( G ) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs . The graph shows the normalized fluorescence intensity of TMRE , an indicator of mitochondrial membrane potential , relative to age - matched control animals . Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples . The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity . All experiments described in this figure were repeated at least three times per genotype . Error bars represent the standard error of the mean ( s . e . m . ) . * p < 0 . 05 , * * p < 0 . 005 by Student t 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 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

(G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.


PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon . ( A ) Mitochondrial ( Mito ) and postmitochondrial supernatant ( Sup ) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc , Complex V β ( Comp V β ) , NADH dehydrogenase ( ubiquinone ) Fe - S protein 3 ( NDUFS3 ) , voltage - dependent anion channel ( VDAC ) and Actin . elav - GAL4 was used to drive expression of the RNAi constructs used in these experiments . ( B ) Densitometry of the MPP - PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software . PINK1 band intensities were normalized to VDAC intensity , and this ratio was in turn normalized to the MPP - PINK1 / VDAC ratio in Control - R animals . ( C ) Quantification of the Rho - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( D ) Quantification of the AFG - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( E ) Quantification of the MPP - PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control . ( F ) Quantification of the Rho - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . ( G ) Quantification of the AFG - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . Results shown are representative of at least three independent subcellular fractionation experiments per genotype . Error bars represent s . e . m . * p < 0 . 05 by Student t test .

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

PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.(A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V β (Comp V β), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. Results shown are representative of at least three independent subcellular fractionation experiments per genotype. Error bars represent s.e.m. *p<0.05 by Student t test.


A ) Mitochondrial fractions isolated from the heads of flies expressing Control - R were divided in half . One of the two samples was treated with 0 . 5 µg / ml Proteinase K and both samples were incubated at 4 ° C for 20 minutes . Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin ( Mfn ) , the inner membrane - associated matrix protein Complex V β ( Comp V β ) , and the matrix luminal protein pyruvate dehydrogenase ( PDH ) . elav - GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments . ( B ) Densitometry of Mfn , Comp V β , and PDH bands from experiments represented by panel A were performed using Fiji software , and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown .

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A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane-associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown.


( C ) Mitochondrial fractions isolated from the heads of flies expressing Control - R and PINK1 - Myc were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( D ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel C were performed as described in B . The Mfn quantification data from panel B is also included for comparison .

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(C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison.


( E ) Mitochondrial fractions isolated from the heads of Lon - R2 and PINK1 - Myc expressing flies were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( F ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel E were performed as described in B . All experiments described were repeated at least three times . Error bars represent s . e . m .

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(E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.


PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon . Immunofluorescent staining of thoracic muscles expressing matrix - targeted YFP ( Mito - YFP ) under control of the sqh genomic promoter , together with either ( A ) Myc - tagged MitochondrialRho ( Miro - Myc ) , ( B ) FLAG - tagged Optic atrophy 1 ( Opa1 - FLAG ) , ( C ) endogenous Cytochrome C ( Cyto C ) , ( D ) Myc - tagged PINK1 ( PINK1 - Myc ) , or ( E ) Lon - RNAi1 ( Lon - R1 ) and PINK1 - Myc . The muscle - specific Mhc - GAL4 driver was used to drive expression of Miro - Myc in panel A and Opa1 - FLAG in panel B , and the muscle - specific Dmef2 - GAL4 driver was used to drive expression of Lon - R1 in panel E . ( F ) We determined the percent volume shared between green objects ( representing the matrix marker Mito - YFP ) and red objects ( representing the mitochondrial proteins analyzed in A - E ) . Red objects that shared at least 60 % of their volume with Mito - YFP were considered to co - localize . Bar represents 2 µm .

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PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon.Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged MitochondrialRho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A-E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.


A ) The eye severity score of flies co - overexpressing PINK1 with Control - R ( n = 74 ) , Lon - R1 ( n = 163 ) , or Lon - R2 ( n = 90 ) using the ey - GAL4 driver .

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A) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver.


( B ) Comparison of the frequency of thoracic indentations in flies expressing the PINK1 - RNAi construct ( PINK1 - R ) in an otherwise wild - type background to the frequency in animals that also bear heterozygous mutations in Lon : PINK1 - RNAi + / + ( n = 306 ) , PINK1 - RNAi LonEP / + ( n = 74 ) , and PINK1 - RNAi LonDf / + ( n = 83 ) . LonEP refers to the LonG3998 P - element allele of Lon , and LonDf refers to the Df ( 3L ) Exel9011 deletion that removes the Lon gene along with several other genes . The Dmef2 - GAL4 driver was used to drive expression of the PINK1 - R construct in experiments described in panel B . Error bars represent s . e . m . * p < 0 . 05 , * * p < 0 . 005 by Student t 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 B-CONTROLLED_VAR 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 O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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

(B) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L)Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.


B Representative images showing co - expression of the stage - specific markers Nestin , Tbr2 , Doublecortin ( DCX ) , and Calbindin ( all in green ) , with the β - Galactosidase ( β - Gal , red ) reporter in 8 - week old BATGAL and Axin2LacZ / + mice . Nuclei are counterstained with DAPI ( in blue ) . Scale bar = 10µm . Insets show a 1 . 5x magnification of selected cells ( position indicated by dashed box ) . Scale bar = 5µm .

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B Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.


C Fraction of Nestin - , Tbr2 - , DCX - and Calbindin - positive cells expressing β - Gal in BATGAL and Axin2LacZ / + animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [ BATGAL : Tbr2 vs . DCX p = 0 . 0056 , DCX vs . Calbindin p < 0 . 0001 ; Axin2LacZ : Nestin vs . Tbr2 p = 0 . 0003 , Tbr2 vs . DCX p = 0 . 0115 , DCX vs . Calbindin p < 0 . 0001 ] ( n = 3 animals per mouse model and marker ) .

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C Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [BATGAL: Tbr2 vs. DCX p=0.0056, DCX vs. Calbindin p<0.0001; Axin2LacZ: Nestin vs. Tbr2 p=0.0003, Tbr2 vs. DCX p=0.0115, DCX vs. Calbindin p<0.0001] (n= 3 animals per mouse model and marker).


