( B ) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin , doxycycline and chloramphenicol ( + antibiotic ) , followed by a 48 hours recovery period ( - antibiotic ) . During both periods , proteins are isolated at 12h , 24h and 48h . Antibodies against subunits of C I ( NDUFB6 ) , C III ( UQCRC1 ) , C IV ( COXI ) , MRPS35 and MRPL37 were used as indicated .

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(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.


( C ) Quantitative analysis of the COXI recovery upon treatment with antibiotics ( Actinonin , DOXY - doxycycline and CAP - chloramphenicol ) as in B . The values represent ratios of recovered ( 48h , - antibiotics ) versus untreated ( 0h ) COXI levels . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) . ( Actinonin and CAP n = 3 , DOXY n = 4 ) .

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

(C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY - doxycycline and CAP - chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonin and CAP n=3, DOXY n=4).


( D ) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunobloting with antibodies against MRPS35 and MRPL12 . The profile of a given mt - RNA is calculated after qRT - PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis . Results of individual experiments are provided for 12S and 16S rRNA , and for mt - mRNAs , results of distribution from two independent experiments using probes against ATP6 , COXIII and ND5 were combined . Error bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 3 ) .

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(D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=3).


( E ) Relative association of 12S rRNA , 16S rRNA and mt - mRNAs with mitoribosomes ( 55S ) in Clpp knockout mitochondria comparing to wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S and 16S n = 3 ; mRNA n = 6 ) .

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(E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).


( B ) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( Clpp + / + ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) . The arrowhead indicates positions of EFG1 .

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(B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1.


( C ) Expression levels of potential CLPP substrates from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( set to 1 ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) .

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(C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4).


( D ) Cycloheximide ( CHX ) chase assay to determine the stability of ERAL1 and EFG1 . Proteins are visualized by Western blot with respective antibodies . Proteins are isolated after 2 , 4 , 6 , 8 , 10 and 12 hours after beginning of chase ( 0 ) . The arrowhead indicates positions of ERAL1 .

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

(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.


( E ) Western blot analysis and subsequent quantification of protein levels in wild type ( C1 , C2 ) and Clpp knockout ( KO1 , KO2 ) MEFs upon Clpx siRNA ( Clpx ) relative to untreated ( Ctrl ) wild type . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 6 ) . The arrowhead indicates positions of either CLPX or ERAL1 , and stars indicate position of cross - reacting bands .

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(E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.


( A ) Sucrose density gradient sedimentation profile ( " short gradients " ) of ribosomal proteins and transcripts from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The migration of ribosomal subunits ( 28S and 39S ) and mitoribosome ( 55S ) was detected by immunoblotting with antibodies against MRPS35 and MRPL12 . 12S rRNA was analysed by Northern blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sucrose density gradient sedimentation profile ("short gradients") of ribosomal proteins and transcripts from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunoblotting with antibodies against MRPS35 and MRPL12. 12S rRNA was analysed by Northern blot.


( B ) Sedimentation profiles of ERAL1 , MRPS28 , EFG1 , CLPP , CLPX , EFTU and MTIF2 in heartmitochondria as in A .

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(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heartmitochondria as in A.


( C ) Relative abundance of proteins bound to the 28S ribosomal subunit ( fractions 3 and up ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( ERAL1 n = 4 ; EFG1 , CLPX , MTIF2 n = 3 ; EFTU n = 2 ) .

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(C) Relative abundance of proteins bound to the 28S ribosomal subunit (fractions 3 and up). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (ERAL1 n=4; EFG1, CLPX, MTIF2 n=3; EFTU n=2).


( D ) Sucrose density gradient sedimentation profile ( " long gradients " as in figure 4D ) of ERAL1 , MRPS35 , MRPL37 , CLPX and EFG1 from control ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) heartmitochondria . The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis ( Input ) . Only fractions 4 - 11 containing the 28S , 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions ( 1 - 3 ) .

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(D) Sucrose density gradient sedimentation profile ("long gradients" as in figure 4D) of ERAL1, MRPS35, MRPL37, CLPX and EFG1 from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis (Input). Only fractions 4 - 11 containing the 28S, 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions (1-3).


( A ) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins . Western blot analysis of input ( I ) , control ( C ) and eluate ( E ) is shown . Asterix indicates the cross - reacting band .

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(A) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins. Western blot analysis of input (I), control (C) and eluate (E) is shown. Asterix indicates the cross-reacting band.


( B ) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit ( MRPS35 ) upon Eral1 siRNA . TOMM20 was used as a loading control .

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(B) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit (MRPS35) upon Eral1 siRNA. TOMM20 was used as a loading control.


( C ) De novo synthesis of mitochondrial proteins in wild type ( Clpp + / + ) and Clpp knockout ( Clpp - / - ) MEFs upon Eral1 siRNA . Proteins were isolated after labeling with 35S - methionine for 1 hour from cells on control siRNA ( Ctrl ) or cells on Eral1 siRNA ( Eral1 ) . Positions of individual mitochondrial encoded proteins are indicated . Coomassie blue staining was used to ensure equal loading .

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(C) De novo synthesis of mitochondrial proteins in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) MEFs upon Eral1 siRNA. Proteins were isolated after labeling with 35S-methionine for 1 hour from cells on control siRNA (Ctrl) or cells on Eral1 siRNA (Eral1). Positions of individual mitochondrial encoded proteins are indicated. Coomassie blue staining was used to ensure equal loading.


( D ) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1 . Levels relative to wild type cells treated with control siRNA ( set to 1 ) are presented . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; ATP5A1 n = 2 ; the rest n = 6 ) . Combined results from 2 - 3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment . Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp - knockout cells upon Eral1 siRNA .

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(D) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1. Levels relative to wild type cells treated with control siRNA (set to 1) are presented. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; ATP5A1 n=2; the rest n=6). Combined results from 2-3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment. Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp-knockout cells upon Eral1 siRNA.


( E ) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs . Cells ( 0 ) were treated with doxycycline for 48 hours ( T ) and upon doxycycline removal allowed to recover for 24 hours ( R ) . ( F ) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA . The values are calculated as ratio between recovered ( R ) to doxycycline untreated ( 0 ) protein levels in Eral1 - knockdown cells ( Eral1 ) and compared to the same results obtained in control cells ( Ctrl ) . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( COXI n = 4 ; NDUFB6 n = 3 ) . Numbers within bars represent statistical difference between control / Eral1 siRNA treated cells and numbers above bars represent difference between + / + ; Eral1 siRNA and - / - ; Eral1 siRNA .

