A - D Relative mRNA levels of ( A ) Pparß / δ , ( B ) Foxo1 , ( C ) citrate synthase , and ( D ) Pgc - 1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . P - values versus WT : Pparβ / δ * * P = 0 . 0016 at 65 days ; Foxo1 * * * P = 0 . 0001 at 105 days , citrate synthase * * * P < 0 . 0001 at 105 days , Pgc - 1α * P = 0 . 0104 at 105 days ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

A-D Relative mRNA levels of (A) Pparß/δ, (B) Foxo1, (C) citrate synthase, and (D) Pgc-1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Pparβ/δ **P = 0.0016 at 65 days; Foxo1 ***P = 0.0001 at 105 days, citrate synthase ***P < 0.0001 at 105 days, Pgc-1α *P = 0.0104 at 105 days (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


E Representative Western blot of PGC - 1α and respective actin that was used for normalization . PGC - 1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age - matched WT . * * P - values versus WT : 0 . 02 ( n = 6 / genotype at 65 days ; n = 5 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

E Representative Western blot of PGC-1α and respective actin that was used for normalization. PGC-1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age-matched WT. **P-values versus WT: 0.02 (n = 6/genotype at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


F Relative mRNA levels of ERRα , Mfn2 , and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . P - values versus WT : ERRα * * * P < 0 . 0001 , Mfn2 * * P = 0 . 002 and Nrf1 * * P = 0 . 009 ( n = 6 - 7 and 7 - 8 for WT and SOD1G86R , respectively , at 65 days ; n = 5 and 6 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

F Relative mRNA levels of ERRα, Mfn2, and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: ERRα ***P < 0.0001, Mfn2 **P = 0.002 and Nrf1 **P = 0.009 (n = 6-7 and 7-8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Mitochondrial DNA ( mtDNA ) was quantified using qPCR . Relative mtDNA levels are expressed as the ratio between mitochondrial - encoded gene Cox1 and the nuclear - encoded gene cyclophilin A . Graphs represent mean fold change ± SEM from age - matched WT for tibialis anterior ( left panel ) and soleus ( right panel ) of WT and SOD1G86R mice . * * * P < 0 . 0001 ( n = 5 and 4 for WT and SOD1G86R , respectively , at 65 days ; n = 6 / genotype at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Mitochondrial DNA (mtDNA) was quantified using qPCR. Relative mtDNA levels are expressed as the ratio between mitochondrial-encoded gene Cox1 and the nuclear-encoded gene cyclophilin A. Graphs represent mean fold change ± SEM from age-matched WT for tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. ***P < 0.0001 (n = 5 and 4 for WT and SOD1G86R, respectively, at 65 days; n = 6/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of Gpx1 were measured by qPCR at the indicated ages in tibialis anterior ( left panel ) and soleus ( right panel ) of WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from age - matched WT . * * * P < 0 . 0001 ( n = 7 and 8 for WT and SOD1G86R , respectively , at 65 days ; n = 9 and 8 for WT and SOD1G86R , respectively , at 105 days , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Relative mRNA levels of Gpx1 were measured by qPCR at the indicated ages in tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. ***P < 0.0001 (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 9 and 8 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).


A - I Relative mRNA levels of ( A ) Pdk4 , ( B ) Pparβ / δ , ( C ) Foxo1 , ( D ) Pfk1 , ( E ) Acsf2 , ( F ) citrate synthase , ( G ) PGC - 1α , ( H ) Mfn2 , and ( I ) Gpx1 were evaluated by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . P - values versus WT : Pdk4 * * * P = 0 . 002 and # # P = 0 . 0039 , Pparβ / δ # P = 0 . 0178 , Foxo1 * * P = 0 . 0033 and # # P = 0 . 0038 , Pfk1 * * * P = 0 . 0002 and # # P = 0 . 0080 , Acsf2 * P = 0 . 0437 , citrate synthase # # # P = 0 . 0004 , Pgc - 1α * * P = 0 . 0084 and # # # P = 0 . 0009 , Mfn2 * P = 0 . 0272 , $ P = 0 . 0145 and # # # P = 0 . 0002 and Gpx1 * * * P < 0 . 0001 and # # # P < 0 . 0001 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

A-I Relative mRNA levels of (A) Pdk4, (B) Pparβ/δ, (C) Foxo1, (D) Pfk1, (E) Acsf2, (F) citrate synthase, (G) PGC-1α, (H) Mfn2, and (I) Gpx1 were evaluated by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. P-values versus WT: Pdk4 ***P = 0.002 and ##P = 0.0039, Pparβ/δ #P = 0.0178, Foxo1 **P = 0.0033 and ##P = 0.0038, Pfk1 ***P = 0.0002 and ##P = 0.0080, Acsf2 *P = 0.0437, citrate synthase ###P = 0.0004, Pgc-1α **P = 0.0084 and ###P = 0.0009, Mfn2 *P = 0.0272, $P = 0.0145 and ###P = 0.0002 and Gpx1 ***P < 0.0001 and ###P < 0.0001 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Grip strength is represented as mean of percent from T0 for each experimental group ± SEM . # # P = 0 . 0045 and * * * P = 0 . 0003 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Grip strength is represented as mean of percent from T0 for each experimental group ± SEM. ##P = 0.0045 and ***P = 0.0003 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of muscular atrophy markers Murf1 and Atg - 1 were measured by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . * * P = 0 . 0079 and * * * P = 0 . 0018 ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Relative mRNA levels of muscular atrophy markers Murf1 and Atg-1 were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.0079 and ***P = 0.0018 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


Relative mRNA levels of denervation markers AChRα , AChRγ , and MuSK were measured by qPCR in tibialis anterior of control ( CT ) or DCA - treated ( DCA ) WT and SOD1G86R mice . Graphs represent mean fold change ± SEM from CT WT group . * * P = 0 . 015 for AChRα , # P = 0 . 028 and * * P = 0 . 074 for AChRγ , # P = 0 . 042 and * * P = 0 . 0029 for MuSK ( n = 9 / genotype in CT groups , n = 9 and 8 for WT and SOD1G86R , respectively , in DCA group , two - way ANOVA followed by Fisher ' s LSD post hoc test ) .

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

Relative mRNA levels of denervation markers AChRα, AChRγ, and MuSK were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.015 for AChRα, #P = 0.028 and **P = 0.074 for AChRγ, #P = 0.042 and **P = 0.0029 for MuSK (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).


( B ) Immunoblot analysis of whole cell samples of the HA - tagged full - length ( FL ) IFT54 , and its deletion mutants as indicated , each expressed in the ift54 null mutant ; wild type ( WT ) and ift54 mutant cells were used as control . Anti - HA was use to visualize the transgenic proteins , antibodies to FLA8 / KIF3B were used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of whole cell samples of the HA-tagged full-length (FL) IFT54, and its deletion mutants as indicated, each expressed in the ift54 null mutant; wild type (WT) and ift54 mutant cells were used as control. Anti-HA was use to visualize the transgenic proteins, antibodies to FLA8/KIF3B were used as a loading control.