D Detection of the corrected total cell fluorescence ( CTCF ) of the β - Gal reporter in Nestin - , Tbr2 - , DCX - and Calbindin - positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [ Nestin vs . Tbr2 p < 0 . 0001 , Tbr2 vs . DCX p = 0 . 0029 , DCX vs . Calbindin p < 0 . 0001 ] ( Nestin : n = 236 cells , Tbr2 : n = 134 cells , DCX : n = 200 cells , Calbindin : n = 300 cells ) .

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D Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX- and Calbindin-positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [Nestin vs. Tbr2 p <0.0001, Tbr2 vs. DCX p=0.0029, DCX vs. Calbindin p<0.0001] (Nestin: n= 236 cells, Tbr2: n= 134 cells, DCX: n= 200 cells, Calbindin: n= 300 cells).


F Quantification of BrdU + cells expressing β - Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [ 0 vs . 7dpi p = 0 . 0036 , 7 vs . 28dpi p = 0 . 0115 , 7 vs . 42dpi p = 0 . 0007 ] ( 0dpi time - point ; n = 4 animals ) , 3dpi ( n = 3 animals ) , 7dpi ( n = 3 animals ) , 14dpi ( n = 4 animals ) , 28dpi ( n = 6 animals ) and 42dpi ( n = 6 animals ) .

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F Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).


G Total cell fluorescence of β - Gal in BrdU + cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [ 0 vs . 3dpi p < 0 . 0001 , 7 vs . 14dpi p < 0 . 0001 , 14 vs . 28dpi p < 0 . 0001 , 28 vs . 42dpi p = 0 . 0333 ] ( 0dpi n = 194 cells , 3dpi n = 245 cells , 7dpi n = 224 cells , 14dpi n = 72 cells , 28dpi n = 103 cells and 42dpi n = 114 cells )

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G Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)


B Representative images of transduced adult - born neurons at 17dpi . CAG - dnLEF - IRES - GFP ( dnLEF , green ) and CAG - RFP ( control , red ) double transduced cells ( arrows ) and CAG - RFP single transduced cells ( arrowheads ) . Scale bar = 20

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B Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm


C Representative reconstructions of control and dnLEF neurons . Scale bar = 20

C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm


D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [ p < 0 . 0001 ] , while the number of branch points remained comparable [ p = 0 . 7914 ] . Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

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D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


E DnLEF neurons displayed basal dendrites [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

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E DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


G Representative images depicting CAG - GFP - IRES - Cre transduced adult - born neurons in control and β - catex3 mice at 17dpi . Scale bar =

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G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm


H Representative reconstructions of control and β - catex3 neurons . Scale bar =

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H Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µm


I Quantification showed decreased dendritic length of β - catex3 neurons [ p < 0 . 0001 ] ; no difference was apparent in number of branch points [ p = 0 . 3256 ] . Sholl analysis displayed a less complex dendritic tree of β - catex3 neurons [ p < 0 . 0001 ] ( control : n = 20 cells from 5 animals , β - catex3 : n = 20 cells from 5 animals ) .

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I Quantification showed decreased dendritic length of β-catex3 neurons [p<0.0001]; no difference was apparent in number of branch points [p=0.3256]. Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons [p<0.0001] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


J The number of basal dendrites was increased in β - catex3 neurons [ p = 0 . 0003 ] ( control : n = 20 cells from 5 animals , β - catex3 : n = 20 cells from 5 animals ) .

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

J The number of basal dendrites was increased in β-catex3 neurons [p=0.0003] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


B Representative reconstructions of control and β - catex3 iDCX neurons at 3dpT . Scale bar = 20µm .

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

B Representative reconstructions of control and β-catex3 iDCX neurons at 3dpT. Scale bar= 20µm.


C Analysis of morphology showed increased dendritic length in β - catex3 iDCX neurons at 3dpT [ p = 0 . 0320 ] . The number of branch points was unaltered [ p = 0 . 0651 ] . Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β - catex3 iDCX mice [ p < 0 . 0001 ] ( control : n = 25 cells from 9 animals ; β - catex3 iDCX : n = 25 cells from 8 animals ) . D Analysis of morphology showed no difference in dendritic length [ p = 0 . 1029 ] and branch point number [ p = 0 . 3726 ] between experimental groups at 13dpT . Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β - catex3 iDCX mice [ p < 0 . 01 ] ( control : n = 23 cells from 9 animals , β - catex3 iDCX : n = 26 cells from 8 animals ) .

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

C Analysis of morphology showed increased dendritic length in β-catex3 iDCX neurons at 3dpT [p=0.0320]. The number of branch points was unaltered [p=0.0651]. Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β-catex3 iDCX mice [p<0.0001] (control: n = 25 cells from 9 animals; β-catex3 iDCX: n = 25 cells from 8 animals). D Analysis of morphology showed no difference in dendritic length [p=0.1029] and branch point number [p=0.3726] between experimental groups at 13dpT. Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β-catex3 iDCX mice [p<0.01] (control: n= 23 cells from 9 animals, β-catex3 iDCX: n= 26 cells from 8 animals).


E Spine densities were comparable between experimental groups at 3dpT [ iML p = 0 . 1512 , oML p = 0 . 3841 ] . 13dpT β - catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [ iML p < 0 . 0001 , oML p = 0 . 0402 ] ( 3dpT : control : n = 20 cells from 3 animals , β - catex3 iDCX : n = 20 cells from 4 animals ; 13dpT control : n = 20 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 7 animals ) . Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT . Scale bars = 1µm .

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

E Spine densities were comparable between experimental groups at 3dpT [iML p=0.1512, oML p=0.3841]. 13dpT β-catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [iML p<0.0001 , oML p=0.0402] (3dpT: control: n= 20 cells from 3 animals, β-catex3 iDCX: n= 20 cells from 4 animals; 13dpT control: n= 20 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 7 animals). Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT. Scale bars= 1µm.