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(E) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs. Cells (0) were treated with doxycycline for 48 hours (T) and upon doxycycline removal allowed to recover for 24 hours (R).(F) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA. The values are calculated as ratio between recovered (R) to doxycycline untreated (0) protein levels in Eral1-knockdown cells (Eral1) and compared to the same results obtained in control cells (Ctrl). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; NDUFB6 n=3). Numbers within bars represent statistical difference between control/Eral1 siRNA treated cells and numbers above bars represent difference between +/+; Eral1 siRNA and -/-; Eral1 siRNA.


a , TG levels in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) and cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , n = 3 ) .

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a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).


b , TG levels in vector - infected ( VEC ) and siAtg5 cells in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM .

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b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.


c , TG levels in wild - type ( WT ) or Atg5 knockout mice embryonic fibroblasts ( Atg5 - / - ) ( * P 0 . 01 or * * P 0 . 0001 , n = 5 ) .

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c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).


d f , Cells from a and b stained with BODIPY 493 / 503 ( d ) . Nuclei are highlighted with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; d ) . Error bars , s . e . m .

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df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.


d f , Cells from a and b stained with oil red O ( e ) or visualized by electron microscopy ( f ) . Right : quantifications of LD number and size ( * P 0 . 01 , * * P 0 . 001 with untreated wild - type cells ; # P 0 . 01 , # # P 0 . 001 with cells in RM ; n = 4 6 ) .

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df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).


a , b , VEC and siAtg5 cells cultured with oleate ( OL ) or in MCDM were examined for their rates of TG synthesis ( a ) and β - oxidation ( b ) as compared to cells in regular medium alone ( * P 0 . 03 , * * P 0 . 004 with VEC cells in the same medium , n = 3 4 ) .

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a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).


c , d , Rates of TG decay in OL ( c ) and MCDM ( d ) ( * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 as compared to VEC cells , n = 3 7 ) .

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c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37).


e , TG levels in wild - type cells treated with dimethyl sulphoxide vehicle ( DMSO ) , 3 - methyladenine ( 3MA ) or diethylumbelliferyl phosphate ( DEUP ) ( * P 0 . 00001 with DMSO - treated cells , # P 0 . 003 with 3MA - treated cells , n = 6 ) . Error bars , s . e . m .

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e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.


f , Co - localization of BODIPY 493 / 503 ( green ) with LAMP1 ( red ) or LC3 ( red ) in hepatocytes in MCDM .

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f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM.


g , High - magnification regions of hepatocytes in MCDM alone ( none ) or treated with vinblastine and stained as labelled . Arrows indicate co - localization .

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g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.


a , Electron micrographs of cultured hepatocytes . dm , double - membrane cytosolic vesicles . Arrows indicate membranes in LDs . Insets show the double membrane in nucleus ( N ) , lipid - containing vesicles ( L ) and mitochondria ( mit ) , but not in LDs .

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a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs.


b , Mouse liver LC3 immunogold . Insets show higher magnification . Arrowheads indicate gold particles ( black ) and LC3 - labelled bilayer membranes ( white ) . c , Percentages of autophagic vacuoles ( AVs ) containing only lipid ( Lipids , * ) , other cargo ( Other ) or mixed cargo ( Mixed ) in cells treated as in b ( * P 0 . 01 , * * P 0 . 001 with cells in RM , n = 4 6 ) . Left : representative examples . Error bars , s . e . m .

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b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.


a , Immunoblots of cellular homogenates ( Hom ) and LDs from fed ( F ) or 24 - h starved ( S ) mice . IκB , inhibitor of the nuclear factor of kappa light polypeptide gene enhancer ; GPDH , glyceraldehyde - 3 - phosphate dehydrogenase .

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a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase.


b , Percentages of autophagic vacuoles ( AVs ) containing only lipid , other cargo or mixed cargo calculated from samples processed as in Supplementary Fig . 16b ( * P 0 . 001 , * * P 0 . 0001 , n = 4 6 ) . ( Supplementary Figure 16 Effects of starvation on hepatic lipid droplets . a , Mouseliverelectron micrographs . Arrows : Lipid - containing double membrane vesicles . b , Electron micrographs of autophagic vacuoles ( AVs ) isolated from mice fed or starved for the indicated time periods . The percentages of double membrane vesicles containing lipid in isolated AVs from 3 - 4 mice per condition are shown ( * P < 0 . 01 as compared to fractions from fed animals ) . ( * ) indicate lipid )

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b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)


c , Immunoblots of Hom , AV and lysosomes ( Lys ) . L - 1 , LAMP1 .

c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1.


d , Immunoblots of liver Hom and LDs from HFD - fed mice .

d, Immunoblots of liver Hom and LDs from HFD-fed mice.


e , Liver homogenate immunoblots . PDI , protein disulphide isomerase .

e, Liver homogenate immunoblots. PDI, protein disulphide isomerase.


f , Total hepatic TG and cholesterol content ( * P 0 . 01 , * * P 0 . 00001 , n = 817 ) .

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f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).


g , Hepatic TG concentration ( * P 0 . 05 , n = 3 ) .

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g, Hepatic TG concentration (*P  0.05, n = 3).


h , Percentage of cellular cholesterol in lysosomes ( * P 0 . 02 , n = 4 ) .

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h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).


i , Immunoblots of homogenates and isolated LDs . Error bars , s . e . m .

i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.


E , F : EM images of negatively stained Dam1c alone ( E ) and in the presence of Bim1 ( F ) , at low salt conditions . The final concentrations of Dam1c and Bim1 were 0 . 3 µM and 0 . 1 µM , respectively . The scale bars are 100 nm , boxes mark representative complexes shown as magnified inserts .

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E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.


B : Chromatogram and SDS - PAGE of elution fractions from gel filtration . Binding of Bim1 to Dam1 ∆ SxIPc lacking the SxIP motif in the C - terminus of Duo1 was examined by analytical size exclusion chromatography . Elution fractions were analyzed by SDS - PAGE .

