunoblot with anti - HA Abs of lysates from parental ( Ctrl ) and tagged lines ( TgCRMPa - HA3 and TgCRMPb - HA3 ) together with inducible - knockdown lines ( TgCRMPa - HA3 _ iKD and TgCRMPb - HA3 _ iKD ) treated with ATc for 0 , 24 or 48h . TgROP5 was used as loading control . Two close bands around 300kDa were detected for TgCRMPa . A ~ 820kDa protein , corresponding to the predicted size for TgCRMPb , was observed together with a ~ 130kDa band .

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

Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.


Quantification of microneme secretion in TgCRMPa - and TgCRMPb - depleted tachyzoites was measured by detecting the processed form ( arrowhead ) of TgMIC2 ( arrow ) in the media . Control , TgCRMPa _ iKD and TgCRMPb _ iKD parasites , ATc - treated ( + ) and untreated ( - ) , were stimulated with propranolol to release microneme contents . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of two independent experiments .

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

Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Co - immunoprecipitation of TgCRMPa and TgCRMPb . Lysates from parasites co - expressing TgCRMPa - FLAG3 and TgCRMPb - HA3 were split and incubated with either anti - FLAG ( left panels ) or anti - HA beads ( right panels ) . Eluates were subjected to SDS - PAGE and immunoblotted with anti - HA and anti - FLAG Abs . Untagged , TgCRMPa - FLAG3 and TgCRMPb - HA3 parasites were used as controls . TgCRMPs robustly associate with each other .

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

Co-immunoprecipitation of TgCRMPa and TgCRMPb. Lysates from parasites co-expressing TgCRMPa-FLAG3 and TgCRMPb-HA3 were split and incubated with either anti-FLAG (left panels) or anti-HA beads (right panels). Eluates were subjected to SDS-PAGE and immunoblotted with anti-HA and anti-FLAG Abs. Untagged, TgCRMPa-FLAG3 and TgCRMPb-HA3 parasites were used as controls. TgCRMPs robustly associate with each other.


Quantification of microneme secretion in control and Tg277910 - depleted tachyzoites was measured as in Fig 2H . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of two independent experiments .

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

Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Immunofluorescence images of extracellular tachyzoites of untagged , TgCRMPa - HA3 and TgCRMPb - HA3 parasites , incubated either with host cell monolayers for 2min , or with ionophore A23187 , to induce natural or artificial conoid extrusion , respectively . Parasites were immunostained with anti - HA Abs ; DNA was labeled by Hoechst . CRMPa and CRMPb consistently accumulate at the tip of extruded conoids ( arrows ) . The apexes of A23187 - treated parasites were magnified on the right , and increased in brightness and contrast to highlight the apical dots . DIC : differential interference contrast . Quantification of the dot pattern upon A23187 treatment shown in ( A ) . Values are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the apical accumulation of TgCRMPa and TgCRMPb ; n = number of parasites analyzed per line .

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

Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.


Immunofluorescence images of extracellular TgCRMPb - HA3 tachyzoites in presence ( - ATc ) or absence ( + 48h ATc ) of TgCRMPa - FLAG3 ( iKD line ) . Parasites were stained with anti - HA Abs to visualize CRMPb . TgCRMPb localization at the tip of the extruded conoid ( arrow ) disappears upon TgCRMPa depletion , but it is still detected in the cytoplasm ( lower panel ) . DNA is labeled by Hoechst . Single focal planes are shown . DIC : differential interference contrast . Quantification of the dot pattern shown in ( C ) . The values are reported as in ( B ) .

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

Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.   Quantification of the dot pattern shown in (C). The values are reported as in (B).


Whole cell lysates from TIR1 - expressing parental line ( Ctrl ) , HA3 - miniAID - TgCRMPa _ iKD ( N - term ) and TgCRMPa - miniAID - HA3 _ iKD ( C - term ) lines were immunoblotted with anti - HA Abs to visualize tagged CRMPa in IAA - treated and untreated samples . CRMPa was undetectable upon 24h incubation with IAA when C - terminally , but not N - terminally , tagged with the miniAID - HA3 , suggesting that the C - terminus is the one exposed towards the cytosol . TgMIC2 was used as loading control and detected with anti - MIC2 Abs . MW : molecular weight standards .

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

Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.