( C ) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated . Wild type ( WT ) cells were used as a control .

O O O O O O O O O O O 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) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated. Wild type (WT) cells were used as a control.


( D ) Representative DIC images of wild type ( WT ) , ift54 and ift54 cells transformed with the deletion constructs as indicated . Arrows indicate cilia bulges . Scale bar , 5 μm .

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(D) Representative DIC images of wild type (WT), ift54 and ift54 cells transformed with the deletion constructs as indicated. Arrows indicate cilia bulges. Scale bar, 5 μm.


( A ) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated ; wild - type ( WT ) cells were used as control . Specimens were stained with anti - HA and anti - α - tubulin antibodies followed by imaging using an epifluorescence microscope . Arrows indicate protein accumulation at ciliary tip ( left ) or proximal end of cilia ( right ) . Scale bars , 5 μm .

(A) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated; wild-type (WT) cells were used as control. Specimens were stained with anti-HA and anti-α-tubulin antibodies followed by imaging using an epifluorescence microscope. Arrows indicate protein accumulation at ciliary tip (left) or proximal end of cilia (right). Scale bars, 5 μm.


( B , C ) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated . Cells were fixed and stained with antibodies against IFT43 ( an IFT - A subunit ) ( B ) or against IFT38 ( an IFT - B subunit ) ( C ) followed by imaging using both DIC and epifluorescence microscopy . IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261 - 275 mutant but at proximal end of cilia in IFT54Δ342 - 356 mutant . Arrows indicate ciliary bulges . WT , wild - type cells . Scale bars , 5 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 O O O O O O O O O O O

(B, C) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated. Cells were fixed and stained with antibodies against IFT43 (an IFT-A subunit) (B) or against IFT38 (an IFT-B subunit) (C) followed by imaging using both DIC and epifluorescence microscopy. IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261-275 mutant but at proximal end of cilia in IFT54Δ342-356 mutant. Arrows indicate ciliary bulges. WT, wild-type cells. Scale bars, 5 μm.


( A ) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC . D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261 - 275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type cells . Arrows indicate ciliary bulges . Scale bar , 5 μm . ( B ) Immunostaining of cells with antibody against kinesin - II subunit FLA10 . FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261 - 275 mutant or at proximal end of cilia in IFT54Δ342 - 356 mutant . WT , wild type cells . The insets show enhanced and / or enlarged images . Arrows indicate ciliary bulges . Scale bar , 5 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 O O O O O O O O O O O O O O O O O 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) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC. D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261-275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. Arrows indicate ciliary bulges. Scale bar, 5 μm. (B) Immunostaining of cells with antibody against kinesin-II subunit FLA10. FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261-275 mutant or at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. The insets show enhanced and/or enlarged images. Arrows indicate ciliary bulges. Scale bar, 5 μm.


( C , D ) Immunoblot analysis of whole cells ( C ) and isolated cilia ( D ) of wild type and the three IFT54 deletion mutants . The membranes were probed with antibodies against IFT complex and motor proteins as indicated . Wild - type levels of the various IFT components were detected in the whole cell samples of the deletion strains ( C ) . IFT121 , IFT38 and FLA8 accumulated in cilia of both IFT54Δ261 - 275 and IFT54Δ342 - 356 mutants . D1bLIC was relatively reduced in IFT54Δ261 - 275 mutant cilia but was substantially increased in IFT54Δ342 - 356 mutant cilia ( D ) . Ratios of protein amounts from one representative experiment are in parentheses .

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

(C, D) Immunoblot analysis of whole cells (C) and isolated cilia (D) of wild type and the three IFT54 deletion mutants. The membranes were probed with antibodies against IFT complex and motor proteins as indicated. Wild-type levels of the various IFT components were detected in the whole cell samples of the deletion strains (C). IFT121, IFT38 and FLA8 accumulated in cilia of both IFT54Δ261-275 and IFT54Δ342-356 mutants. D1bLIC was relatively reduced in IFT54Δ261-275 mutant cilia but was substantially increased in IFT54Δ342-356 mutant cilia (D). Ratios of protein amounts from one representative experiment are in parentheses.


( A ) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43 - YFP . An ift43 rescue strain expressing IFT43 - YFP ( as a control ) and the IFT54Δ342 - 356 mutant expressing IFT43 - YFP were analyzed by live imaging via TIRF microscopy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43-YFP. An ift43 rescue strain expressing IFT43-YFP (as a control) and the IFT54Δ342-356 mutant expressing IFT43-YFP were analyzed by live imaging via TIRF microscopy.


( B - C ) Velocities ( B ) and frequencies ( C ) of anterograde and retrograde IFT of IFT43 - YFP in control and the IFT54Δ342 - 356 mutant . n represents the number of cilia assayed from three independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , * * * , p < 0 . 0001 .

(B-C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of IFT43-YFP in control and the IFT54Δ342-356 mutant. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, ***, p<0.0001.


( D ) Deletion of residues 342 - 356 of IFT54 increases its interaction with kinesin - II . Cell extracts from wild type ( WT ) , ift54 expressing HA - tagged full - length ( FL ) IFT54 or IFT54Δ342 - 356 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies . The normalized ratios of FLA8 and FLA10 in the immunoprecipitates ( IFT54 versus the mutant ) are 1 : 3 . 90 and 1 : 3 . 13 , respectively .

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

(D) Deletion of residues 342-356 of IFT54 increases its interaction with kinesin-II. Cell extracts from wild type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ342-356 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 versus the mutant) are 1:3.90 and 1:3.13, respectively.


( E ) ATP treatment induces dissociation of IFT54 but not IFT54Δ342 - 356 mutant from kinesin - II . Immunoprecipitation experiments were performed as shown in ( D ) in the presence or absence of ATP . The normalized ratios of FLA8 and FLA10 in the immunoprecipitates ( IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 37 : 1 . 70 : 1 . 38 and 1 : 0 . 35 : 1 . 47 : 1 . 45 , respectively .

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

(E) ATP treatment induces dissociation of IFT54 but not IFT54Δ342-356 mutant from kinesin-II. Immunoprecipitation experiments were performed as shown in (D) in the presence or absence of ATP. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.37:1.70:1.38 and 1:0.35:1.47:1.45, respectively.


( F ) Analysis of the interaction of IFT54 and IFT54Δ342 - 356 mutant with kinesin - II by GST - pulldown assay . Bacterial expressed GST , GST - tagged IFT54 or IFT54Δ342 - 356 mutant was mixed respectively with recombinant kinesin - II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting . The normalized ratios of KAP , FLA10 and FLA8 in the pull - down samples ( GST - IFT54 without ATP / with ATP / mutant without ATP / with ATP ) are 1 : 0 . 51 : 1 . 31 : 1 . 44 , 1 : 0 . 55 : 1 . 29 : 1 . 19 and 1 : 0 . 50 : 1 . 31 : 1 . 29 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of the interaction of IFT54 and IFT54Δ342-356 mutant with kinesin-II by GST-pulldown assay. Bacterial expressed GST, GST-tagged IFT54 or IFT54Δ342-356 mutant was mixed respectively with recombinant kinesin-II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting. The normalized ratios of KAP, FLA10 and FLA8 in the pull-down samples (GST-IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.51:1.31:1.44, 1:0.55:1.29:1.19 and 1:0.50:1.31:1.29, respectively.