B: Chromatogram and SDS-PAGE of elution fractions from gel filtration. Binding of Bim1 to Dam1∆SxIPc lacking the SxIP motif in the C-terminus of Duo1 was examined by analytical size exclusion chromatography. Elution fractions were analyzed by SDS-PAGE.


C : Solid phase binding assay to test for binding of different Dam1 complexes to Bim1 . Recombinant GST - Bim1185 - 344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1 ∆ SxIPc . Input and pull down samples were analyzed by SDS - PAGE .

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C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.


D : Pull down assay to compare binding of Dam1WTc and Dam1 ∆ SxIPc to immobilized GST - Bim1185 - 344 . Soluble cell lysates from yeast strains expressing the respective Duo1 - 6xFlag alleles were incubated with GST - Bim1185 - 344 immobilized on beads . Duo1 - 6xFlag bound to GST - Bim1185 - 344 was analyzed by western blot . An anti - Pgk1 antibody was used to confirm equal protein amounts in input samples .

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D: Pull down assay to compare binding of Dam1WTc and Dam1∆SxIPc to immobilized GST-Bim1185-344. Soluble cell lysates from yeast strains expressing the respective Duo1-6xFlag alleles were incubated with GST-Bim1185-344 immobilized on beads. Duo1-6xFlag bound to GST-Bim1185-344 was analyzed by western blot. An anti-Pgk1 antibody was used to confirm equal protein amounts in input samples.


C : Representative images from fluorescence live cell microscopy of Dad1 - GFP strains with Duo1WT - 6xFlag or Duo1 ∆ SxIP - 6xFlag . Displayed cells are in either metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . D : Quantification of signal intensities of Dad1 - GFP at metaphase and anaphase kinetochore clusters of Duo1WT - 6xFlag and Duo1 ∆ SxIP - 6xFlag strains . Bars represent the mean + / - standard deviation . Each spot represents the value measured for a single kinetochore cluster . Values were normalized to the mean value of metaphase clusters of the Duo1WT - 6xFlag strain . A one - way ANOVA test with Sidak ' s multiple comparisons test was used to calculate p values . n ≥ 76 kinetochore clusters .

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C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters.


F : Analysis of cell cycle progression of yeast strains with different Duo1 alleles . Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot ( left ) . An antibody against Pgk1 was used to confirm equal protein amounts in samples . The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS ( right ) . Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content , respectively . An intermediate DNA content is detected for cells in S phase .

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F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase.


B : Representative images from live cell microscopy of Dam1WT and dam1 - 19 strains with Dad1 - GFP . Displayed cells are in metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . C : Quantification of Dad1 - GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1 - 19 strains . Bars represent the mean + / - standard deviation . Each spot represents the value measured for a single kinetochore cluster . Values were normalized to the mean value of metaphase clusters of the Dam1WT strain . p - values were obtained by a one - way ANOVA test with Sidak ' s multiple comparisons test . n ≥ 86 kinetochore clusters .

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B: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters.


E , F : GST - Bim1185 - 344 pull down assays . Binding of immobilized GST - Bim1185 - 344 to Duo1 - 6xFlag was analyzed in context of ( E ) selective inhibition of Mps1 ( mps1 - 737 ) or overexpression of Mps1 from a galactose - inducible promotor ( F ) . For inhibition of Mps1 , cells were grown at the restrictive temperature of 37 ° C . Overexpression of Mps1 was achieved by growing cells in medium containing galactose . GST - Bim1185 - 344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions . Binding of Duo1 was analyzed by western blot . Pgk1 indicates equal protein amounts in all samples .

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E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1 (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing galactose. GST-Bim1185-344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by western blot. Pgk1 indicates equal protein amounts in all samples.


G : Live cell microscopy of a Dad1 - GFP strain under conditions of Mps1 overexpression . The localization of Dam1c was visualized by Dad1 - GFP in either an Mps1WT or Mps1 overexpression setup ( pGal - Mps1 ) . Ndc80 - mRuby2 was used as reference point for the localization of kinetochores . Images show representative metaphase cells from both strains . The area in the white dashed box is shown as magnification in the lower left corner . White arrowheads point at nuclear microtubules decorated by Dad1 - GFP . Please note that brightness and contrast of the Dad1 - GFP image in the lower row were optimized to make the nuclear microtubules clearly visible . Scale bar : 2 μm .

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G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules decorated by Dad1-GFP. Please note that brightness and contrast of the Dad1-GFP image in the lower row were optimized to make the nuclear microtubules clearly visible. Scale bar: 2 μm.


A , B : 5 µM , 7 . 5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1 - Bik1 complex were analyzed by size exclusion chromatography . SDS - PAGE analysis of elution fractions is shown in B . High molecular weight species of Dam1WTc were eluted after 0 . 9 - 1 . 0 ml ( see chromatogram in A ) .

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A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A).


B : Pull down assay for analysis of Dam1c - Bim1 binding to Ndc80c . Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre - formed Dam1c - Bim1 complex with increasing amounts of Bim1 . Input and elution samples were analyzed by SDS - PAGE . LC and HC denote the light and heavy chains of the M2 anti - Flag antibody used for immobilization of Ndc80c on beads . Protein names in blue , red and green refer to Dam1c , Ndc80c and Bim1 , respectively .

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B: Pull down assay for analysis of Dam1c-Bim1 binding to Ndc80c. Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre-formed Dam1c-Bim1 complex with increasing amounts of Bim1. Input and elution samples were analyzed by SDS-PAGE. LC and HC denote the light and heavy chains of the M2 anti-Flag antibody used for immobilization of Ndc80c on beads. Protein names in blue, red and green refer to Dam1c, Ndc80c and Bim1, respectively.


D : Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80 - FRB - GFP background . Ndc80WT or Ndc80 ∆ 490 - 510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1 ∆ SxIP allele ( at DUO1 locus ) . Cells were spotted on YEPD medium without and with 1 µg / ml Rapamycin and incubated at the indicated temperatures .

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D: Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.


B : Serial dilution assay of yeast strains with 6xFlag - tagged Duo1WT or Duo1 ∆ SxIP and Ask1WT , Ask12A or Ask12D . Cells were grown at the indicated temperatures and images were taken two days ( 25 ° C , 30 ° C and 37 ° C ) or four days ( 16 ° C ) after spotting .

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B: Serial dilution assay of yeast strains with 6xFlag-tagged Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting.


E - H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected ( E ) , 2 months after intraductal injection with MCF7 , arrows point to undilated ductal tips ( F ) , MM134 , dotted line highlight the subtending duct ( G ) , or SUM44 cells ( H ) , ( n > 6 ) scale bar , 1 mm .

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E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm.


I Ex vivo luminescence images of bones , brain , lungs and liver from MM134 engrafted mice 1 month post injection ( top ) and ovaries ( bottom ) 12 months post injection . Scale bar , 1 cm . J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis . Number of mice analyzed MM134 and SUM44 for 30 - 180 : N = 5 , 10 , 180 - 270 days : N = 9 , 5 , 270 - 360 days : N = 14 , 20 , respectively . Dashed line indicates background levels .

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I Ex vivo luminescence images of bones, brain, lungs and liver from MM134 engrafted mice 1 month post injection (top) and ovaries (bottom) 12 months post injection. Scale bar, 1 cm. J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis. Number of mice analyzed MM134 and SUM44 for 30-180: N=5, 10, 180-270 days: N=9, 5, 270-360 days: N=14, 20, respectively. Dashed line indicates background levels.


E , F Representative micrographs of IHC for Ki67 , ER , and PR on histological sections of MM134 ( E ) and SUM44 ( F ) xenografts 5 months after intraductal injection counter stained with hematoxylin . Scale bars , 200 μm . Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for > 1000 cells counted on 3 glands .

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E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.


G Representative micrographs of PR - IHC on glands from 5 - month - old MM134 xenografts from hosts treated for 2 months with vehicle ( left ) or E2 ( right ) pellets . Scale bars , 200 μm . Right , bar plot showing percentage of PR + cells in MM134 and SUM44 xenografts with n > 1000 cells per condition on glands from 3 different mice . Data represent mean ± SEM unpaired , Student ' s t - test , two - tailed ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001.


D Representative RNAscope image of sections from patient ILCs ( n = 5 ) stained with anti - LOXL1 probes ( red ) and counterstained with DAPI ( blue ) . Scale bars ; 50 μm .

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D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1 probes (red) and counterstained with DAPI (blue). Scale bars; 50 μm.


E Representative immunofluorescence image of primary ILC ( n = 3 ) stained with anti - LOXL1 ( green ) and anti - ER ( red ) antibodies counterstained with DAPI ( blue ) . Scale bars ; 50 μm and right inset 30 μm .

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E Representative immunofluorescence image of primary ILC (n=3) stained with anti-LOXL1 (green) and anti-ER (red) antibodies counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.


E Radiance - based tumor growth curves ( mean ± SE ) for T47D treated with vehicle ( n = 8 ) or BAPN ( n = 12 ) 8 days after intraductal injections . n . s not significant .

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E Radiance-based tumor growth curves (mean ± SE) for T47D treated with vehicle (n=8) or BAPN (n=12) 8 days after intraductal injections. n.s not significant.


F Radiance - based tumor growth curves for ( mean ± SE ) ILC ER + PDXs T125 and T137 treated with vehicle ( n = 6 , 7 ) or BAPN ( n = 7 , 8 ) 66 days after engraftment . * * p < 0 . 01 .

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F Radiance-based tumor growth curves for (mean ± SE) ILC ER+ PDXs T125 and T137 treated with vehicle (n=6, 7) or BAPN (n=7, 8) 66 days after engraftment. ** p < 0.01.


G Radiance - based tumor growth curves ( mean ± SE ) for non - ILC ER + PDXs T99 and T157 treated with vehicle ( n = 7 , 7 ) or BAPN ( n = 8 , 12 ) , 8 days after intraductal injection 3 weeks after intraductal injections . n . s not significant .

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G Radiance-based tumor growth curves (mean ± SE) for non-ILC ER+ PDXs T99 and T157 treated with vehicle (n=7, 7) or BAPN (n=8, 12), 8 days after intraductal injection 3 weeks after intraductal injections. n.s not significant.


E Representative fluorescent stereo - micrographs of glands injected with MM134 : RFP - luc2 cells ( left , top and bottom ) infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 ( bottom ) , n = 5 and 3 . Scale bar , 1000 μm . F Barplot showing the ratio of GFP / RFP signal from fluorescent stereo - micrographs of glands engrafted with MM134 cells , n = 8 . Statistical analysis by paired Student t - test , two - tailed . * p < 0 . 05 .

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E Representative fluorescent stereo-micrographs of glands injected with MM134:RFP-luc2 cells (left, top and bottom) infected with either GFP:sh-scramble (top) or GFP+ shLOXL1 (bottom), n=5 and 3. Scale bar, 1000 μm. F Barplot showing the ratio of GFP/RFP signal from fluorescent stereo-micrographs of glands engrafted with MM134 cells, n=8. Statistical analysis by paired Student t-test, two-tailed. *p < 0.05.


H Representative fluorescent stereo - micrographs of glands engrafted with SUM44 : RFPluc2 cells infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 , n = 6 . Scale bar , 1000 μm . I Barplot ( mean ± SEM ) showing quantification of the ratio GFP / RFP from fluorescent stereo - micrographs of SUM44 . Statistical analysis by unpaired Student ' s t - test , two - tailed , n = 7 sh - scramble and n = 6 for sh - LOXL1 , * * * * p < 0 . 0001 .

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H Representative fluorescent stereo-micrographs of glands engrafted with SUM44:RFPluc2 cells infected with either GFP:sh-scramble (top) or GFP+ shLOXL1, n=6. Scale bar, 1000 μm. I Barplot (mean ± SEM) showing quantification of the ratio GFP/RFP from fluorescent stereo-micrographs of SUM44. Statistical analysis by unpaired Student's t-test, two-tailed, n=7 sh-scramble and n=6 for sh-LOXL1, ****p < 0.0001.


J , K Representative photomicrographs of picrosirius red stained histological sections for sh - scramble or sh - LOXL1 MM134 ( J ) and SUM44 ( K ) xenografts . Scale bars , 100 μm .

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J, K Representative photomicrographs of picrosirius red stained histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.


Relative fold repression for let - 7a reporter in RAW 264 . 7 cells during LPS treatment ( mean + / - s . e . m . , n = 3 ) ( B ) . The schematic representation of the constructs used for luciferase assays are shown .

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Relative fold repression for let-7a reporter in RAW 264.7 cells during LPS treatment (mean+/- s.e.m., n=3) (B). The schematic representation of the constructs used for luciferase assays are shown.


Relative levels of pro - inflammatory cytokines TNF - ɑ and IL - 6 mRNA , a target of let - 7a miRNA , have been plotted against time of LPS stimulation , 18S rRNA was used for normalization ( mean + / - s . e . m . , n = 2 ( IL - 6 ) , n = 3 ( TNF - α ) ) ( C ) .

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Relative levels of pro-inflammatory cytokines TNF-ɑ and IL-6 mRNA, a target of let-7a miRNA, have been plotted against time of LPS stimulation, 18S rRNA was used for normalization (mean+/- s.e.m., n=2 (IL-6), n=3 (TNF-α)) (C).


Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Ago2 was pulled down using anti - FLAG beads and the levels of Ago - associated let - 7a miRNA were measured by qRT - PCR . Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti - HA antibody and used for normalization of amounts of miRNA detected by qRT - PCR . Levels of cellular let - 7a were measured by Northern blot and U6 RNA was used as loading control . Relative levels of cellular and Ago2 bound let - 7a were plotted ( mean + / - s . e . m . , n = 3 ) ( D ) .

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Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Ago2 was pulled down using anti-FLAG beads and the levels of Ago-associated let-7a miRNA were measured by qRT-PCR. Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti-HA antibody and used for normalization of amounts of miRNA detected by qRT-PCR. Levels of cellular let-7a were measured by Northern blot and U6 RNA was used as loading control. Relative levels of cellular and Ago2 bound let-7a were plotted (mean+/- s.e.m., n=3) (D).


Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Phosphorylated Ago2 ( pY - Ago2 ) level was measured using 4G10 antibody specific for phosphorylated tyrosine . Phosphorylated Ago2 ( pY - Ago2 ) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down ( E , upper panel ) . Relative 4G10 intensities were plotted from three independent experiments ( mean + / - s . e . m ) ( E , lower panel ) .

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Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Phosphorylated Ago2 (pY-Ago2) level was measured using 4G10 antibody specific for phosphorylated tyrosine. Phosphorylated Ago2 (pY-Ago2) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down (E, upper panel). Relative 4G10 intensities were plotted from three independent experiments (mean+/- s.e.m) (E, lower panel).


Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Primary cells ( PEC ) were isolated from BALB / c mice and endogenous Ago2 was immunoprecipitated with anti - Ago2 ( eIF2C2 ) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody . Relative intensities of phospho - Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes ( mean + / - s . e . m . , n = 3 ) ( F , upper panel ) . Similar experiments were done with FH - Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH - Ago1 and FH - Ago3 were measured in RAW 264 . 7 cells ( F , lower panel ) .

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Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Primary cells (PEC) were isolated from BALB/c mice and endogenous Ago2 was immunoprecipitated with anti-Ago2 (eIF2C2) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody. Relative intensities of phospho-Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes (mean+/- s.e.m., n=3) (F, upper panel). Similar experiments were done with FH-Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH-Ago1 and FH-Ago3 were measured in RAW 264.7 cells (F, lower panel).


Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 cells . Knockdown of PP2A , MKP1 , MKP3 or SHP1 / 2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs . β - Actin was used as loading control ( G ) .

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Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Knockdown of PP2A, MKP1, MKP3 or SHP1/2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs. β-Actin was used as loading control (G).


Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 cells . Luciferase based let - 7a miRNA reporter assay was done in LPS - treated cells downregulated for specific phosphatases . Levels at 0 hrs time point were taken as units ( mean + / - s . e . m . , n = 4 ) ( H ) .

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Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Luciferase based let-7a miRNA reporter assay was done in LPS-treated cells downregulated for specific phosphatases. Levels at 0 hrs time point were taken as units (mean +/- s.e.m., n=4) (H).


Effect of knockdown of different phosphatases on miRNA - association of Ago2 in 24hrs LPS treated RAW 264 . 7 cells . Ago2 associated let - 7a was estimated by qRT - PCR in LPS treated cells depleted for specific phosphatases . let - 7a miRNA level was normalized against respective FH - Ago2 bands ( mean + / - s . e . m . , n = 3 ) ( I ) .

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Effect of knockdown of different phosphatases on miRNA-association of Ago2 in 24hrs LPS treated RAW 264.7 cells. Ago2 associated let-7a was estimated by qRT-PCR in LPS treated cells depleted for specific phosphatases. let-7a miRNA level was normalized against respective FH-Ago2 bands (mean+/- s.e.m., n=3) (I).


Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody . HA - Ago2 was detected with anti - HA antibody ( J ) . Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments .

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Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody. HA-Ago2 was detected with anti-HA antibody (J). Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments.


Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Cellular miRNA levels ( K , left panel mean + / - s . e . m . , n = 3 ) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT - PCR and plotted ( K , right panel , mean + / - s . e . m . , n = 3 ) .

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Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Cellular miRNA levels (K, left panel mean +/- s.e.m., n=3) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT-PCR and plotted (K, right panel, mean+/- s.e.m., n=3).


Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Ago2 associated IL - 1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs ( mean + / - s . e . m . , n = 3 ) ( L ) .

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Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Ago2 associated IL-1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs (mean+/- s.e.m., n=3) (L).


Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264 . 7 cells after LPS stimulation ( A ) .

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Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).


Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation . The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount ( B ) .

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Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).


Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on miRNA activity . Change in fold repression for a let - 7a reporter upon LPS exposure in control and OA treated cells ( mean + / - s . e . m . , n = 3 ) ( C ) .

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Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).


Schematic representation of the in vitro phosphatase assay ( upper panel ) . PP2A was immunoprecipitated from 24hrs LPS treated RAW 264 . 7 cells and were incubated in vitro with wild type or phosphorylation defective FH - Ago2 mutant ( Ago2Y529F ) isolated from HEK293 cells transfected with respective expression constructs . Phosphorylated Ago2 ( pYAgo2 ) levels were measured by western blot analysis using anti - phospho Tyrosine specific 4G10 antibody . Relative intensities were quantified by densitometric analysis and mentioned below the respective panels . FH - Ago2 was detected by anti - HA antibody ( Lower panel ) .

Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).


Defective de novo miRNP formation in cells pre - treated with PP2A inhibitor . Ago2 associated ( F ) and total ( G ) miR - 122 levels were measured by qRT - PCR and normalized against immuneprecipitated FH - Ago2 content and U6 RNA respectively . Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted . All samples were treated for 24 hrs with LPS ( mean + / - s . e . m . , n = 4 ) .

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Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).


PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Expression of PTPA after LPS stimulation in RAW 264 . 7 cells as well as in PEC isolated from BALB / c mice . β - actin is used as loading control ( H ) .

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PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).


PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264 . 7 cells ( I ) . Phosphorylated Ago2 level was measured by western blot in HA - immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH - Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody ( I , left panel ) . The PTPA was detected with anti PTPA antibody , β - Actin was used as loading control ( I , right panel ) .

PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).


Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs . Ago2 - associated miRNA ( J , left panel ) ( mean + / - s . e . m . , n = 3 ) and mRNA levels ( J , right panel ) ( mean + / - s . e . m . , n = 2 ) were also quantified using qRT - PCR .

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Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs. Ago2-associated miRNA (J, left panel) (mean+/- s.e.m., n=3) and mRNA levels (J, right panel) (mean+/- s.e.m., n=2) were also quantified using qRT-PCR.


PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264 . 7 cells treated with LPS for 24 hrs and was incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells and upon the assay reaction , the phospho - Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody ( K , right panel ) .

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PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).


Expression of different cytokines and phosphatases in RAW 264 . 7 cells and PEC infected with Leishmania donovani ( Ld ) parasites . Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264 . 7 ( B , left panel ) and mouse peritoneal macrophage cells PEC ( B , right panel ) .

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Expression of different cytokines and phosphatases in RAW 264.7 cells and PEC infected with Leishmania donovani (Ld) parasites. Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264.7 ( B, left panel) and mouse peritoneal macrophage cells PEC (B, right panel).


Cytokine mRNA levels like IL - 10 , TNF - α and IL - 6 were quantified after 24 hrs of Ld infection in RAW 264 . 7 ( C , upper panel mean + / - s . e . m . , n = 3 ) and mouse peritoneal macrophage PEC ( mean + / - s . e . m . , n = 3 ) ( C , Lower panel ) .

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Cytokine mRNA levels like IL-10, TNF-α and IL-6 were quantified after 24 hrs of Ld infection in RAW 264.7 (C, upper panel mean +/- s.e.m., n=3) and mouse peritoneal macrophage PEC (mean+/- s.e.m., n=3) (C, Lower panel).


Effect of Ld infection on Ago2 phosphorylation and its miRNA association . RAW 264 . 7 cells were transfected with FH - Ago2 expression construct and infection was done for various time points followed by FH - Ago2 pull down using anti - FLAG beads . Phosphorylated Ago2 levels were checked during the course of Ld infection ( D ) .

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Effect of Ld infection on Ago2 phosphorylation and its miRNA association. RAW 264.7 cells were transfected with FH-Ago2 expression construct and infection was done for various time points followed by FH-Ago2 pull down using anti-FLAG beads. Phosphorylated Ago2 levels were checked during the course of Ld infection (D).


Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let - 7a level measurement in primary macrophages ( mean + / - s . e . m . , n = 2 ) ( E , left panel ) and in RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) ( E , right panel ) .

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Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let-7a level measurement in primary macrophages (mean+/- s.e.m., n=2) (E, left panel) and in RAW 264.7 cells (mean+/- s.e.m., n=3) (E, right panel) .


Schematic representation of in vitro phosphatase assay ( F , upper panel ) . PP2A , MKP1 , MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody ( F , lower panel ) and measured by densitometry . BC ; Bead Control .

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Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. BC; Bead Control.


PP2A MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted ( mean + / - s . e . m . , n = 3 ) ( G ) . BC ; Bead Control .

PP2A MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted (mean+/- s.e.m., n=3) (G). BC; Bead Control.


Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells ( H ) .

Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H).


Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Ago2 associated let - 7a levels were also estimated and plotted ( mean + / - s . e . m . , n = 4 ) ( I ) .

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Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Ago2 associated let-7a levels were also estimated and plotted (mean+/- s.e.m., n=4) (I).


Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment ( 100 nM ) were detected . Cells were pre - treated with OA ( 100 nM ) for 2 hrs before infection ( J ) . β - Actin was used as loading control .

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Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control.


Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells and mouse PEC . Phospho - Ago2 level was measured in OA pre - treated ; Ld infected RAW 264 . 7 cells expressing FH - Ago2 and in PEC post 24 hrs of infection with or without OA pre - treatment . Phosphorylated Ago2 levels were also checked ( K ) .

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Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).


Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots . Alix was used at MVB marker , while Calnexin was used as ER marker ( L ) .

Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots. Alix was used at MVB marker, while Calnexin was used as ER marker (L).


RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . OptiprepR fractions , positive for MVB ( fraction number 2 , 3 , 4 ) and ER ( fraction number 7 , 8 , 9 ) markers were pooled separately and Ago2 was immunoprecipitated using Anti - FLAG beads . Phosphorylated Ago2 levels were quantified in western blot done with phospho - tyrosine specific 4G10 antibody ( M ) .

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RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M).


Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . PP2A percentage intensity plot for each fraction was done for control and infected samples ( N ) .

Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. PP2A percentage intensity plot for each fraction was done for control and infected samples (N).


Effect of PP2A downregulation or inhibition on pro and anti - inflammatory cytokine levels in Ld infected macrophage . IL - 10 levels were measured after Ld infection in PP2A knocked - down RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) and in PEC ( mean + / - s . e . m . , n = 3 ) . IL - 6 was also measured in RAW cells after OA treatment ( mean + / - s . e . m . , n = 5 ) and also in PEC pre - treated with OA ( mean + / - s . e . m . , n = 3 ) . Values obtained at 0 hrs ( non - infected ) or non - OA treated samples were considered as unit in each case . IL - 6 protein level ( n = 5 ) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock - down RAW 264 . 7 cells .

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Effect of PP2A downregulation or inhibition on pro and anti-inflammatory cytokine levels in Ld infected macrophage. IL-10 levels were measured after Ld infection in PP2A knocked-down RAW 264.7 cells (mean+/- s.e.m., n=3) and in PEC (mean+/- s.e.m., n=3). IL-6 was also measured in RAW cells after OA treatment (mean+/- s.e.m., n=5) and also in PEC pre-treated with OA (mean+/- s.e.m., n=3). Values obtained at 0 hrs (non-infected) or non-OA treated samples were considered as unit in each case. IL-6 protein level (n=5) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock-down RAW 264.7 cells.


Effect of PP2A knock - down on parasite internalization Internalized parasites were imaged in RAW 264 . 7 cells stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 ( red ) ( B ) .

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Effect of PP2A knock-down on parasite internalization Internalized parasites were imaged in RAW 264.7 cells stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 (red) (B).


Effect of PP2A knock - down on parasite internalization and cellular IL - 1β level . Number of Ld internalized was measured in control and PP2A knock - down cells and relative numbers of parasites within infected cells were plotted ( C , left panel , ( mean + / - s . e . m . , n = 20 cells ) . IL - 1β mRNA levels were also quantified in those cells by qRT - PCR done for total cellular RNA [ C , right panel , ( mean + / - s . e . m . , n = 3 ) ] . Scale bar 20 µm . 4x Zoomed insets are shown .

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Effect of PP2A knock-down on parasite internalization and cellular IL-1β level. Number of Ld internalized was measured in control and PP2A knock-down cells and relative numbers of parasites within infected cells were plotted (C, left panel, (mean+/- s.e.m., n=20 cells). IL-1β mRNA levels were also quantified in those cells by qRT-PCR done for total cellular RNA [C, right panel, (mean+/- s.e.m., n=3)]. Scale bar 20 µm. 4x Zoomed insets are shown.


Effect of PP2A knockdown on IL - 10 level in Ld - infected cells expressing phosphorylation defective mutant of Ago2 . In RAW 264 . 7 cells , depleted for endogeneous Ago2 ( using a specific siRNA against the 3 ' UTR of Ago2 mRNA ) , either the wild type or Y529F mutant of FH - Ago2 ( without having the 3 ' UTR of Ago2 ) was expressed in siCon or siPP2A treated RAW 264 . 7 cells . IL - 10 mRNA level was measured subsequent to Ld infection . Relative IL - 10 levels were plotted . Expression levels in non - infected siCon treated cells in each case were designated as unit ( mean + / - s . e . m . , n = 3 ) .

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Effect of PP2A knockdown on IL-10 level in Ld-infected cells expressing phosphorylation defective mutant of Ago2. In RAW 264.7 cells, depleted for endogeneous Ago2 (using a specific siRNA against the 3'UTR of Ago2 mRNA), either the wild type or Y529F mutant of FH-Ago2 (without having the 3'UTR of Ago2) was expressed in siCon or siPP2A treated RAW 264.7 cells. IL-10 mRNA level was measured subsequent to Ld infection. Relative IL-10 levels were plotted. Expression levels in non-infected siCon treated cells in each case were designated as unit (mean+/- s.e.m., n=3).


Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . In images obtained , Ago2 was detected with α - HA ( detected at 405nm ) and Leishmania was stained for GP63 ( detected at 564nm ) ( E ) .

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Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. In images obtained, Ago2 was detected with α-HA (detected at 405nm) and Leishmania was stained for GP63 (detected at 564nm) (E).


Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted . Scale bar 10 µm . RNA was isolated from different experimental sets and TNF - α , IL - 1β and IL - 10 mRNA levels were estimated ( mean + / - s . e . m . , n = 3 ) ( F ) .

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Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted. Scale bar 10 µm. RNA was isolated from different experimental sets and TNF-α, IL-1β and IL-10 mRNA levels were estimated (mean +/- s.e.m., n=3) (F).


Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . RAW 264 . 7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs . RAW 264 . 7 cells were stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 ( red ) ( G , left panel ) . Number of internalized parasites per 100 macrophages were calculated and relative values were plotted ( G , right panel ) ( mean + / - s . e . m . , n = 20 ) . Scale bar 20 µm .

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 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

Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. RAW 264.7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs. RAW 264.7 cells were stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 (red) (G, left panel). Number of internalized parasites per 100 macrophages were calculated and relative values were plotted (G, right panel) (mean+/- s.e.m., n=20). Scale bar 20 µm.


LPS induced miRNA export from mammalian macrophage cells . A schematic diagram of the experiments has been described in left panel of ( A ) . Characterization and quantification of exosomes derived from Control and LPS - activated RAW 264 . 7 cells by Nanoparticle Tracking Analysis ( NTA ) done for isolated exosomes . Relative size distributions are shown ( A , right panel ) .

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

LPS induced miRNA export from mammalian macrophage cells. A schematic diagram of the experiments has been described in left panel of (A). Characterization and quantification of exosomes derived from Control and LPS-activated RAW 264.7 cells by Nanoparticle Tracking Analysis (NTA) done for isolated exosomes. Relative size distributions are shown (A, right panel).


Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264 . 7 cells . RNA content was normalized against total exosomal proteins ( B ) . β - Actin as loading control for HuR western blot .

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

Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264.7 cells. RNA content was normalized against total exosomal proteins (B). β-Actin as loading control for HuR western blot.