Immunofluorescence images of extracellular A23187 - treated parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa , and immunostained as in ( A ) . TgCRMPa accumulates at the tip of extruded conoids ( arrows ) in triton - permeabilized ( + ) or non - permeabilized ( - ) parasites . DIC : differential interference contrast . Single focal planes are shown . Quantification of the dot pattern upon natural ( + H F Fibroblasts ) or artificial ( + A23187 ) conoid extrusion in parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa . Values are reported as in ( B ) .

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

Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.   Quantification of the dot pattern upon natural (+H F Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).


Ultrastructure Expansion Microscopy of extracellular tachyzoites , either untagged or co - expressing TgCRMPa - HA3 / TgCRMPa - TY2 and TgCRMPb - HA3 together or in pairwise combination with TgNd6 - TY2 . Parasites were treated with A23187 prior to fixation and preparation for U - ExM , and stained with anti - HA , anti - TY and anti - α / β tubulin Abs to label CRMPs , Nd6 / CRMPa and microtubules , respectively . Shown are maximum intensity projections of z - stack confocal images . CRMPs overlap with Nd6 at the tip of the extruded conoid ( yellow selection ) . A magnified image of the apical tip of each parasite is shown on the right .

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

Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.


Extent of colocalization between TgCRMPa - TY2 and TgCRMPb - HA3 , and TgCRMPs - HA3 with TgNd6 - TY2 in the apical dot shown in B . Pearson ' s correlation coefficient was measured using the Fiji - JACoP plugin . Values are expressed as mean ± SD . Three - five parasites per line were analyzed . Quantification of the dot pattern for TgCRMPa - HA3 and TgCRMPb - HA3 in TgNd9 _ iKD lines . CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A . TgCRMPs were still found in the apical dot in absence of TgNd9 ( + ATc ) . Numbers are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the tip accumulation of TgCRMPa and TgCRMPb . The number of parasites ( n ) analyzed for each line is reported at the top of the column .

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

Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed. Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.


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

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

Immuno-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 .

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

(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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

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

(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 ) .

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 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

(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 .

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

(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 .

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

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 .

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

(I) 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.


Western blot analysis and quantitation of Sel1L - Hrd1 ERAD proteins in the livers of WT mice at 3 , 9 and 24 weeks of age ( n = 3 - 6 per group , 2 independent repeats ) . α - tubulin , loading controls for Western blot analysis .

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

Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of WT mice at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.


Western blot analysis and quantitation of Sel1L - Hrd1 ERAD proteins in the livers of 10 - week - old WT mice under fasted ( overnight ) or ad libitum fed conditions ( n = 3 - 6 per group , 2 independent repeats ) . α - tubulin , loading controls for Western blot analysis .

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

Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of 10-week-old WT mice under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.


Western blot analysis and quantitation of ERAD proteins in Sel1Lf / f and Sel1LAlb livers ( n = 4 per group , 3 independent repeats ) . Hsp90 loading controls for Western blot analysis .

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

Western blot analysis and quantitation of ERAD proteins in Sel1Lf/f and Sel1LAlb livers (n=4 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.


Growth curves of male ( n = 10 each ) and female ( n = 7 each ) mice .

Growth curves of male (n=10 each) and female (n=7 each) mice.


Representative images ( E ) and nose - to - anus length ( F ) of male mice at 6 weeks of age ( n = 6 - 10 per group ) .

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

Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group).


Organ - to - body - weight ratios of liver and kidney in 6 - week - old male mice ( n = 6 per group , 3 independent repeats ) .

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

Organ-to-body-weight ratios of liver and kidney in 6-week-old male mice (n=6 per group, 3 independent repeats).


Daily food intake ( g / d ) normalized to gram of body weight ( gbw ) ( n = 3 per group , measured over 3 days ) .

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

Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days).


Representative estrus cycle mapping in 2 - 4 - month old females ( n = 6 per group ) .

Representative estrus cycle mapping in 2-4-month old females (n=6 per group).


H & E images of paraffin - embedded liver sections from 9 - week - old mice ( n = 4 per group , 3 independent repeats ) .

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

H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).


Western blot analysis of cell death ( cleaved and pro - caspase - 3 ) in livers of 9 - week - old mice , with WT ileum as positive control ( n = 3 per group , 3 independent repeats ) . Hsp90 loading controls for Western blot analysis .