( G ) Interaction of IFT54 with kinesin - II is conserved in mammalian cells . Cell extracts from HEK293T cells expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ535 - 549 ( corresponding deletion mutant of IFT54Δ342 - 356 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP and KIF3A antibodies , respectively . Please note , the deletion mutant had an increased interaction with kinesin - II subunit KIF3A relative to the control . The normalized ratio of KIF3A in the immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 1 . 76 .

(G) Interaction of IFT54 with kinesin-II is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ535-549 (corresponding deletion mutant of IFT54Δ342-356) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and KIF3A antibodies, respectively. Please note, the deletion mutant had an increased interaction with kinesin-II subunit KIF3A relative to the control. The normalized ratio of KIF3A in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:1.76.


( A ) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC - YFP . Wild - type ( WT ) and IFT54Δ261 - 275 mutant cells expressing D1bLIC - YFP were analyzed by live imaging via TIRF microscopy .

O O O O O O O O O O O O O O O O O O O O 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) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC-YFP. Wild-type (WT) and IFT54Δ261-275 mutant cells expressing D1bLIC-YFP were analyzed by live imaging via TIRF microscopy.


( B , C ) Velocities ( B ) and frequencies ( C ) of anterograde and retrograde IFT of D1bLIC - YFP . n represents the number of cilia assayed from four independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , n . s . : no significance ; * * * , p < 0 . 0001 .

(B, C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of D1bLIC-YFP. n represents the number of cilia assayed from four independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( D ) Kymograms of IFT43 - YFP of WT and mutant cilia .

(D) Kymograms of IFT43-YFP of WT and mutant cilia.


( E , F ) Velocities ( E ) and frequencies ( F ) of anterograde and retrograde IFT of IFT43 - YFP . n represents the number of cilia assayed from three independent experiments . Values show the mean ± S . D . Unpaired two - tailed Student ' s t test analysis , n . s . : no significance ; * * * , p < 0 . 0001 .

(E, F) Velocities (E) and frequencies (F) of anterograde and retrograde IFT of IFT43-YFP. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.


( G ) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits . Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine , tryptophan , histidine , and adenine ( - 4 ) or lacking leucine and tryptophan ( - 2 ) . DHC1bT , DHC1b tail domain ; Empty AD or BD vectors were used as control .

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(G) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits. Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or lacking leucine and tryptophan (-2). DHC1bT, DHC1b tail domain; Empty AD or BD vectors were used as control.


( H ) Interaction of IFT54 and IFT54Δ261 - 275 with D1bLIC by GST - pulldown assay . Bacterial expressed GST , GST - IFT54 or GST - IFT54Δ261 - 275 were mixed respectively with His - D1bLIC followed by GST pulldown and immunoblotting with anti - GST and anti - His antibodies . The normalized ratio of His - D1bLIC in the pull - down samples ( GST - IFT54 versus mutant ) is 1 : 0 . 32 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interaction of IFT54 and IFT54Δ261-275 with D1bLIC by GST-pulldown assay. Bacterial expressed GST, GST-IFT54 or GST-IFT54Δ261-275 were mixed respectively with His-D1bLIC followed by GST pulldown and immunoblotting with anti-GST and anti-His antibodies. The normalized ratio of His-D1bLIC in the pull-down samples (GST-IFT54 versus mutant) is 1:0.32.


( I ) Co - immunoprecipitation of IFT54 and IFT54Δ261 - 275 with D1bLIC . Cell extracts from wild - type ( WT ) , ift54 expressing HA - tagged full - length ( FL ) IFT54 or IFT54Δ261 - 275 mutant were subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies . Please note , IFT54Δ261 - 275 mutant exhibited weaker interaction with D1bLIC relative to the control . The normalized ratio of D1bLIC in the immunoprecipitates ( IFT54 - HA versus mutant ) is 1 : 0 . 37 .

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(I) Co-immunoprecipitation of IFT54 and IFT54Δ261-275 with D1bLIC. Cell extracts from wild-type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ261-275 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. Please note, IFT54Δ261-275 mutant exhibited weaker interaction with D1bLIC relative to the control. The normalized ratio of D1bLIC in the immunoprecipitates (IFT54-HA versus mutant) is 1:0.37.


( J ) Interaction of IFT54 with D1bLIC is conserved in mammalian cells . Cell extracts from HEK293T cells expressing eGFP , eGFP tagged TRAF3IP1 or TRAF3IP1Δ460 - 474 ( corresponding deletion mutant of IFT54Δ261 - 275 ) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti - GFP and DYNC2LI1 , human homologue of D1bLIC , antibodies , respectively . Please note , the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control . The normalized ratio of DYNC2LI1 in the immunoprecipitates ( TRAF3IP1 versus mutant ) is 1 : 0 . 49 .

(J) Interaction of IFT54 with D1bLIC is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ460-474 (corresponding deletion mutant of IFT54Δ261-275) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and DYNC2LI1, human homologue of D1bLIC, antibodies, respectively. Please note, the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control. The normalized ratio of DYNC2LI1 in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:0.49.


( A ) EM analysis of KMnO4 - fixed pex3 atg1 and WT cells grown for 16 h on MM - M / G . The inset shows a cluster of vesicles ( enlarged from the boxed region ) .

O O O O O O O O O O O O O O O O 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) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region).


( B ) iEM analysis of pex3 atg1 cells using α - Pex14 antibodies .

(B) iEM analysis of pex3 atg1 cells using α-Pex14 antibodies.


( C and D ) FM images of pex3 atg1 cells producing Pex14 - mCherry and the ER marker BiPN30 - eGFP - HDEL ( C ) , or Pex14 - mGFP complemented with Mitotracker orange staining ( D ) .

O O O O O O O O O O O O O O O O O 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 and D) FM images of pex3 atg1 cells producing Pex14-mCherry and the ER marker BiPN30-eGFP-HDEL (C), or Pex14-mGFP complemented with Mitotracker orange staining (D).


( E ) Electron tomography analysis of a serial - sectioned pex3 atg1 cell ( a ) containing a perinuclear membrane cluster ( b , arrows ) . ( d - f ) 10 - nm - thin digital slices through the tomogram reconstruction ( viewing direction indicated in c ) revealed vesicles ( black arrows ) and reticular structures ( red arrows ) . The surface - rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d . CW , cell wall ; M , mitochondrion ; N , nucleus ; P , peroxisome ; V , vacuole . Bars : ( A ) 500 nm ; ( A , inset ; and B ) 100 nm ; ( C and D ) 2 . 5 µm ; ( E ) 250 nm .

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(E) Electron tomography analysis of a serial-sectioned pex3 atg1 cell (a) containing a perinuclear membrane cluster (b, arrows). (d-f) 10-nm-thin digital slices through the tomogram reconstruction (viewing direction indicated in c) revealed vesicles (black arrows) and reticular structures (red arrows). The surface-rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d. CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bars: (A) 500 nm; (A, inset; and B) 100 nm; (C and D) 2.5 µm; (E) 250 nm.


( A ) iEM analysis of pex3 cells using α - Pex14 antibodies , identifying structures ( arrows ) in the vicinity of mitochondria . CW , cell wall ; M , mitochondrion . ( B ) Quantification of Pex14 - mGFP spots in pex3 and pex3 atg1 cells .

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(A) iEM analysis of pex3 cells using α-Pex14 antibodies, identifying structures (arrows) in the vicinity of mitochondria. CW, cell wall; M, mitochondrion. (B) Quantification of Pex14-mGFP spots in pex3 and pex3 atg1 cells.


( C ) FM images of pex3 atg1 or pex3 cells , producing Pex14 - mGFP complemented with FM4 - 64 vacuolar staining . The inset ( enlarged from the boxed region ) shows optimized intensities for pex3 cells , highlighting the Pex14 - mGFP spot and vacuolar mGFP .

(C) FM images of pex3 atg1 or pex3 cells, producing Pex14-mGFP complemented with FM4-64 vacuolar staining. The inset (enlarged from the boxed region) shows optimized intensities for pex3 cells, highlighting the Pex14-mGFP spot and vacuolar mGFP.


( D ) WB analysis of cells grown for 16 h on MM - M / G using α - Pex11 or α - Pex14 antibodies . Pyruvate carboxylase 1 ( Pyc1 ) was used as a loading control . Error bars indicate SEM . Bars : ( A ) 100 nm ; ( C ) 1 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

(D) WB analysis of cells grown for 16 h on MM-M/G using α-Pex11 or α-Pex14 antibodies. Pyruvate carboxylase 1 (Pyc1) was used as a loading control. Error bars indicate SEM. Bars: (A) 100 nm; (C) 1 µm.


( A ) FM images of pex3 atg1 cells grown for 16 h on MM - M / G . Cells produced Pex14 - mCherry and C - terminal mGFP fusions of the indicated proteins .

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(A) FM images of pex3 atg1 cells grown for 16 h on MM-M/G. Cells produced Pex14-mCherry and C-terminal mGFP fusions of the indicated proteins.


( B ) WB analysis of WT ( 1 ) and pex3 atg1 cells ( 2 ) , grown for 6 or 16 h on MM - M / G .

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(B) WB analysis of WT (1) and pex3 atg1 cells (2), grown for 6 or 16 h on MM-M/G.


( C ) FM images showing mGFP - fluorescence in pex3 atg1 cells producing Pex14 - mCherry ( control ) or Pex14 - mCherry together with the indicated mGFP fusion protein . Cells were grown for 6 h on MM - M / G .

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(C) FM images showing mGFP-fluorescence in pex3 atg1 cells producing Pex14-mCherry (control) or Pex14-mCherry together with the indicated mGFP fusion protein. Cells were grown for 6 h on MM-M/G.


( D and E ) iEM of pex3 atg1 cells using α - Pex5 ( D ) or α - alcohol oxidase antibodies ( E ) .

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(D and E) iEM of pex3 atg1 cells using α-Pex5 (D) or α-alcohol oxidase antibodies (E).


( F ) Cell fractionation of the two indicated strains ( seperated by the horizontal broken line ) . Postnuclear supernatants ( PNS ) were subjected to differential centrifugation resulting in a 30 , 000 g organelle pellet ( P ) and supernatant fraction ( S ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell fractionation of the two indicated strains (seperated by the horizontal broken line). Postnuclear supernatants (PNS) were subjected to differential centrifugation resulting in a 30,000 g organelle pellet (P) and supernatant fraction (S).


( G ) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top ( 1 ) to bottom ( 10 ) fractions .

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

(G) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top (1) to bottom (10) fractions.


( H ) Colocalization of Pex8 - GFP and Pex14 - mCherry in pex10 cells .

(H) Colocalization of Pex8-GFP and Pex14-mCherry in pex10 cells.


( A ) FM images of pex3 cells with Pex14 - mCherry upon Pex3 - eGFP reintroduction after shifting cells from MM - Glu with ammonium sulfate to MM - M / G with methylamine .

O 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 B-CONTROLLED_VAR O

(A) FM images of pex3 cells with Pex14-mCherry upon Pex3-eGFP reintroduction after shifting cells from MM-Glu with ammonium sulfate to MM-M/G with methylamine.


( B - E ) Live cell FM images of pex3 atg1 cells upon Pex3 reintroduction . Shown are pex3 atg1 cells producing Pex14 - mCherry and PAMOPEX3 - eGFP ( B ) , PAMOPEX3 . PEX10 - GFP ( C ) , PAMOPEX3 . PMP47 - GFP ( D ) , or PAMOPEX3 . PTEFGFP - SKL ( E ) . Cells were grown similar to the method in A . The arrows in B - D indicate the first detectable GFP signal . Bars , 2 . 5 µm .

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(B-E) Live cell FM images of pex3 atg1 cells upon Pex3 reintroduction. Shown are pex3 atg1 cells producing Pex14-mCherry and PAMOPEX3-eGFP (B), PAMOPEX3.PEX10-GFP (C), PAMOPEX3.PMP47-GFP (D), or PAMOPEX3.PTEFGFP-SKL (E). Cells were grown similar to the method in A. The arrows in B-D indicate the first detectable GFP signal. Bars, 2.5 µm.


( A and B ) WB analysis ( A ) and protein quantification ( B ) in WT ( lane 1 ; blue ) , pex3 atg1 ( lane 2 ; red ) , and pex3 atg1 - PAOXPEX19 ( lane 3 ; green ) cells grown for 6 h on MM - M / G . The protein levels in WT were set to 100 % .

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(A and B) WB analysis (A) and protein quantification (B) in WT (lane 1; blue), pex3 atg1 (lane 2; red), and pex3 atg1-PAOXPEX19 (lane 3; green) cells grown for 6 h on MM-M/G. The protein levels in WT were set to 100%.


( C and D ) FM images ( C ) and quantification ( D ) of Pmp47 - mGFP and Pex10 - mGFP in pex3 atg1 ( 1 ; red ) and pex3 atg1 - PAOXPex19 ( 2 ; green ) cells grown for 6 h on MM - M / G . Control cells in D did not produce mGFP . Significance indications : n . s . , P < 0 . 10 ; * , 0 . 10 > P > 0 . 05 ; * * , 0 . 05 > P > 0 . 01 ; * * * , P < 0 . 01 . Error bars indicate SD .

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(C and D) FM images (C) and quantification (D) of Pmp47-mGFP and Pex10-mGFP in pex3 atg1 (1; red) and pex3 atg1-PAOXPex19 (2; green) cells grown for 6 h on MM-M/G. Control cells in D did not produce mGFP. Significance indications: n.s., P < 0.10; *, 0.10 > P > 0.05; **, 0.05 > P > 0.01; ***, P < 0.01. Error bars indicate SD.