Binding of miRNAs with HuR after LPS exposure was measured . Amount of RNA was normalized against HuR content . Anti GFP - antibody was used for immunoprecipitation control ( C ) . Values are mean + / - s . e . m . and , n =

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

Binding of miRNAs with HuR after LPS exposure was measured. Amount of RNA was normalized against HuR content. Anti GFP-antibody was used for immunoprecipitation control (C). Values are mean+/- s.e.m. and, n=3


Effect of GW4869 , the inhibitor of exosome mediated miRNA export , on exosomal miRNA content released by LPS stimulated cells . The level of miRNAs , let - 7a and miR - 155 were measured in exosomes ( EV ) released from LPS activated cell after GW4869 treatment . Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit . Values are mean + / - s . e . m . and , n =

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Effect of GW4869, the inhibitor of exosome mediated miRNA export, on exosomal miRNA content released by LPS stimulated cells. The level of miRNAs, let-7a and miR-155 were measured in exosomes (EV) released from LPS activated cell after GW4869 treatment. Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit. Values are mean+/- s.e.m. and, n=3


Effect of GW4869 on cellular cytokine levels in LPS treated cells . The effect of GW4869 on cellular expression of pro - inflammatory cytokines IL - 1β and TNF - α in LPS - activated macrophages were measured and normalized against 18S rRNA . Values in naive RAW 264 . 7 cells without GW4869 and LPS treatment were set as unit . Values are mean + / - s . e . m . and , n =

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

Effect of GW4869 on cellular cytokine levels in LPS treated cells. The effect of GW4869 on cellular expression of pro-inflammatory cytokines IL-1β and TNF-α in LPS-activated macrophages were measured and normalized against 18S rRNA. Values in naive RAW 264.7 cells without GW4869 and LPS treatment were set as unit. Values are mean+/- s.e.m. and, n=3


Effect of siRNA - mediated downregulation of protein HuR on cellular expression of pro - inflammatory cytokines . In the left panel of F , effect of siRNA treatment on cellular cytokine mRNA levels in LPS - stimulated RAW 264 . 7 cells is shown . Relative levels measured against 18S rRNA are plotted . The effect of HuR depletion on cellular miR - 155 content has been measured and relative values normalized against U6 snRNA are plotted ( right panel , F ) .

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Effect of siRNA-mediated downregulation of protein HuR on cellular expression of pro-inflammatory cytokines. In the left panel of F, effect of siRNA treatment on cellular cytokine mRNA levels in LPS-stimulated RAW 264.7 cells is shown. Relative levels measured against 18S rRNA are plotted. The effect of HuR depletion on cellular miR-155 content has been measured and relative values normalized against U6 snRNA are plotted (right panel, F).


The amount of let - 7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 ( G ) . Values are mean + / - s . e . m . and , n =

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

The amount of let-7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 (G) .Values are mean+/- s.e.m. and, n=3


Effect of HuR expression on inflammatory responses in macrophage cells . Effect of ectopic expression of HuR on exosomal let - 7a content in naive macrophage cells ( H , right panel ) . HuR over expression was checked by western blot ( H , left panel ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n =

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

Effect of HuR expression on inflammatory responses in macrophage cells. Effect of ectopic expression of HuR on exosomal let-7a content in naive macrophage cells (H, right panel). HuR over expression was checked by western blot (H, left panel). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3


Effect of HuR expression on inflammatory responses in macrophage cells . The effect of HuR expression on production of pro - inflammatory cytokine TNF - α in RAW 264 . 7 cells was measured . mRNA and protein level of TNF - α was quantified . Values obtained with pCINeo expression and without LPS treatment was taken as unit ( I ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n =

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

Effect of HuR expression on inflammatory responses in macrophage cells. The effect of HuR expression on production of pro-inflammatory cytokine TNF-α in RAW 264.7 cells was measured. mRNA and protein level of TNF-α was quantified. Values obtained with pCINeo expression and without LPS treatment was taken as unit (I). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3


Effect of HA - HuR expression on p38 mediated activation of downstream signalling events . The Levels of p - p38 and p - MSK1 have been monitored by western blotting done with cell extracts prepared from HA - HuR and control plasmid transfected , untreated and LPS treated RAW 264 . 7 cells . β - Actin blot was used as loading control .

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Effect of HA-HuR expression on p38 mediated activation of downstream signalling events. The Levels of p-p38 and p-MSK1 have been monitored by western blotting done with cell extracts prepared from HA-HuR and control plasmid transfected, untreated and LPS treated RAW 264.7 cells. β-Actin blot was used as loading control.


Downregulation of HuR in Ld - infected macrophages . HuR levels were monitored against time in Ld - infected primary macrophage cells ( PEC ) from BALB / c mice ( K , left upper panel ) as well as in Ld - infected RAW 264 . 7 cells ( K , upper right panel ) . HuR levels were also monitored against time in L . major - infected RAW 264 . 7 cells ( K , lower left panel ) and L . mexicana - infected RAW 264 . 7 cells ( K , lower right panel ) .

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Downregulation of HuR in Ld-infected macrophages. HuR levels were monitored against time in Ld-infected primary macrophage cells (PEC) from BALB/c mice (K, left upper panel) as well as in Ld-infected RAW 264.7 cells (K, upper right panel) .HuR levels were also monitored against time in L. major-infected RAW 264.7 cells (K, lower left panel) and L. mexicana-infected RAW 264.7 cells (K, lower right panel).


Downregulation of HuR in Ld - infected macrophages . Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld - infected RAW 264 . 7 cells . In parallel assays levels of pro - inflammatory cytokine TNF - α was measured in Ld - infected cells either with no treatment or pre - treated with MG132 ( L ) . Values are mean + / - s . e . m . and , n = 3 .

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 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

Downregulation of HuR in Ld-infected macrophages. Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld-infected RAW 264.7 cells. In parallel assays levels of pro-inflammatory cytokine TNF-α was measured in Ld-infected cells either with no treatment or pre-treated with MG132 (L). Values are mean+/- s.e.m. and, n=3.


Downregulation of HuR in Ld - infected macrophages . The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined . Input samples were monitored for HuR downregulation by western blot . Presence of Alix ( marker for early endosomes ) and calnexin ( marker protein for ER ) were used to confirm separation of organelles during density gradient fractionation . Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection ( M upper panel ) . Densitometric analysis of individual fractions was done and the respective values were plotted ( M , lower panel ) .

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

Downregulation of HuR in Ld-infected macrophages. The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined. Input samples were monitored for HuR downregulation by western blot. Presence of Alix (marker for early endosomes) and calnexin (marker protein for ER) were used to confirm separation of organelles during density gradient fractionation. Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection (M upper panel). Densitometric analysis of individual fractions was done and the respective values were plotted (M, lower panel).


effect of HA - HuR expression on PP2A protein

effect of HA-HuR expression on PP2A protein levels


effect of HA - HuR expression on PP2A RNA levels were scored .

effect of HA-HuR expression on PP2A RNA levels were scored.