Western blot analysis of cell death (cleaved and pro-caspase-3) in livers of 9-week-old mice, with WT ileum as positive control (n=3 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.


Representative transmission electron microscope ( TEM ) images obtained from 9 - week - old female mice livers ( n = 10 - 12 cells from one mouse each ) . N , nucleus ; mito , mitochondria ; ER , endoplasmic reticulum .

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

Representative transmission electron microscope (TEM) images obtained from 9-week-old female mice livers (n=10-12 cells from one mouse each). N, nucleus; mito, mitochondria; ER, endoplasmic reticulum.


Volcano plot depicting transcriptomics data from the livers of 9 - week - old Sel1Lf / f and Sel1LAlb mice ( n = 3 per group ) ; dotted line marks p = 0 . 05 ; black dots represent fold change > 2 .

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

Volcano plot depicting transcriptomics data from the livers of 9-week-old Sel1Lf/f and Sel1LAlb mice (n=3 per group); dotted line marks p=0.05; black dots represent fold change>2.


qPCR analyses of Fgf21 expression in 9 - week - old livers ( n = 3 - 6 per group , 3 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

qPCR analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). Ribosomal L32, loading control for qPCR analysis.


Western blot analyses of Fgf21 expression in 9 - week - old livers ( n = 3 - 6 per group , 3 independent repeats ) . α - tubulin , loading control for Western blot analysis .

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

Western blot analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). α-tubulin, loading control for Western blot analysis.


ELISA analysis of Fgf21 in serum from 8 - 9 - week - old mice ( n = 6 - 7 per group ) .

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

ELISA analysis of Fgf21 in serum from 8-9-week-old mice (n=6-7 per group).


Protein levels of Sel1L ( left panel ) and mRNA levels of Fgf21 ( right panel ) in primary mouse hepatocytes isolated from the tamoxifen - inducible Sel1L - knockout Sel1LERCre mice ( 2 independent repeats ) . Hsp90 , loading control for Western blot analysis . Ribosomal L32 , loading control for qPCR analysis .

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

Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats). Hsp90 , loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis.


Acute loss - of - function model where 8 - week - old Sel1Lf / f mice were injected i . v . with either AAV8 - Cre or control AAV8 - GFP : Western blot analysis of hepatic and control adipose Sel1L protein ( n = 3 per group ) ; Hsp90 loading control for Western blot analysis .

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

Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: Western blot analysis of hepatic and control adipose Sel1L protein (n=3 per group); Hsp90 loading control for Western blot analysis.


Acute loss - of - function model where 8 - week - old Sel1Lf / f mice were injected i . v . with either AAV8 - Cre or control AAV8 - GFP : qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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

Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers ( n = 3 per group ) .

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

Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers (n=3 per group).


Scatter plot depicting the logarithmic fold - change ( FC ) for 16 , 402 genes in Sel1LAlb and Fgf21 transgenic ( Tg ) livers ( n = 3 per group ) ; genes that are highly upregulated or downregulated in both datasets are marked in red and blue , respectively ; genes that are upregulated unique to each data set ( e . g . Derl3 for Sel1L - Hrd1 ERAD - deficient liver ) are marked in green .

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

Scatter plot depicting the logarithmic fold-change (FC) for 16,402 genes in Sel1LAlb and Fgf21 transgenic (Tg) livers (n=3 per group); genes that are highly upregulated or downregulated in both datasets are marked in red and blue, respectively; genes that are upregulated unique to each data set (e.g. Derl3 for Sel1L-Hrd1 ERAD-deficient liver) are marked in green.


Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : qPCR analysis of Fgf21 mRNA 3 weeks after injection ( n = 4 per group ) . Ribosomal L32 , loading control for qPCR analysis .

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

Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: qPCR analysis of Fgf21 mRNA 3 weeks after injection (n=4 per group). Ribosomal L32, loading control for qPCR analysis.


Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : ELISA analysis of Fgf21 in serum ( K ) 3 weeks after injection ( n = 4 per group ) .

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

Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: ELISA analysis of Fgf21 in serum (K) 3 weeks after injection (n=4 per group).


Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : Weight gain curve after injection ( n = 7 per group ) .

Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: Weight gain curve after injection (n=7 per group).


Western blot analysis of p - Stat5 in livers of 9 - week - old mice ( n = 3 per group , 3 independent repeats ) , with quantitation of the ratio of p - to total Stat5 shown below the blot . Hsp90 , loading control for Western blot analysis .