( E and F ) iEM analysis of pex19 ( E ) and pex19 atg1 ( F ) cells using α - Pex14 antibodies .

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(E and F) iEM analysis of pex19 (E) and pex19 atg1 (F) cells using α-Pex14 antibodies.


( G ) EM analysis of KMnO4 - fixed pex3 atg1 pex25 cells grown for 16 h on MM - M / G - showing membrane vesicles ( arrows ) .

(G) EM analysis of KMnO4-fixed pex3 atg1 pex25 cells grown for 16 h on MM-M/G-showing membrane vesicles (arrows).


( H ) FM image of pex3 atg1 pex25 cells producing Pex14 - mGFP . CW , cell wall ; M , mitochondrion ; N , nucleus ; V , vacuole .

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(H) FM image of pex3 atg1 pex25 cells producing Pex14-mGFP. CW, cell wall; M, mitochondrion; N, nucleus; V, vacuole.


Immunoblot of media and lysate collected from HEK293T cells overexpressing ERdj3WT or ERdj3KDEL . Fresh media was conditioned on cells for 24 h prior to harvest .

Immunoblot of media and lysate collected from HEK293T cells overexpressing ERdj3WT or ERdj3KDEL. Fresh media was conditioned on cells for 24 h prior to harvest.


qPCR analysis of ERdj3 mRNA in HEK293T cells treated with or without thapsigargin ( Tg ; 6 h , 100 nM ) . Error bars represent the mean ± 95 % confidence interval as calculated in DataAssist 2 . 0 ( n = 3 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O 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 ERdj3 mRNA in HEK293T cells treated with or without thapsigargin (Tg; 6 h, 100 nM). Error bars represent the mean ± 95% confidence interval as calculated in DataAssist 2.0 (n = 3).


Representative immunoblot of lysates and media collected from HEK293T - Rex cells stably expressing non - silencing ( NS ) or ERdj3 shRNA . Cells were treated with the indicated concentration of Tg in fresh media for 16 h . Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T - Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C . * indicates a pvalue < 0 . 05 ; ( 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 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 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

Representative immunoblot of lysates and media collected from HEK293T-Rex cells stably expressing non-silencing (NS) or ERdj3 shRNA. Cells were treated with the indicated concentration of Tg in fresh media for 16 h. Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T-Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C. *indicates a pvalue < 0.05; (n = 3).


Representative autoradiogram of [ 35S ] - labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg ( 1 µM ) . The experimental paradigm for labeling is shown above . Quantification of fraction intracellular ( dark grey ) and extracellular ( light grey ) [ 35S ] - labeled ERdj3 following a 4 h chase relative to total [ 35S ] - labeled ERdj3 at 4 h ( as shown in Figure 1E ) . Error bars represent standard error from biological replicates ( n = 3 ) . Quantification of total [ 35S ] - labeled ERdj3 in media collected from cells pretreated with vehicle or Tg , as shown in Figure 1E . The media [ 35S ] - labeled ERdj3 was measured by densitometry and is normalized to the amount of media [ 35S ] - labeled ERdj3 at 4 h from vehicle - treated cells . Error bars represent standard error from biological replicates . * indicates a pvalue < 0 . 05 ; ( 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 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 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

Representative autoradiogram of [35S]-labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg (1 µM). The experimental paradigm for labeling is shown above. Quantification of fraction intracellular (dark grey) and extracellular (light grey) [35S]-labeled ERdj3 following a 4 h chase relative to total [35S]-labeled ERdj3 at 4 h (as shown in Figure 1E). Error bars represent standard error from biological replicates (n = 3). Quantification of total [35S]-labeled ERdj3 in media collected from cells pretreated with vehicle or Tg, as shown in Figure 1E. The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 4 h from vehicle-treated cells. Error bars represent standard error from biological replicates. *indicates a pvalue < 0.05; (n = 3).


Representative immunoblot and quantification of sera collected at the indicated times from DBA / 2J mice fed a high fat ( 60 % fat ) diet after a 18 h fast . A schematic of the fasting / refeeding experimental setup is shown above . The quantification was normalized for each mouse to the amount of ERdj3 in the serum draw immediately prior the fast . * indicates a p - value < 0 . 05 ; ( 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 O

Representative immunoblot and quantification of sera collected at the indicated times from DBA/2J mice fed a high fat (60% fat) diet after a 18 h fast. A schematic of the fasting/refeeding experimental setup is shown above. The quantification was normalized for each mouse to the amount of ERdj3 in the serum draw immediately prior the fast. *indicates a p-value < 0.05; (n = 6).


Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [ by 1 g / mL dox ] and / or ATF6 [ by 10 M TMP ] , as indicated ( Shoulders et al , 2013 ) . Immunoblots of the ATF6 target protein BiP ( reactive with the KDEL antibody ) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.


Representative autoradiogram of [ 35S ] - labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [ by 1 g / mL dox ] and / or ATF6 [ by 10 M TMP ] ( Shoulders et al , 2013 ) . The experimental protocol is shown above . Quantification of relative amounts of [ 35S ] - labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B ( n = 3 ) . The media [ 35S ] - labeled ERdj3 was measured by densitometry and is normalized to the amount of media [ 35S ] - labeled ERdj3 at 8 h from vehicle - treated cells . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 ; * * * indicates p - value < 0 . 001 as compared to the vehicle - treated condition . Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B . The fraction of ERdj3 secreted was calculated by normalizing the recovered [ 35S ] - labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [ 35S ] - labeled ERdj3 signal in the media and lysates at t = 0 h . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 as compared to the vehicle - treated condition . Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B . The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [ 35S ] - labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [ 35S ] - labeled ERdj3 signal in the media and lysates at t = 0 h . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 as compared to the vehicle - treated 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 autoradiogram of [35S]-labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP] (Shoulders et al, 2013). The experimental protocol is shown above. Quantification of relative amounts of [35S]-labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B (n=3). The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 8 h from vehicle-treated cells. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01; *** indicates p-value < 0.001 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B. The fraction of ERdj3 secreted was calculated by normalizing the recovered [35S]-labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B. The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [35S]-labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.


Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native ( N ) or guanidinium hydrochloride ( GdnHCl ) - denatured ( D ) RNAse A covalently coupled to sepharose resin . Resin was incubated with media for 2 h at ambient temperature , followed by washing and elution in Laemmli ' s reducing buffer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNAse A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and elution in Laemmli's reducing buffer.


Plot showing the time - dependent increase in ThioT fluorescence ( ex : 440 nm , em : 485 nm ) of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation in the presence or absence ( black ) of recombinant ERdj3 ( RERdj3 ) . The RERdj3 concentrations and molar ratios of RERdj3 to A1 - 40 are as follows : Violet : 1 . 5 nM , 1 : 6561 ; light blue : 4 . 6 nM , 1 : 2187 ; dark blue : 14 nM , 1 : 729 ; dark green : 41 nM , 1 : 243 ; light green : 120 nM , 1 : 81 ; yellow : 370 nM , 1 : 27 ; orange : 1 . 1 M , 1 : 9 ; red : 3 . 3 M , 1 : 3 , or maroon : 10 M , 1 : 1 . All traces represent the average of three replicates .