F Representative images of recombined ( GFP + ) neurons co - expressing Prox1 ( grey ) as a marker for neuronal fate and the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . G Fraction of DCX expressing neurons is slightly reduced in β - catex3 iDCX at 3dpT [ p = 0 . 0338 ] , while fraction of Calbindin expressing cells is slightly increased in β - catex3 iDCX at 3dpT [ p = 0 . 0345 ] . At 13dpT no difference is visible in stage specific marker expression [ DCX p = 0 . 8564 , Calbindin p = 0 . 0841 ] ( 3dpT : control : n = 12 animals , β - catex3 iDCX : n = 12 animals , 13dpT : control : n = 10 animals , β - catex3 iDCX : n = 14 animals ) .

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

F Representative images of recombined (GFP+) neurons co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar = 10µm. G Fraction of DCX expressing neurons is slightly reduced in β-catex3 iDCX at 3dpT [p=0.0338], while fraction of Calbindin expressing cells is slightly increased in β-catex3 iDCX at 3dpT [p=0.0345]. At 13dpT no difference is visible in stage specific marker expression [DCX p=0.8564, Calbindin p=0.0841] (3dpT: control: n= 12 animals, β-catex3 iDCX: n= 12 animals, 13dpT: control: n= 10 animals, β-catex3 iDCX: n= 14 animals).


B Expression of DCX and Calbindin in BrdU + neurons was comparable between experimental groups at 3dpT and 13dpT ( control : n = 5 animals , β - catex3 iDCX : n = 5 animals ) .

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

B Expression of DCX and Calbindin in BrdU+ neurons was comparable between experimental groups at 3dpT and 13dpT (control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


C Representative reconstructions of control and β - catex3 iDCX neurons expressing RFP at 3dpT . Scale bar = 20µm .

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

C Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 3dpT. Scale bar= 20µm.


D Quantification of dendritic length [ p = 0 . 0003 ] , branch points [ p = 0 . 0433 ] , and Sholl analysis [ p < 0 . 0001 ] showed a higher dendritic complexity of β - catex3 iDCX neurons at 3dpT ( control : n = 22 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

D Quantification of dendritic length [p=0.0003], branch points [p=0.0433], and Sholl analysis [p<0.0001] showed a higher dendritic complexity of β-catex3 iDCX neurons at 3dpT (control: n= 22 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


E Representative reconstructions of control and β - catex3 iDCX neurons expressing RFP at 13dpT . Scale bar = 20µm .

E Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 13dpT. Scale bar= 20µm.


F Quantification of dendritic length [ p < 0 . 0001 ] , and branch points [ p = 0 . 0495 ] , and Sholl analysis [ p < 0 . 0001 ] showed a lower dendritic complexity of β - catex3 iDCX neurons at 13dpT ( control : n = 20 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

F Quantification of dendritic length [p<0.0001], and branch points [p=0.0495], and Sholl analysis [p<0.0001] showed a lower dendritic complexity of β-catex3 iDCX neurons at 13dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


A Representative overview images and quantification of the β - Galactosidase reporter signal in the DG of 8 - week old , 24 - week old and 36 - week old BATGAL mice . Note the decrease in reporter signal with increasing age ( n = 4 animals per age , 200 - 300 cells per animal ) [ 8 weeks vs 24 weeks p = 0 . 0268 , 8 weeks vs 36 weeks p = 0 . 0268 ] . Scale bar = 100µm .

A Representative overview images and quantification of the β-Galactosidase reporter signal in the DG of 8-week old, 24-week old and 36-week old BATGAL mice. Note the decrease in reporter signal with increasing age (n= 4 animals per age, 200-300 cells per animal) [8 weeks vs 24 weeks p=0.0268, 8 weeks vs 36 weeks p=0.0268]. Scale bar= 100µm.


B Representative images of BrdU + cells ( red ) expressing DCX ( grey ) or β - Galactosidase ( green ) at 14 days post BrdU injection ( dpBrdU ) , 28dpBrdU and 42dpBrdu in 8 - week old and 24 - week old BATGAL mice . Scale bar = 10µm . C Percentage of BrdU + cells expressing DCX in 8 - week old and 24 - week old BATGAL mice ( 14dpi : n = 3 animals , 28dpi : n = 3 animals , 42dpi : n = 3 animals ) . Note the prolonged expression of DCX in 24 - week old mice [ 28dpBrdU p = 0 . 0155 ] . D Percentage of BrdU + cells expressing the β - Galactosidase reporter in 8 - week old and 24 - week old BATGAL mice ( 14dpi : n = 3 animals , 28dpi : n = 3 animals , 42dpi : n = 3 animals ) . Note the reduced reporter expression in 24 - week old mice [ 28dpBrdU p = 0 . 0117 , 42dpBrdU p = 0 . 0034 ] .

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

B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm. C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155]. D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034].


B Representative reconstructions of control and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

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

B Representative reconstructions of control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar = 20µm.


C Dendritic length [ p < 0 . 0001 ] and number of branch points [ p < 0 . 0001 ] was increased in 24 - week - old β - catex3 iDCX compared to 24 - week - old controls at 3dpT ( control : n = 21 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 6 animals ) . At 13dpT dendritic length [ p = 0 . 5788 ] and number of branch points [ p = 0 . 4978 ] were comparable ( control : n = 21 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 8 animals ) .

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

C Dendritic length [p<0.0001] and number of branch points [p<0.0001] was increased in 24-week-old β-catex3 iDCX compared to 24-week-old controls at 3dpT (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 6 animals). At 13dpT dendritic length [p=0.5788] and number of branch points [p=0.4978] were comparable (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 8 animals).


D Representative images showing dendritic segment of the inner molecular layer of the DG in 24 - week old mice at 13dpT . Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24 - week old β - catex3 iDCX mice at 13dpT [ iML p = 0 . 0047 , oML p < 0 . 0001 ] ( control n = 19 cells from 4 animals , β - catex3 iDCX : n = 20 cells from 4 animals ) . Scale bars = 1µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images showing dendritic segment of the inner molecular layer of the DG in 24-week old mice at 13dpT. Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24-week old β-catex3 iDCX mice at 13dpT [iML p=0.0047, oML p<0.0001] (control n=19 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals). Scale bars = 1µm.