Western blot analysis of p-Stat5 in livers of 9-week-old mice (n=3 per group, 3 independent repeats), with quantitation of the ratio of p- to total Stat5 shown below the blot. Hsp90, loading control for Western blot analysis.


qPCR analysis of p - Stat5 - associated growth genes in the livers of 9 - week - old mice ( n = 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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

qPCR analysis of p-Stat5-associated growth genes in the livers of 9-week-old mice (n=6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


Z ambulatory activity of 9 - week - old male mice as measured over 24 hr ( n = 4 males per group ) .

Z ambulatory activity of 9-week-old male mice as measured over 24 hr (n=4 males per group).


Blood glucose levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

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

Blood glucose levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


serum insulin levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

serum insulin levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


serum triglyceride ( TG ) levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

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

serum triglyceride (TG) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


serum cholesterol ( CHOL ) levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

serum cholesterol (CHOL) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


H & E images of inguinal white adipose tissue ( iWAT ) from 8 - week - old mice ( n = 3 per group , 2 independent repeats ) .

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

H&E images of inguinal white adipose tissue (iWAT) from 8-week-old mice (n=3 per group, 2 independent repeats).


qPCR analysis of browning - related genes in iWAT ( n = 3 - 6 per group ) . Ribosomal L32 , loading control for qPCR analysis .

qPCR analysis of browning-related genes in iWAT (n=3-6 per group). Ribosomal L32, loading control for qPCR analysis.


Western blot analysis of Ucp1 in iWAT of 8 - week - old mice ( n = 3 - 4 per group , 3 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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

Western blot analysis of Ucp1 in iWAT of 8-week-old mice (n=3-4 per group, 3 independent repeats). Hsp90, loading control for Western blot analysis.


Weight gain curve of male mice after 60 % high fat diet ( HFD ) starting at 5 weeks of age ( n = 4 per group , 2 independent repeats ) .

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

Weight gain curve of male mice after 60% high fat diet (HFD) starting at 5 weeks of age (n=4 per group, 2 independent repeats).


Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding ( n = 4 per group ) .

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

Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).


Western blot analysis of Crebh , Crebh - N , Pparα and Xbp1s in whole cell lysates ( A ) and nuclear extracts ( B ) in WT and Sel1LAlb livers ( n = 3 per group , 3 independent repeats ) with quantitation shown in ( C ) . Hsp90 lamin , loading controls for Western blot analysis .

Western blot analysis of Crebh, Crebh-N, Pparα and Xbp1s in whole cell lysates (A) and nuclear extracts (B) in WT and Sel1LAlb livers (n=3 per group, 3 independent repeats) with quantitation shown in (C). Hsp90 lamin, loading controls for Western blot analysis.


qPCR analysis of Crebh , Ppara and Xbp1s in WT and Sel1LAlb livers ( n = 4 - 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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

qPCR analysis of Crebh, Ppara and Xbp1s in WT and Sel1LAlb livers (n=4-6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F - G ( n = 3 per group ) . Hsp90 , loading controls for Western blot analysis .

Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F-G (n=3 per group). Hsp90 , loading controls for Western blot analysis.


Western blot analysis of Crebh protein in 9 - week - old mice ( n = 3 per group , 2 independent repeats ) . WT mice injected i . p . with tunicamycin ( Tm , 1 . 5 µg / g body weight ) for 72 hours were included as a control . Hsp90 loading controls for Western blot analysis .

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

Western blot analysis of Crebh protein in 9-week-old mice (n=3 per group, 2 independent repeats). WT mice injected i.p. with tunicamycin (Tm, 1.5 µg/g body weight) for 72 hours were included as a control. Hsp90 loading controls for Western blot analysis.


Western blot analysis of Crebh in the livers of 9 - week - old mice after NP40 - detergent fractionation into NP40 soluble ( NP40S ) and pellet ( NP40P ) ( n = 3 per group , 2 independent repeats ) . Hsp90 H2A loading controls for Western blot analysis .

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

Western blot analysis of Crebh in the livers of 9-week-old mice after NP40-detergent fractionation into NP40 soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats). Hsp90 H2A loading controls for Western blot analysis.