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

Plot showing the time-dependent increase in ThioT fluorescence (ex:440 nm, em:485 nm) of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to A1-40 are as follows: Violet: 1.5 nM, 1:6561; light blue: 4.6 nM, 1:2187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 M, 1:9; red: 3.3 M, 1:3, or maroon: 10 M, 1:1. All traces represent the average of three replicates.


Plot showing the time - dependent increase in ThioT fluorescence of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation in the presence or absence of BSA ( 14 µg / mL ) or RERdj3 ( 14 µg / mL ; 370 nM , RERdj3 : A = 1 : 27 ) . All traces represent the average of three replicates .

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

Plot showing the time-dependent increase in ThioT fluorescence of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence of BSA (14 µg/mL) or RERdj3 (14 µg/mL; 370 nM, RERdj3: A=1:27). All traces represent the average of three replicates.


Immunoblot of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation for 90 h in the presence or absence of RERdj3 ( 14 µg / mL ; 370 nM , RERdj3 : A1 - 40 = 1 : 27 ) or BSA ( 14 µg / mL ) . The reaction solution in the well was collected ( Nonadhered ) and separated into soluble and insoluble fractions by centrifugation . These samples , as well as the washed wells of the plate ( Adhered ) , were denatured in 8 M GdnHCl with sonication and analyzed by SDS - PAGE / immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation for 90 h in the presence or absence of RERdj3 (14 µg/mL; 370 nM, RERdj3: A1-40 =1:27) or BSA (14 µg/mL). The reaction solution in the well was collected (Nonadhered) and separated into soluble and insoluble fractions by centrifugation. These samples, as well as the washed wells of the plate (Adhered), were denatured in 8 M GdnHCl with sonication and analyzed by SDS-PAGE/immunoblotting.


Representative images under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T - Rex or conditioned media prepared from HEK293T - Rex cells overexpressing ERdj3WT in the presence or absence of TPrP ( 600 ng / mL ) , as described in Figure 4A . Arrows indicate representative cells containing vacuoles . Insets show 544X magnification of a typical region of the image . Quantification of vacuole formation from images as shown in Figure 4B . Equal area images comprising ~ 1200 cells per well were counted for each sample . Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package ( Schindelin et al , 2012 ) . * * * indicates a p - value < 0 . 001 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T-Rex or conditioned media prepared from HEK293T-Rex cells overexpressing ERdj3WT in the presence or absence of TPrP (600 ng/mL), as described in Figure 4A. Arrows indicate representative cells containing vacuoles. Insets show 544X magnification of a typical region of the image. Quantification of vacuole formation from images as shown in Figure 4B. Equal area images comprising ~1200 cells per well were counted for each sample. Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package (Schindelin et al, 2012). *** indicates a p-value < 0.001 (n = 3).


Immunoblot of M2 anti - FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT , FTTTRA25T , and / or ERdj3WT either as a co - incubation or co - expression experiment as shown in Figure 5A . Beads were washed in RIPA buffer prior to elution . Media inputs ( 1 : 400 ) are shown as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of M2 anti-FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT, FTTTRA25T, and/or ERdj3WT either as a co-incubation or co-expression experiment as shown in Figure 5A. Beads were washed in RIPA buffer prior to elution. Media inputs (1:400) are shown as a control.


Immunoblot of M2 anti - FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated . Media inputs ( 1 : 400 ) are shown as a control .

O O O O O O O O O O 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 of M2 anti-FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated. Media inputs (1:400) are shown as a control.


Immunoblot of M2 anti - FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis , a kinetic stabilizer of TTR tetramers . Media inputs ( 1 : 400 ) are shown as a control .

O 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

Immunoblot of M2 anti-FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis, a kinetic stabilizer of TTR tetramers. Media inputs (1:400) are shown as a control.


Immunoblot of 6E10 anti - APP immunopurifications from the conditioned media of CHO cells or CHO - derived APP751 - expressing ( 7WD10 ) cells overexpressing ERdj3WT as indicated . Cells were replated together as indicated 24 h after transfection at equal stoichiometry , and media conditioned for 36 h . In this case , ERdj3 transfected CHO cells replated with mock - transfected 7WD10 cells serves as the co - incubation condition , while mock - transfected CHO cells replated with ERdj3 - transfected 7WD10 cells comprise the co - expression condition . Media inputs ( 1 : 150 ) are shown as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT as indicated. Cells were replated together as indicated 24 h after transfection at equal stoichiometry, and media conditioned for 36 h. In this case, ERdj3 transfected CHO cells replated with mock-transfected 7WD10 cells serves as the co-incubation condition, while mock-transfected CHO cells replated with ERdj3-transfected 7WD10 cells comprise the co-expression condition. Media inputs (1:150) are shown as a control.


Representative immunoblot of M2 anti - FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co - overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q . Media contained vehicle or trimethoprim ( TMP , 10 M , 16 h ; activates ATF6 ) , as indicated . Media inputs ( 1 : 400 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP - dependent activation of ATF6 . Bar graph depicting quantification of the change in the amount of ERdj3 co - immunoprecipitating with TTRA25T following activation of ATF6 , as in Figure 6A . The ERdj3 / TTR ratio is normalized to the ratio in the absence of ATF6 activation . * indicates a p - value < 0 . 05 ; 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co-overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q. Media contained vehicle or trimethoprim (TMP, 10 M, 16 h; activates ATF6), as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP-dependent activation of ATF6. Bar graph depicting quantification of the change in the amount of ERdj3 co-immunoprecipitating with TTRA25T following activation of ATF6, as in Figure 6A. The ERdj3/TTR ratio is normalized to the ratio in the absence of ATF6 activation. *indicates a p-value < 0.05; n = 3.


Representative immunoblot of M2 anti - FLAG immunopurification of media conditioned for 24 h on HEK293T cells co - overexpressing FTTTRA25T , ERdj3WT , ERdj3H53Q , and / or BiP , as indicated . Media inputs ( 1 : 400 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression .

Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293T cells co-overexpressing FTTTRA25T, ERdj3WT, ERdj3H53Q, and/or BiP, as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.


Quantification of immunoblots of M2 anti - FLAG immunopurification of media collected from cells co - overexpressing either ERdj3WT or ERdj3H53Q , FTTTRA25T , and BiP , as in Figure 6C . The ERdj3 / TTR ratio is normalized to the ratio in the absence of BiP overexpression . * * indicates a p - value < 0 . 01 ; n = 5 .

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

Quantification of immunoblots of M2 anti-FLAG immunopurification of media collected from cells co-overexpressing either ERdj3WT or ERdj3H53Q, FTTTRA25T, and BiP, as in Figure 6C. The ERdj3/TTR ratio is normalized to the ratio in the absence of BiP overexpression. **indicates a p-value < 0.01; n = 5.


Immunoblot of 6E10 anti - APP immunopurifications from the conditioned media of CHO cells or CHO - derived APP751 - expressing ( 7WD10 ) cells overexpressing ERdj3WT , ERdj3H53Q , and BiP as indicated . Media inputs ( 1 : 100 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression .

Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT, ERdj3H53Q, and BiP as indicated. Media inputs (1:100) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.


Number of genes detected on protein and mRNA level in each tissue . The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues . The full classification is provided in the text .

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

Number of genes detected on protein and mRNA level in each tissue. The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues. The full classification is provided in the text.


Abundance distribution of all transcripts detected in all tissues ( grey ) ; the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange .

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

Abundance distribution of all transcripts detected in all tissues (grey); the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange.


Distribution of global transcript and protein abundance in all tissues . It is apparent that the dynamic range of protein expression ( iBAQ scale ) exceeds that of mRNA expression ( FPKM scale ; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers ) .

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

Distribution of global transcript and protein abundance in all tissues. It is apparent that the dynamic range of protein expression (iBAQ scale) exceeds that of mRNA expression (FPKM scale; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers).


Protein to mRNA abundance plot for brain tissue . The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs .

O O O O O O O O O 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 to mRNA abundance plot for brain tissue. The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs.


Ranked abundance plot of proteins and transcripts in human heart . While the 10 most abundant transcripts cover almost 70 % of all transcripts in this tissue , the corresponding proteins only represent about 20 % of the total protein .

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

Ranked abundance plot of proteins and transcripts in human heart. While the 10 most abundant transcripts cover almost 70% of all transcripts in this tissue, the corresponding proteins only represent about 20% of the total protein.


Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins . Regardless of the tissue , the fraction of shared genes rarely exceeds 20 % .

Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins. Regardless of the tissue, the fraction of shared genes rarely exceeds 20%.


Correlation analysis of protein to RNA abundance ( in log10 scale ) across tissues , resulting in almost 90 % positive correlations . The protein highlighted in the next panel are marked .

Correlation analysis of protein to RNA abundance (in log10 scale) across tissues, resulting in almost 90% positive correlations. The protein highlighted in the next panel are marked.


Examples for proteins that show high ( SYK , left panel ) or no ( EIF4A3 , right panel ) correlation of protein / RNA ratios across tissues . While the former indicates that different tissues express different quantities of SYK , EIF4A3 expression appears to be similar in all tissues .

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

Examples for proteins that show high (SYK, left panel) or no (EIF4A3, right panel) correlation of protein/RNA ratios across tissues. While the former indicates that different tissues express different quantities of SYK, EIF4A3 expression appears to be similar in all tissues.


Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues . It is apparent that proteomes correlate stronger across tissues than transcriptomes .

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

Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues. It is apparent that proteomes correlate stronger across tissues than transcriptomes.


Average cellular compositions of tissues highlighted in panel ( B ) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types .

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

Average cellular compositions of tissues highlighted in panel (B) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types.


Fluorescent microscopy of cultured Gfp - Lc3 GMPs ( a ) and HSCs ( b )

Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b)


Electron microscopy images of WT HSCs . Arrowheads indicate autophagic vesicles .

Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles.


d , GFP - LC3 loss in cultured Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percentage of GFP - LC3 MFI in - BafA compared to + BafA conditions .

O O O O O O 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 B-CONTROLLED_VAR O O

d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions.


GFP - LC3 levels in HSCs of fed and starved Gfp - Lc3 mice ( n = 3 ) . Results are expressed as percent of GFP - LC3 MFI in fed mice . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Apoptosis levels in cultured WT HSCs ( n = 3 - 5 ) . Results are expressed as percent caspase activation compared to + cytokines conditions .

Apoptosis levels in cultured WT HSCs (n = 3-5). Results are expressed as percent caspase activation compared to +cytokines conditions.


Apoptosis levels in cultured control ( Cnt ) and Atg12cKO HSCs and GMPs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines Cnt HSCs ( 100 % ) .

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

Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%).


Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice ( n = 4 - 8 mice per group ) . Results are expressed as percent of HSCs in fed mice . Data are means ± s . d . Hatching indicates starved conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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

Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4-8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.


Status of the autophagy machinery in WT HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( set to 0 ) .

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

Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0).


b , c , qRT - PCR analyses of pro - autophagic genes in WT ( b ) and FoxO3a − / − ( c ) HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( b ) or FoxO3a + / + HSCs ( c ) ( set to 0 ) .

b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0).


Autophagy flux in cultured FoxO3a − / − : : Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percent GFP - LC3 MFI in - BafA vs . + BafA conditions , and normalized to FoxO3a + / + − : : Gfp - Lc3 HSCs . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs . Arrowheads indicate autophagic vesicles .

Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs. Arrowheads indicate autophagic vesicles.


a , qRT - PCR analyses of pro - autophagic genes in old HSCs ( n = 3 - 5 ) . Results are expressed as log2 fold expression compared to young HSCs ( set to 0 ) .

O O O O O O O O O O O O O O O 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, qRT-PCR analyses of pro-autophagic genes in old HSCs (n = 3-5). Results are expressed as log2 fold expression compared to young HSCs (set to 0).


Apoptosis levels in cultured young and old HSCs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines young HSCs . Hatching indicates - cytokines conditions .

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Apoptosis levels in cultured young and old HSCs (n = 3). Results are expressed as percent caspase activation compared to +cytokines young HSCs. Hatching indicates -cytokines conditions.


2 - NBD glucose uptake in cultured young and old HSCs ( n = 3 ) .

2-NBD glucose uptake in cultured young and old HSCs (n = 3).


Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate ( M - Pyr ) ( n = 3 ) . Colonies were counted at day 10 and normalized to - BafA conditions . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


( D ) Representative immunoblots ( n = 3 ) showing phosphorylation state of Smad2 and JNK during TGF - β - induced EMT in NMuMG cells . Lamin B serves as loading control .

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(D) Representative immunoblots (n=3) showing phosphorylation state of Smad2 and JNK during TGF-β-induced EMT in NMuMG cells. Lamin B serves as loading control.


( E ) Quantification of one set of representative immunoblots ( in figure D ) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF - β - induced EMT in NMuMG .

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(E) Quantification of one set of representative immunoblots (in figure D) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF-β-induced EMT in NMuMG.


( F ) Representative immunohistochemistry image for pSmad2 and pJNK in non - invasive ( n = 34 ) and invasive ( n = 14 ) breast cancer samples . Scale bars for 10X and 40X images are 200 µm and 50 µm respectively .

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(F) Representative immunohistochemistry image for pSmad2 and pJNK in non-invasive (n=34) and invasive (n=14) breast cancer samples. Scale bars for 10X and 40X images are 200 µm and 50 µm respectively.


( H ) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells ( d0 ) and cells treated with TGF - β for seven days in the presence of JNK inhibitor ( d7 JNKi ) or control ( d7 DMSO ) . Staining was performed with antibodies against Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . It is important to note that the untreated epithelial cells are highly proliferative and appear small in size , round in shape and adhere to surrounding cells whereas day 7 TGF - β - treated ( mesenchymal ) cells are much bigger in size , elongated in shape and detached from the nearby cells . Scale bar , 100 μm , 63X magnification .