E Representative images of recombined ( GFP + , green ) neurons in 24 - week old animals at 13dpT co - expressing Prox1 ( grey ) as a marker for neuronal fate and the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . F Quantification of marker expression . The fraction of Calbindin expressing cells was increased in the recombined population in β - catex3 iDCX at 3dpT [ p = 0 . 0081 ] and 13dpT [ p = 0 . 0259 ] ( 3dpT : control : n = 4 animals , β - catex3 iDCX : n = 9 animals , 13dpT : control : n = 8 animals , β - catex3 iDCX : n = 10 animals ) .

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

E Representative images of recombined (GFP+, green) neurons in 24-week old animals at 13dpT co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. F Quantification of marker expression. The fraction of Calbindin expressing cells was increased in the recombined population in β-catex3 iDCX at 3dpT [p=0.0081] and 13dpT [p=0.0259] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 9 animals, 13dpT: control: n= 8 animals, β-catex3 iDCX: n= 10 animals).


B Representative images of recombined ( GFP + , green ) BrdU + ( grey ) neurons in 24 - week old control and β - catex3 iDCX animals at 13dpT expressing the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . C Quantification of DCX and Calbindin expression in BrdU + recombined cells . At 13dpT , labeled neurons in β - catex3 iDCX animals show a shift towards a more mature marker profile [ DCX p = 0 . 0238 , Calbindin p = 0 . 0714 ] ( 3dpT : control : n = 4 animals , β - catex3 iDCX : n = 8 animals ; 13dpT control : n = 5 animals , β - catex3 iDCX : n = 5 animals ) .

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

B Representative images of recombined (GFP+, green) BrdU+ (grey) neurons in 24-week old control and β-catex3 iDCX animals at 13dpT expressing the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. C Quantification of DCX and Calbindin expression in BrdU+ recombined cells. At 13dpT, labeled neurons in β-catex3 iDCX animals show a shift towards a more mature marker profile [DCX p=0.0238, Calbindin p=0.0714] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 8 animals; 13dpT control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


D Representative reconstructions of RFP - birthdated control and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

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

D Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar= 20µm.


E Quantification of dendritic length [ p = 0 . 0007 ] , branch points [ p = 0 . 3909 ] , and Sholl analysis [ p = 0 . 0001 ] showed increased complexity of birthdated β - catex3 iDCX cells at 3dpT ( control : n = 20 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

E Quantification of dendritic length [p=0.0007], branch points [p=0.3909], and Sholl analysis [p=0.0001] showed increased complexity of birthdated β-catex3 iDCX cells at 3dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


F Representative reconstructions of RFP - birthdated control and β - catex3 iDCX neurons in 24 - week old mice at 13dpT . Scale bar = 20µm .

F Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 13dpT. Scale bar= 20µm.


G Quantification of dendritic length [ p = 0 . 1857 ] , branch points [ p = 0 . 1331 ] , and Sholl analysis [ p = 0 . 1501 ] showed no difference between β - catex3 iDCX and control cells at 13dpT ( control : n = 18 cells from 4 animals , β - catex3 iDCX : n = 20 cells from 4 animals )

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

G Quantification of dendritic length [p=0.1857], branch points [p=0.1331], and Sholl analysis [p=0.1501] showed no difference between β-catex3 iDCX and control cells at 13dpT (control: n= 18 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals)


A TRAF6 expression in differentiating CD4 + T cells . Naïve CD4 + T cells were obtained from wild type C57BL / 6 mice by FACS and activated with anti - CD3 / CD28 ( 1µg , and 2µg / ml ) for the indicated times in the presence of distinct Thelper lineage - directing cytokines or under neutral activation conditions ( Th0 ) . After total RNA extraction and cDNA conversion , RT - PCR determined of Traf6 mRNA in differentiating Th0 , Th1 , Th17 and iTregs was determined .

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

A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined.


B TRAF6 mRNA expression by human Tregs and non - Treg CD4 + T cell . Human Tregs ( CD3 + / CD4 + / CD8 - / CD25HIGH / CD127low / CD39 + ) and non - Treg CD4 + T cells ( CD3 + / CD4 + / CD8 - / CD25 - ) were obtained from the peripheral blood of healthy donors by FACS after Ficoll - Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4 + T cells . TRAF6 mRNA was measured by qRT - PCR .

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

B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR.


C , D Evidence of lymphoproliferative disease in Traf6fl / flFoxp3Cre + mice . ( C ) Spleens and lymph nodes were recovered from Traf6fl / flFoxp3Cre + mice and Traf6fl / fl littermates at 8 weeks of age ( Scale bars : 5 mm ) . ( D ) The cellularity of the lymphoid tissues of Traf6fl / flFoxp3Cre + mice and their Traf6fl / fl Foxp3Cre - littermates was determined ( 8 mice / group ) .

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C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group).


E , H Effect of Treg - specific TRAF6 - deficiency on baseline T cell activation . The frequencies of effector cells ( CD44high / CD62Llow ) , memory cells ( CD44high / CD62Lhigh ) and naïve cells ( CD44low / CD62Lhigh ) in the CD4 + T cell compartments of Traf6fl / fl and Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).


F , I Effect of Treg - specific TRAF6 - deficiency on baseline T cell activation . The frequencies of effector cells ( CD44high / CD62Llow ) , memory cells ( CD44high / CD62Lhigh ) and naïve cells ( CD44low / CD62Lhigh ) in the CD8 + T cell compartments of Traf6fl / fl Foxp3Cre - ( wild type ) and Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).


G , J Impact of TRAF6 - expression on in vitro Treg differentiation . naïve CD4 + T cells were isolated from Traf6fl / flFoxp3Cre + and Traf6fl / fl mice and differentiated into iTregs . Conditions of sub - optimal TGFβ concentrations ( 0 . 5ng / ml , 0 . 05ng / ml ) were tested as well and intracellular FOXP3 was measured after 4 days .

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G, J Impact of TRAF6-expression on in vitro Treg differentiation. naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.


A In vivo suppressive function of WT and Traf6fl / flFoxp3Cre + Tregs . Tregs from the indicated mice ( CD45 . 2 + ) were isolated by FACS , as were naïve ( CD62Lhigh / CD25 - ) CD4 + responder T cells from congenically distinct ( CD45 . 1 + ) donor mice . Tregs and Tresponders were mixed at a 1 : 5 ( 2x105 : 10x105 ) ratio before injection into Rag2 - / - mice . 7 days later , spleens were harvested , and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry .

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A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry.


B Implanted B16 melanoma growth in Traf6fl / flFoxp3Cre + and wild type ( Traf6fl / flFoxp3Cre - ) mice . 1x105 B16F10 cells were injected subcutaneously ( s . c . ) into the shaved flanks of the indicated mice ( n = 5 / group ) . Tumor volumes were monitored every 3 days .

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B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days.


C , D Proinflammatory cytokine production in tumor - bearing mice with and without Treg - specific TRAF6 expression . Cell suspensions of the tumor - draining lymph node and tumor - infiltrating leukocytes ( TILs ) were recovered after 21 days of tumor growth . Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL - 17 and flow cytometry analysis .

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C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.


E , F FOXP3 expression by CD4 + T cells in tumor bearing Traf6fl / flFoxp3Cre + and wild type ( Traf6fl / flFoxp3Cre - ) mice . The frequency of FOXP3 + cells in the CD4 + cells of tumor - draining lymph nodes ( dLN ) , peripheral , non - tumor draining lymph nodes ( pLN ) , spleens , and TILs were determined by flow cytometry .

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E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.


A Assessment of K63 - type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants . 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6 , the enzymatically deficient L74H or C70A TRAF6 mutants , HA - FOXP3 , and FLAG - labeled ubiquitin molecules possessing a single lysine residue ( K63 ) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63 - type only . Cells were lysed and proteins modified by K63 - ubiquitin chains were recovered from lysates by pulling down using a bead - immobilized K63 - specific TUBE reagent . The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre - IP whole cell lysate ( WCL ) .

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A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).


B Co - immunoprecipitation ( co - IP ) of FOXP3 with TRAF6 and its facilitation of K63 - linked ubiquitination . 293T cells expressing the indicated constructs encoding HA - FOXP3 and FLAG - TRAF6 were lysed and incubated with bead immobilized anti - FLAG antibodies . FOXP3 molecules co - IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti - HA antibodies .

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B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies.


C 293T cells were transfected with the indicated combinations of expression constructs encoding HA - FOXP3 , Myc - TRAF6 , and FLAG - tagged ubiquitin molecules - either wild type ( FLAG - Ub ) , or a variant with a K - to - R mutation at either lysine residue 48 ( K48R ) or 63 ( K63R ) responsible for preventing K48 - and K63 - type polyubiquitination , respectively . .

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C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .


D Endogenous , reciprocal co - IP of FOXP3 and TRAF6 in murine iTreg lysate . Naïve CD62Lhigh / CD25 - / CD4 + T cells were obtained by FACS and activated with anti - CD3 / CD28 antibodies ( 1ug and 2ug / ml , respectively ) in the presence of IL - 2 and TGFβ ( 100U / ml and 5ng / ml , respectively ) for four days before lysis . Antibodies against TRAF6 ( left ) or FOXP3 ( right ) were used to pull down target proteins and their interaction partners , which were resolved under denaturing conditions and visualized by immunoblotting .

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D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.


E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl / flFoxp3Cre + and Traf6wt / wtFoxp3Cre + mice . CD4 + / YFP + cells from Traf6fl / flFoxp3Cre + ( " Traf6 - / - " ) or wild type Traf6wt / wtFoxp3Cre + mice ( " Traf6 + / + " ) were isolated by FACS , lysed and either total FOXP3 ( right ) or K63 - ubiquitin - modified proteins ( left ) were immunoprecipitated . Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti - K63 - ubiquitin and FOXP3 , respectively , and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate .

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E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.


A Immunoblot analysis of TRAF6 - mediated ubiquitination of wild type , and mutant FOXP3 molecules . 293T cell lines were transfected with a normal HA - tagged Foxp3 construct , another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines ( 20R ) , or one of 20 single lysine - containing constructs with only the indicated lysine residue available for modification . These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags , respectively . Negative controls did not receive TRAF6 or expressed FOXP3 alone . Labeled FOXP3 proteins were pulled down from cell lysates ( anti - HA ) and ubiquitinated species were visualized by immunoblotting for FLAG .

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A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.


B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6 . 293T cells were transfected with combinations of expression constructs encoding Myc - TRAF6 , FLAG - ubiquitin , and an HA - tagged FOXP3 molecule that was either normal ( HA - FOXP3 ) or possessed a K - to - R mutation at residue 262 ( K262R ) . FOXP3 proteins were pulled down with anti - HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA , respectively .

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B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.


A Impact of K63 - ubiquitination loss on FOXP3 ' s gene silencing capacity . Jurkat T cells transfected with a wild type Foxp3 expression vector , one encoding the K262R mutant , or an empty ( control ) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter . After activation with PMA and ionomycin for 8 hours , luciferase activity was assayed .

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A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.


B IL - 2 production in the presence of wild type and K63 - ubiquitination resistant FOXP3 . Naïve CD4 + T cells isolated from Traf6fl / flCD4Cre + or WT mice were activated overnight by anti - CD3 / CD28 antibodies , then transduced by retroviruses carrying normal Foxp3 , the K262R mutant , or an empty vector . Ires - GFP in the retroviral vector serves as an internal control . GFP + cells were sorted out 48 hours post - transduction . The sorted cells were cultured for one additional day . Culture supernatants were collected for measuring the levels of IL - 2 by ELISA .

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B IL-2 production in the presence of wild type and K63-ubiquitination resistant FOXP3. Naïve CD4+ T cells isolated from Traf6fl/flCD4Cre+ or WT mice were activated overnight by anti-CD3/CD28 antibodies, then transduced by retroviruses carrying normal Foxp3, the K262R mutant, or an empty vector. Ires-GFP in the retroviral vector serves as an internal control. GFP+ cells were sorted out 48 hours post-transduction. The sorted cells were cultured for one additional day. Culture supernatants were collected for measuring the levels of IL-2 by ELISA.


C Cellular distribution of wild type and K262R FOXP3 . Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant . The relative overlap of FOXP3 protein signal and DAPI - stained nuclei was observed by fluorescence microscopy . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) .

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C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


D Immunostaining of FOXP3 in Traf6fl / flCD4Cre + and wild type ( Traf6fl / flCD4Cre - ) derived suspensions of lymph node and spleen cells . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) .

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D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


E Quantification of FOXP3 distribution in murine Tregs . The degree of FOXP3 - nuclear coIocalization in CD4 + / FOXP3 + cells isolated from the lymph nodes and spleens of Traf6fl / flCD4Cre + and wild type ( Traf6fl / flCD4Cre - ) mice was determined by Imagestream analysis , and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown ( red : nuclear stain , green : FOXP3 , Scale bars : 10 um ) .

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E Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).


The in vitro suppressive potency of wild type - and K262R Foxp3 - expressing T cells . Naïve CD4 + T cells were purified from Thy1 . 1 + BALB / c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262 . These engineered " Tregs " were then co - cultured with naïve responder CD4 + T cells stained with CFSE at the indicated ratios and activated with anti - CD3 / CD28 antibodies . Dilution of CFSE signal by the responder populations ( i . e . proliferation ) was assessed by flow cytometry .

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The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.


C In vivo suppression of colitis by wild type - and K262R mutant - Foxp3 expressing T cells . 2x105 naïve CD4 + transductants from A were mixed with 1x106 colitogenic naïve CD4 + T cells from wild type mice and transferred i . v . into Rag2 - / - mice . Rag2 - / - mice receiving no cell transfers , naïve CD4 + T cells alone , or purified wild type Tregs served as controls ( n = 6 / group ) . Recipient mouse body weights were monitored weekly for each group .

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C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.


D , E After 8 weeks , colons were excised , fixed in buffered formalin , and processed for H and E staining ( D ) and histopathology scoring ( E ) . Scale bars shown in 1 mm ) .

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D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).


( B ) Representative IF images in CHME3 stably expressing Flag alone control or the Flag - tagged EB1 constructs depicted in A co - stained for Tyrosinated - MTs ( Tyr - MTs ) , and the nucleus ( Hoechst ) . Scale bar , 10 μm .

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(B) Representative IF images in CHME3 stably expressing Flag alone control or the Flag-tagged EB1 constructs depicted in A co-stained for Tyrosinated-MTs (Tyr-MTs), and the nucleus (Hoechst). Scale bar, 10 μm.


( C ) HIV - 1 - VSV - luc infection is enhanced in CHME3 depleted of endogenous EB1 and stably expressing full - length ( FL ) EB1 , but not in control Flag or any of the other EB1 constructs depicted in A .

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(C) HIV-1-VSV-luc infection is enhanced in CHME3 depleted of endogenous EB1 and stably expressing full-length (FL) EB1, but not in control Flag or any of the other EB1 constructs depicted in A.


( D ) Increasing amount of FL - EB1 , but not ∆ EEY results in a dose - dependent enhancement of HIV - 1 - VSV - luc infection in transiently transfected CHME3 cells .

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(D) Increasing amount of FL-EB1, but not ∆EEY results in a dose-dependent enhancement of HIV-1-VSV-luc infection in transiently transfected CHME3 cells.


HIV - 1 - VSV - luc ( E ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS analysis , respectively .

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HIV-1-VSV-luc (E) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.


HIV - 1 - VSV - ZsGreen at various dilutions ( corresponding to multiplicities of infection ( m . o . i ) 0 . 06 , 0 . 03 and 0 . 02 ) ( F ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS analysis , respectively .

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HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.


( G ) HIV - 1 - WT - luc infection is decreased in CLIP170 depleted CHME3 cells .

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(G) HIV-1-WT-luc infection is decreased in CLIP170 depleted CHME3 cells.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 cells either treated with control or CLIP170 siRNAs ( A ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS analysis of cells ( n = 3 ) showing ∼ 9 % ( Control siRNA ) and 3 % ( CLIP170 siRNA ) shift ( A ) from green ( uncleaved CCF2 ) to blue ( cleaved CCF2 ) . % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot .

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Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


HIV - 1 - VSV - luc infection is enhanced in Flag - CLIP170 , but not in Flag control , expressing CHME3 cells .

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HIV-1-VSV-luc infection is enhanced in Flag-CLIP170, but not in Flag control, expressing CHME3 cells.


Molecular weight markers ( in kDa ) are shown to the right of WBs in C .

Molecular weight markers (in kDa) are shown to the right of WBs in C.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 cells expressing Flag control or Flag - CLIP170 ( D ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS analysis of cells ( n = 3 ) showing no difference ( D ) from green ( uncleaved CCF2 ) to blue ( cleaved CCF2 ) . % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot .

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Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


( E ) The percentage fused double - labelled ( GFP - Vpr + / S15 - tomato - ) HIV - 1 - DL - WT are not affected in CHME3 cells depleted of CLIP170 . Data represent mean of ≥ 895 virus particles analyzed in ≥ 11 fields of view ( ≥ 17 cells ) for each condition . One - way ANOVA was used to calculate statistical significance ( mean ± SEM ) .

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(E) The percentage fused double-labelled (GFP-Vpr+/S15-tomato-) HIV-1-DL-WT are not affected in CHME3 cells depleted of CLIP170. Data represent mean of ≥895 virus particles analyzed in ≥11 fields of view (≥17 cells) for each condition. One-way ANOVA was used to calculate statistical significance (mean ±SEM).


( F ) Percentage HIV - 1 - WT - GFP - Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h . p . i , scatterplot with mean of ≥ 354 virus particles quantified in ≥ 13 cells per sample . Statistical significance was determined by one - way ANOVA * = p < 0 . 05 , * * * = p < 0 . 001 .

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(F) Percentage HIV-1-WT-GFP-Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h.p.i, scatterplot with mean of ≥354 virus particles quantified in ≥13 cells per sample. Statistical significance was determined by one-way ANOVA * = p<0.05, *** = p<0.001.


Infected cells in F analyzed by live cell imaging . Scatterplots representing displacement over time ( velocity ) Each dot represents one viral particle tracked , red line indicates the mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t test . * * * = p < 0 . 001 .

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Infected cells in F analyzed by live cell imaging. Scatterplots representing displacement over time (velocity) Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


Infected cells in F analyzed by live cell imaging . cumulative distance travelled over time ( speed ) ( H ) . Each dot represents one viral particle tracked , red line indicates the mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t test . * * * = p < 0 . 001 .

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Infected cells in F analyzed by live cell imaging. cumulative distance travelled over time (speed) (H). Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


( I ) The induction of Ac - MTs is blocked in HIV - 1 - VSV - luc ( Virus ) infected , but not in uninfected ( NI ) or Mock infected CHME3 depleted of EB1 , but not CLIP170 at 6 h . p . i . Tyrosinated MTs ( Tyr - MTs ) are not affected . Representative fields are shown . Similar results were obtained in three independent experiments . Scale bar , 50 μm .

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(I) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were either lysed and analyzed by WB Molecular weight markers ( in kDa ) are shown to the right of WBs in A .

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CHME3 treated with negative control, EB1 or CLIP170 siRNAs were either lysed and analyzed by WB Molecular weight markers (in kDa) are shown to the right of WBs in A.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were infected with GFP - Vpr / S15 - tomato - labeled VSV - G pseudotyped HIV - 1 ( B ) . Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean .

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CHME3 treated with negative control, EB1 or CLIP170 siRNAs were infected with GFP-Vpr/S15-tomato-labeled VSV-G pseudotyped HIV-1 (B). Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean.


( C - D ) Amounts of pelleted p24 CA normalized to the input CA at 3 h . p . i . with HIV - 1 - VSV - luc in 293A ( C ) or CHME3 ( D ) treated with control , EB1 or CLIP170 siRNAs . Mean + / - SEM from three independent experiments is shown . Statistical significance was determined by one - way ANOVA . Error bars = standard error of the mean ( * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 ) .

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(C-D) Amounts of pelleted p24 CA normalized to the input CA at 3 h.p.i. with HIV-1-VSV-luc in 293A (C) or CHME3 (D) treated with control, EB1 or CLIP170 siRNAs. Mean +/- SEM from three independent experiments is shown. Statistical significance was determined by one-way ANOVA. Error bars = standard error of the mean (* = p-value ≤0.05, ** = p-value ≤0.01, *** = p-value ≤0.001).


Representative ( n = 3 ) WB analysis demonstrating that while GAPDH - HA control do not bind ( A FEZ1 - Flag control pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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Representative (n=3) WB analysis demonstrating that while GAPDH-HA control do not bind (A FEZ1-Flag control pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB analysis demonstrating that while EB1 - Flag do not bind pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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Representative (n=3) WB analysis demonstrating that while EB1-Flag do not bind pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB analysis demonstrating that while Flag - CLIP170 pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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Representative (n=3) WB analysis demonstrating that while Flag-CLIP170 pelleted with in vitro assembled HIV-1 CA-NC complexes.


( D ) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates ( n = 3 ) from C . Statistical significance was determined by t test ( mean ± SEM ) * * * = p < 0 . 001 .

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(D) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates (n=3) from C. Statistical significance was determined by t test (mean ±SEM) *** = p<0.001.


( E ) Representative WB analysis ( n = 3 ) showing the levels of HIV - 1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation . Fractions 1 - 3 contain free p24 not associated with the intact cores , while fractions # 8 and # 9 harbouring intact HIV - 1 cores .

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(E) Representative WB analysis (n=3) showing the levels of HIV-1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation. Fractions 1-3 contain free p24 not associated with the intact cores, while fractions #8 and # 9 harbouring intact HIV-1 cores.


( F ) Flag - CLIP170 , but not Flag control , also binds to fractions containing intact HIV - 1 cores ( Virus # 8 and # 9 ) but not with Mock fractions .

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(F) Flag-CLIP170, but not Flag control, also binds to fractions containing intact HIV-1 cores (Virus #8 and #9) but not with Mock fractions.


( G ) HIV - 1 - WT - luc infection is increased in CHME3 cells stably expressing Flag - CLIP170 , but not control Flag or Flag - CLIP170ΔCAPgly . Molecular weight markers ( in kDa ) are shown to the right of WBs .

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(G) HIV-1-WT-luc infection is increased in CHME3 cells stably expressing Flag-CLIP170, but not control Flag or Flag-CLIP170ΔCAPgly. Molecular weight markers (in kDa) are shown to the right of WBs.


( H ) Representative images ( n = 3 ) of CHME3 expressing Flag - CLIP170 or Flag - CLIP170ΔCAPgly stained for Flag and Tyr - MTs . Scale bar , 10 μm .

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(H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.


( A ) Super resolution microscopy ( SIM ) imaging of Flag - tagged proteins , cyclophilin A ( CypA - Flag ) , EB1 - Flag , or Flag - CLIP170 bound to in vitro assembled CA - NC complexes , stained with anti - p24 AG3 . 0 ( Red ) , anti - Flag or anti - CLIP170 ( Green ) . Scale bar , 2 μm . Representative images from different fields of view are shown .

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(A) Super resolution microscopy (SIM) imaging of Flag-tagged proteins, cyclophilin A (CypA-Flag), EB1-Flag, or Flag-CLIP170 bound to in vitro assembled CA-NC complexes, stained with anti-p24 AG3.0 (Red), anti-Flag or anti-CLIP170 (Green). Scale bar, 2 μm. Representative images from different fields of view are shown.