Representative confocal images of Crebh in the liver cryosections of 8 - week - old mice ( zoomed out versions in Figure EV3F ) . Note that a fraction of hepatocytes is binucleated . Yellow arrows represent Crebh staining inside in the hepatocyte nucleus .

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

Representative confocal images of Crebh in the liver cryosections of 8-week-old mice (zoomed out versions in Figure EV3F). Note that a fraction of hepatocytes is binucleated. Yellow arrows represent Crebh staining inside in the hepatocyte nucleus.


ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9 - week - old mice , normalized first to 5 % input group and then to no - antibody ChIP samples ( n = 3 pooled per group , 2 independent repeats ) .

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

ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9-week-old mice, normalized first to 5% input group and then to no-antibody ChIP samples (n=3 pooled per group, 2 independent repeats).


Western blot analysis of SEL1L , HRD1 and CREBH proteins in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . , β - actin loading controls for Western blot analysis .

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

Western blot analysis of SEL1L, HRD1 and CREBH proteins in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. , β-actin loading controls for Western blot analysis.


qPCR analysis of SEL1L , HRD1 and FGF21 ( K ) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . Ribosomal L32 , loading control for qPCR analysis .

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qPCR analysis of SEL1L, HRD1 and FGF21 (K) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. Ribosomal L32, loading control for qPCR analysis.


Western blot analysis of Crebh protein half - life in transfected WT and HRD1 - / - HEK293T cells treated with cycloheximide ( CHX ) for indicated times . The decay of protein from one experiment is shown below . Hsp90 , loading control for Western blot analysis .

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Western blot analysis of Crebh protein half-life in transfected WT and HRD1-/- HEK293T cells treated with cycloheximide (CHX) for indicated times. The decay of protein from one experiment is shown below. Hsp90, loading control for Western blot analysis.


Western blot analysis and quantitation of Crebh in Crebh - transfected WT ( B ) and HRD1 - / - ( C ) HEK293T cells pre - treated with the proteasomal inhibitor bortezomib ( BTZ ) or lysosomal inhibitor chloroquine ( CHQ ) for 2 hours and then with CHX for additional 1 hour ( n = 2 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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

Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


Western blot analysis of Crebh ubiquitination following immunoprecipitation ( IP ) of Crebh - Flag and Crebh - N - Flag in HEK293T cells transfected with indicated plasmids . Samples were boiled with SDS before IP for denaturing IP and not so for native IP . These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation . Hsp90 , loading control for Western blot analysis .

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Western blot analysis of Crebh ubiquitination following immunoprecipitation (IP) of Crebh-Flag and Crebh-N-Flag in HEK293T cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation. Hsp90, loading control for Western blot analysis.


Western blot analysis of endoglycosidase H ( endoH ) - sensitivity of Crebh in the livers of 9 - week - old mice ; ' r ' refers to endoH - cleavage - resistant species and ' s ' refers to endoH - cleavage - sensitive species ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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Western blot analysis of endoglycosidase H (endoH)-sensitivity of Crebh in the livers of 9-week-old mice; 'r' refers to endoH-cleavage-resistant species and 's' refers to endoH-cleavage-sensitive species (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection ( n = 3 mice each , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection (n=3 mice each, 2 independent repeats). Hsp90, loading control for Western blot analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR analysis of Crebh and Fgf21 mRNA 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of Crebh and Fgf21 mRNA 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . ELISA analysis of circulating Fgf21 ( C ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) .

5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. ELISA analysis of circulating Fgf21 (C) 5 weeks post injection (n=3 per group, 2 independent repeats).


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR analysis of hepatic growth - associated genes 5 weeks after injection ( n = 6 per group ) . Ribosomal L32 , loading control for qPCR analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of hepatic growth-associated genes 5 weeks after injection (n=6 per group). Ribosomal L32, loading control for qPCR analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Weight gain 6 weeks post injection ( n = 10 per group ) .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Weight gain 6 weeks post injection (n=10 per group).


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Insulin tolerance test ( ITT ) 5 - weeks after injection ( n = 10 per group ) .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot analysis ( H ) of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis (H) of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from 10 - week - old Sel1Lf / f and Sel1LAlb mice under overnight fasted or fed states .

Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA in the livers of 10 - week - old Sel1Lf / f mice .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice.


during fasting - feeding Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers of 10 - week - old Sel1Lf / f and Sel1LAlb mice .