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(H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification.


( I ) mRNA levels for classical EMT markers in cells treated as in ( F ) were measured by qRT - PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

(I) mRNA levels for classical EMT markers in cells treated as in (F) were measured by qRT-PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


( J ) Transwell migration assay for NMuMG cells treated as in G . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


. ( E ) ChIP assay using H3K27me3 - specific antibody in epithelial cells ( day 0 ) ) or NMuMG cells treated for seven days with TGF - β ( mesenchymal ) and DMSO or JNKi . qRT - PCRs were performed for indicated gene promoters and enrichments are plotted on the right y - axis as ratio of precipitated DNA ( bound ) to total input DNA . On the left y - axis RNA levels of analyzed genes derived from RNA - seq data ( fold change to untreated normalized tag counts ) are plotted . Mean and SEM is plotted from two independent biological replicates .

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. (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates.


( F ) FAIRE assay was performed in cells treated as in ( D ) and qRT - PCRs were performed for the same gene promoters and plotted as in ( E ) .

(F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).


JNK - induced transcription factors are critically required for EMT . ( A ) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors . Right panel shows RNA levels ( normalized tag counts ) for these factors .

JNK-induced transcription factors are critically required for EMT. (A) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors. Right panel shows RNA levels (normalized tag counts) for these factors.


( B ) Same overlap as in ( A ) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT . Right panel displays RNA levels as in ( A ) for 10 transcription factors identified in this overlap .

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(B) Same overlap as in (A) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT. Right panel displays RNA levels as in (A) for 10 transcription factors identified in this overlap.


( C ) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days ( d4 ) of siRNA - mediated depletion of factors compared to non - targeting control ( NTC ) . Staining was performed with antibodies against epithelial markers E - cadherin and ZO1 , mesenchymal markers Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . Scale bar , 100 μm , 40X magnification . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(C) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days (d4) of siRNA-mediated depletion of factors compared to non-targeting control (NTC). Staining was performed with antibodies against epithelial markers E-cadherin and ZO1, mesenchymal markers Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. Scale bar, 100 μm, 40X magnification. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Depletion of Prkrir , Pcbp4 and Tsc22d1 rescues TGF - β - induced reduction in the rate of cell division . ( A ) Lineage trees tracked by live imaging under control , Non treated control ( NTC ) and siRNA - mediated depletion of factors ( X = cell death / cells could not be tracked till the end ) .

Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end).


( B ) Average number of rounds of division per clone in NTC compared with control and siRNA - mediated depletion of factors ( ANOVA , Tukeys post - test , * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).


PRKRIR , PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate . ( A ) Scratch assay in MDA - MB - 231 cells treated for four days with DMSO or JNKi . Scale bar , 200 μm ; 20 X magnification . Mean and SEM is plotted from three independent biological replicates .

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PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.


( B ) mRNA levels for key EMT marker genes in MDA - MB - 231 treated with DMSO or JNKi for 4 days were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological

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(B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates


( C ) mRNA levels for transcription factors in isogenic epithelial ( MDA - MB - 361 ) and mesenchymal ( MDA - MB - 231 ) breast cancer cells were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(C) mRNA levels for transcription factors in isogenic epithelial (MDA-MB-361) and mesenchymal (MDA-MB-231) breast cancer cells were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


( D ) Scratch assay in MDA - MB - 231 cells transfected with NTC or siRNA against PRKRIR , NFIL3 , PCBP4 and TSC22D1 for 4 days as in A . Mean and SEM is plotted from three independent biological replicates as bar plot on right .

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(D) Scratch assay in MDA-MB-231 cells transfected with NTC or siRNA against PRKRIR, NFIL3, PCBP4 and TSC22D1 for 4 days as in A. Mean and SEM is plotted from three independent biological replicates as bar plot on right .


( E ) Invasion assay in MDA - MB - 231 cells treated as in ( D ) . Mean and SEM is plotted from three independent biological replicates .

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(E) Invasion assay in MDA-MB-231 cells treated as in (D). Mean and SEM is plotted from three independent biological replicates.


( F ) mRNA levels of key EMT marker genes in MDA - MB - 231 and MDA - MB - 231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor ( SP600125 ) for 24 hours were measured by qRT - PCRs relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( C ) mRNA levels for representative genes highlighted in ( A ) for cells treated like in ( Fig 5D ) were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


F ) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag - HA tagged Tsc22d1 and detection using an anti - Flag antibody , Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus . Y - axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions . SEM is derived from independent biological replicates .

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F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates.


( G ) ChIP assay using anti - Flag antibody following expression of Flag - HA tagged TSC22D1 in MDA - MB - 231 cells . Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y - axis as ratio of precipitated DNA ( bound ) to total input DNA and then further divided by the same obtained in the empty vector transfected cells . SEM is derived from independent biological replicates .

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(G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates.


( H ) mRNA levels for PRKRIR , PCBP4 , TSC22D1 in non - invasive ( DCIS ) ( n = 20 ) and invasive tumor ( n = 20 ) samples were measured by qRT PCRs relative to TBP and plotted on the y - axis . ZEB1 was used as an established positive control for invasive tumors . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration . ( A ) RT - qPCR analysis for key EMT markers in embryonic stem cells ( ES ) , neuronal progenitors ( NP ) and terminally differentiated neurons ( TN ) . mRNA levels were measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( B ) Western blot analyzing total JNK and phospho - JNK ( p - JNK ) during neuronal differentiation of stem cells . β - actin serve as a loading control .

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(B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control.


( C ) RNA levels of Pcbp4 , Prkrir and Tsc22d1 during neuronal differentiation as shown in A , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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(C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( D ) RNA levels of Pcbp4 , Prkrir , Tsc22d1 , represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 ( JNKi ) for 6 hours .

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(D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours.


( E ) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in ( A ) , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

(E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( F ) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA - seq data from Ventricular Zone ( VZ ) , Sub - Ventricular Zone ( SVZ ) and Cortical plate ( CP ) of E14 . 5 mouse Cortex .

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(F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex.


( G ) RNA levels of genes shown in Figure ( E ) , represented by average normalized tag counts derived from RNA - seq data of TGF - β - induced EMT in NMuMG cells and plotted on the y - axis . Error bars represent SEM from three biological replicates .

(G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates.


( I ) A representative image from immunofluorescence analysis performed with anti - GFP and anti - Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain . The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining . y axis represent percentage of cells above or below the Tbr1 stain region . Error bars represent SEM from 3 independent biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( A - B ) qRT - PCR analysis of TGF ligands , TGF receptors , and TGF target expression in control and FRS2 knockdown HASMCs . ( NS : not significant compared to control , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) . - actin was used for sample loading normalization . Histogram of qRT - PCR results are representative of three independent experiments .

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(A-B) qRT-PCR analysis of TGF ligands, TGF receptors, and TGF target expression in control and FRS2 knockdown HASMCs. (NS: not significant compared to control, *p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test). -actin was used for sample loading normalization. Histogram of qRT-PCR results are representative of three independent experiments.