( B ) WB analysis confirming protein expression in samples from A and C . Molecular weight markers ( in kDa ) are shown to the right of WBs .

(B) WB analysis confirming protein expression in samples from A and C. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) Quantification ( n = 3 ) of the association of each protein with 97 to 137 CA - NC complexes , using untransfected lysates ( Control ) and EB1 - Flag as controls for non - specific background staining .

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(C) Quantification (n=3) of the association of each protein with 97 to 137 CA-NC complexes, using untransfected lysates (Control) and EB1-Flag as controls for non-specific background staining.


( D ) Lysates from untransfected , CypA - Flag - or Flag - CLIP170 - transfected cells were subjected to CA - NC binding assays and WB analysis .

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(D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis.


( E ) Levels of CypA or CLIP170 bound to WT or R18L CA - NC assemblies in samples from D were quantified using Fiji ( n = 4 experimental replicates ; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA - NC assembly , normalized to WT CA - NC arbitrarily set to 1 ) . Statistical significance was determined by t test ( mean ± SEM ) . * * * = p < 0 . 001 .

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(E) Levels of CypA or CLIP170 bound to WT or R18L CA-NC assemblies in samples from D were quantified using Fiji (n = 4 experimental replicates; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA-NC assembly, normalized to WT CA-NC arbitrarily set to 1). Statistical significance was determined by t test (mean ±SEM). *** = p<0.001.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) , but not HA - tagged GAPDH ( GAPDH ) binds to Flag - CLIP170 ( C ) in 293T cells . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv), but not HA-tagged GAPDH (GAPDH) binds to Flag-CLIP170 (C) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) endogenous forms of various human SxIP - containing + TIPs , but not vimentin ( D ) in 293T cells . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) endogenous forms of various human SxIP-containing +TIPs, but not vimentin (D) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) or to Flag - tagged SxIP fragment ( Flag - Clasp2M ) ( E ) in 293T cells . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) or to Flag-tagged SxIP fragment (Flag-Clasp2M) (E) in 293T cells. Results are representative of three experimental replicates.


HIV - 1 - VSV - luc ( Virus ) , but not uninfected ( NI ) or Mock , infection induces the formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH , as shown by WB analysis ( F )

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HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH, as shown by WB analysis (F)


HIV - 1 - VSV - luc ( Virus ) , but not uninfected ( NI ) or Mock , infection induces the formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH or imaging at 6 h . p . i ( G ) . Scale bar , 15 μm . Representative fields are shown . Results are representative of three experimental replicates .

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HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH or imaging at 6 h.p.i (G). Scale bar, 15 μm. Representative fields are shown. Results are representative of three experimental replicates.


( H ) GAPDH - MHRhiv expression in CHME3 reduces infection with HIV - 1 - WT - luc . Statistical significance was determined by t test . * = p < 0 . 05 . Data are mean values from three independent experiments + / - SEM . Molecular weight markers ( in kDa ) are shown to the right of WBs .

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(H) GAPDH-MHRhiv expression in CHME3 reduces infection with HIV-1-WT-luc. Statistical significance was determined by t test. * = p<0.05. Data are mean values from three independent experiments +/- SEM. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) Anti - HA co - IP in CHME3 expressing GAPDH - MHRhiv , GAPDH - MHRsiv or GAPDH - MHRmulv showing binding to endogenous CLIP170 . Results are representative of three experimental replicates .

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(C) Anti-HA co-IP in CHME3 expressing GAPDH-MHRhiv, GAPDH-MHRsiv or GAPDH-MHRmulv showing binding to endogenous CLIP170. Results are representative of three experimental replicates.


Anti - HA co - IP in Rhesus macaque FRhK4 ( D ) cells expressing GAPDH alongside GAPDH -

Anti-HA co-IP in Rhesus macaque FRhK4 (D) cells expressing GAPDH alongside GAPDH-MHRsiv


Anti - HA co - IP in rat fibroblast Rat2 ( E ) cells expressing GAPDH alongside GAPDH - MHRmulv Anti - EB1 co - IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells . Results are representative of three experimental replicates .

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Anti-HA co-IP in rat fibroblast Rat2 (E) cells expressing GAPDH alongside GAPDH-MHRmulv Anti-EB1 co-IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells. Results are representative of three experimental replicates.


FRhK4 cells infected with SIV - VSV - luc ( F ) stained for Ac - MTs , Tyr - MTs , and the nucleus ( Hoechst ) at 6 h . p .

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FRhK4 cells infected with SIV-VSV-luc (F) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i


Rat2 cells infected with MuLV - VSV - luc ( G ) stained for Ac - MTs , Tyr - MTs , and the nucleus ( Hoechst ) at 6 h . p . i ,

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Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i,


FRhK4 cells infected with SIV - VSV - luc analyzed by

FRhK4 cells infected with SIV-VSV-luc analyzed by WB


Rat2 cells infected with MuLV - VSV - luc analyzed by

Rat2 cells infected with MuLV-VSV-luc analyzed by WB


Measurements of SIV - VSV - luc infectivity in FRhK4 ( J ) cells depleted of CLIP170 . Statistical significance was determined by t test . * * * = p < 0 . 001 . Data are mean values from two independent experiments + / - SEM .

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Measurements of SIV-VSV-luc infectivity in FRhK4 (J) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM.


Measurements of MuLV - VSV - luc infectivity in Rat2 ( K ) cells depleted of CLIP170 .

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Measurements of MuLV-VSV-luc infectivity in Rat2 (K) cells depleted of CLIP170.


( L ) 293T transfected with GAPDH - MHRhiv or R162Q mutant ( GAPDH - MHRhivQ ) were subjected to anti - HA co - IP . Representative WB analysis is shown ( n = 2 ) . Molecular weight markers ( in kDa ) are shown to the right of WBs .

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

(L) 293T transfected with GAPDH-MHRhiv or R162Q mutant (GAPDH-MHRhivQ) were subjected to anti-HA co-IP. Representative WB analysis is shown (n = 2). Molecular weight markers (in kDa) are shown to the right of WBs.