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during fasting-feeding Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice.


during fasting - feeding Serum Fgf21 levels in 10 - week - old Sel1Lf / f and Sel1LAlb mice .

during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Serum Fgf21 levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


( A ) Time series of TIRF microscopyimages showing GFP - dynein ( green in merge ) localising to the plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . Protein concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 and 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ratio ) .

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(A) Time series of TIRF microscopyimages showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( B ) individual and dual colour kymographs showing GFP - dynein ( green in merge ) localising to the plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . Protein concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 and 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ratio ) .

(B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( C ) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP - dynein ( green in merge ) and Alexa647 - EB3 ( red in merge ) on dynamic Alexa568 - microtubules ( blue in merge ) . Concentrations were 10 nM GFP - dynein , 10 nM human dynactin , 10 nM Alexa647 - EB3 , and 17 . 5 µM tubulin .

(C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin.


( D , E ) Dual and single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in the absence of either dynactin ( D ) . Concentrations of the proteins present as in A .

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(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) . Concentrations of the proteins present as in A.


( D , E ) Dual and single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in the absence of EB proteins ( E ) . Concentrations of the proteins present as in A .

(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of EB proteins (E). Concentrations of the proteins present as in A.


( F ) Averaged fluorescence intensity profiles of GFP - dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 ( blue squares , as in A ) , in the absence of dynactin ( red circles , as in D ) , or in the absence of an EB protein ( black triangles , as in E ) . Mean values from at least two separate experiments per condition ( with a total of at least 30 kymographs ) are shown ; error bars are s . e .

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(F) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 (blue squares, as in A), in the absence of dynactin (red circles, as in D), or in the absence of an EB protein (black triangles, as in E). Mean values from at least two separate experiments per condition (with a total of at least 30 kymographs) are shown; error bars are s. e.


( G ) Kymographs of GFP - dyneinmicrotubule end tracking in the absence of ATP ( in contrast to all other experiments that contain ATP ) . Other conditions as in A . For merged kymographs , microtubule plus and minus ends are labelled by ( + ) and ( − ) . Experiments were performed at 30 ° C .

(G) Kymographs of GFP-dyneinmicrotubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP - dynein showing end tracking , processive motility and diffusive and static binding in the presence of all DDB components and EB1 . Protein concentrations are 10 nM GFP - dynein ( green in merge ) , 20 nM human dynactin , 20 nM EB1 , 200 nM Alexa647 - BicD2 - N ( magenta in merge ) , and 17 . 5 µM Alexa568 - tubulin ( blue in merge ) . Alexa647 - BicD2 - N often co - localises with processively moving and occasionally with statically bound GFP - dynein , but not with plus end - tracking GFP - dynein .

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(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein.


( B ) Averaged fluorescence intensity profiles of GFP - dynein ( green squares ) and Alexa647 - BicD2 - N ( magenta circles ) at growing microtubule plus ends . Mean values from three separate experiments per condition ( with a total of at least 75 kymographs ) are shown ; error bars are s . e .

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(B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e.


( C , D ) Dual and single colour kymographs of an Atto565 - microtubule ( magenta ) in the presence of GFP - dynein ( green ) , human dynactin and EB1 , and either ( C ) in the absence or ( D ) presence of 5 µM BicD2 - N ; other conditions as in A . The increased BicD2 - N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP - dynein .

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(C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein.


( E ) Averaged fluorescence intensity profiles of GFP - dynein at growing microtubule plus ends for the condition without BicD2 - N ( red circles , as in C ) or with 5 µM BicD2 - N ( black squares , as in D ) . Mean values from three separate experiments per condition ( with a total of at least 106 kymographs ) are shown ; error bars are s . e . Microtubule plus and minus ends in merged kymographs are labelled by ( + ) and ( − ) . Experiments were performed at 30 ° C .

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(E) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the condition without BicD2-N (red circles, as in C) or with 5 µM BicD2-N (black squares, as in D). Mean values from three separate experiments per condition (with a total of at least 106 kymographs) are shown; error bars are s. e. Microtubule plus and minus ends in merged kymographs are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured . The protein concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 5 µM BicD2 - N , 20 nM EB1 and 17 . 5 µM Atto647 - tubulin .

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(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin.