Fission is associated with ultrastructural changes in mitochondria . EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission . Cortical cultures were exposed to aged SNOC control ( A - D ) or 200 μM SNOC ( E - L ) for 6 h in presence of 80 μM zVAD ‐ fmk methyl ester . A slice from each tilt series is shown ( A , E I ) , followed by orthogonal views of 3D reconstructions after segmentation of OMM ( blue ) IMM ( gray ) , with cristae ( various arbitrary colors ) ( B - D , F - H J - L ) . For clarity , 10 representative cristae are shown in ( B , D , F , G , J K ) , while cristae are displayed in ( C , H L ) . control mitochondrion ( A - D ) is elongated with a matrix , indicating no swelling , 34 cristae . NO ‐ exposed mitochondrion in ( E - H ) is dividing , lower mitochondrion of couplet shows severe damage , recognized by an outer rupture , a regionally confined inner blowout , slight matrix swelling cristae fragmentation . Each of daughter mitochondria is shorter rounder than controls . Unlike upper daughter , few of cristae extend throughout lower mitochondrion . Additionally , cristae are considerably smaller regionally confined . lower mitochondrion of couplet contains 82 cristae , while upper contains only 39 , indicating cristae fragmentation in former . NO ‐ exposed mitochondrial triplet in ( I - L ) displays further damage , including degradation , accumulation of an electron dense mass , greater cristae fragmentation , apparent mitochondrial fragmentation . cristae are smaller than controls , with 73 in top mitochondrion , 112 in middle , 47 in bottom . Scale bars , 400 nm . See Supplementary Video 3 , 4 5 of tomographic reconstruction .

O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.


( A ) Projection image of two neurons expressing Mito ‐ DsRed2 ( red ) MyrPalm ‐ CFP ( blue ) . were high ‐ pass filtered z ‐ stacks were projected over maximum .

O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

(A) Projection image of two neurons expressing Mito‐DsRed2 (red) and MyrPalm‐CFP (blue). Images were high‐pass filtered and z‐stacks were projected over maximum intensity.


( B ) Changes in mitochondrial dynamics were evaluated as mean skeletal of mitochondria or ( C ) as mitochondrial numbers .

O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O

(B) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or (C) as mitochondrial numbers.


( D ) Magnified representative frames of time ‐ lapse recording of within rectangle of ( A ) indicate mitochondrial at time 0 , 20 min 1 h . These time points are depicted in ( B ) ( C ) by red arrows . Data are representative of 26 neurons , which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons ( six experiments ) . Scale bar , 10 μm . See Supplementary Video 6 .

O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O 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-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

(D) Magnified representative frames of the time‐lapse recording of the area within the white rectangle of (A) indicate mitochondrial morphology at time 0, 20 min and 1 h. These time points are depicted in (B) and (C) by red arrows. Data are representative of 26 neurons, which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons (six experiments). Scale bar, 10 μm. See Supplementary Video 6.


( E ) Autophagy in neurites at 6 h after SNOC exposure . Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in left image . arrowhead in right panel indicates wrappers characteristic of autophagosomes . arrowheads in insets show double membranes of autophagosome those of mitochondria within autophagosome . Scale bars , 500 nm .

O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O

(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.


( A ) Mitochondrial fission is linked to a drop in ATP . Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors ( 2 μM rotenone plus 2 μg / ml oligomycin ; Rot / Olig ) . ATP concentrations are shown as mean ± s . e . m . normalized to plating density of neurons ( n = 6 )

O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)


( B ) [ ATP ] / [ ADP ] ratios in purified corticalneurons exposed to increasing SNOC concentrations ( n = 4 ) .

O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O

(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).


( C ) bar graph depicts ethidine density in arbitrary units of aged fresh SNOC treated neurons at 2 . 5 h .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O

(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.


( D ) Cortical neurons expressing Mito ‐ GFP ( green ) were exposed to aged or fresh 200 μM SNOC solution . After 2 . 5 h cultures were loaded with hydroethidine . are representative of more that 20 neurons analyzed ( in D ) for each condition from at least three independent experiments . Scale bar , 10 μm .

O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.


( E ) Reduced glutathione ( GSH ) partially blocks mitochondrial fission . Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester , washed once then exposed to 35 μM SNOC . Mitochondrial fission was scored after 1 h . Data are means ± s . e . m . of quintuplicate samples from one representative experiment of a total of three ( † † † significance at P0 . 001 or n . s . , † not significant compared to control ; * significance at P0 . 05 ) .

O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).


( A ) Representative of mitochondrial before after SNOC ( 175 μM ; 7 h ) or rotenone ( 30 nM ; 2 h ) treatments a comparison of effects of Aβ25 - 35 versus Aβ35 - 25 ( 10 μM , 6 h ) , as indicated . Scale bar , 20 μm .

O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.


( B ) of mitochondrial fission at 18 h ( † † † † significance at P0 . 05 0 . 001 as compared to control pcDNA3 transfection ; * , * * * * * significance at P0 . 05 , 0 . 01 0 . 001 , respectively , compared to SNOC treated , pcDNA3 transfected neurons ; n = 3 independent experiments ) . Dying neurons were recognized by their shrunken condensed nuclei after Hoechst 33342 .

O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O 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-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O

(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.


( C ) Enforced Drp1 or Fis1 expression ( 60 h ) evokes fission neuronal ( * , * * * * * significance at P0 . 05 , 0 . 01 0 . 001 , respectively , compared to pcDNA3 transfected neurons ; n = 3 independent experiments ) .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O 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-GENEPROD O B-CELL_TYPE O O O O O O O O

(C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments).


( D ) Rotenone induces dose ‐ dependent mitochondrial fission . fraction of neurons displaying fissioned mitochondria is shown as mean ± s . e . m . ( * significance at P0 . 05 , n = 4 ) .

O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O B-SUBCELLULAR 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) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).


( E ) Mfn1 or Drp1K38A inhibits 100 nM rotenone ‐ induced mitochondrial fission ( 4 h ) ( 48 h ) . Data indicate means ± s . e . m . of triplicate measurements ( † † † † † significance at P0 . 01 0 . 001 as compared to untreated control ; * * * significance at P0 . 001 as compared to rotenone treatment with pcDNA3 transfection ; n = 3 ) .

O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-GENEPROD O O O O O O O

(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).


( F ) Mitochondrial fission by Aβ35 - 25 or Aβ25 - 35 exposure . fraction of neurons exhibiting fragmented mitochondria is shown as mean ± s . e . m . ( † † significance at P0 . 01 compared to pcDNA3 transfected , Aβ35 − 25 treated control ; * * * significance at P0 . 05 0 . 001 , respectively , compared to pcDNA3 transfected , Aβ25 - 35 treated neurons ; n = 3 ) .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O

(F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).


A , B Co - immunoprecipitation ( IP ) of EBNA2 ( A ) PLK1 ( B ) using EBNA2 - , PLK1 - specific antibodies , or isotype - matched control antibodies performed on total lysates ( L ) of lymphoblastoid long - term B cultures ( LCLs ) infected with recombinant EBV 2089 , which is on EBV B95 -

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O 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-ORGANISM O O O O O B-ORGANISM O O

A, B Co-immunoprecipitation (IP) of EBNA2 (A) and PLK1 (B) using EBNA2-, PLK1-specific antibodies, or isotype-matched control antibodies performed on total cell lysates (L) of lymphoblastoid long-term B cell cultures (LCLs) infected with recombinant EBV strain 2089, which is based on EBV strain B95-8


GFP - tagged EBNA2 fragments to co - precipitate endogenous PLK1 . Total lysates immunoprecipitates ( IP ) were analyzed by Western blotting ( WB ) .

B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O

GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB).


( H ) Co - immunoprecipitation of transfected EBNA2 wt or S379A endogenous PLK1 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O

(H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.


( D ) EBNA2 candidate phosphorylation mutants were expressed in DG75 B tested for PLK1 binding by co - immunoprecipitations followed by Western blotting .

(D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting.


( F ) GST EBNA2 fragment 246 - 487 wt mutant S457A / T465V were treated with recombinant active PLK1 ( + ) in presence of [ γ - 32P ] ATP in vitro or left untreated ( - ) . CRS , an artificial PLK1 substrate was used as a positive control .

O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.


( H ) HA - tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B tested for activation of an EBNA2 / CBF1 responsive promoter reporter luciferase plasmid . Activation of reporter gene is shown as relative response normalized to Renilla luciferase activity shown relative to wt activity .

O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-CELL_LINE B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity.


( A ) Flag - tagged active ( PLK1 ) kinase inactive PLK1 ( K82M ) were co - expressed with HA - EBNA2 wt , docking site mutant ( S379A ) phosphorylation mutant ( S457A / T465V ) tested for activation of an EBNA2 / CBF1 responsive promoter reporter in DG75 . Activation of reporter firefly luciferase gene is shown as relative response normalized to Renilla luciferase activity shown relative to EBNA2 wt activity .

O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O B-ORGANISM B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O

(A) Flag-tagged active (PLK1) and kinase inactive PLK1 (K82M) were co-expressed with HA-EBNA2 wt, docking site mutant (S379A) and phosphorylation mutant (S457A/T465V) and tested for activation of an EBNA2/CBF1 responsive promoter reporter in DG75 cells. Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity.


( B ) 30 µg of extracts that were produced for luciferase assays were tested for expression using Flag - , HA - GAPDH - specific antibodies .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O

(B) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag-, HA- and GAPDH-specific antibodies.


( C ) Expression of EBNA2 , LMP1 , PLK1 , MYC , GAPDH was analyzed by Western blotting immunostaining of whole extracts of lines established from 3 donors .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(C) Expression of EBNA2, LMP1, PLK1, MYC, and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors.


( B ) Survival curve of humanized mice infected with 105 GRU EBV wt ( n = 13 ) , EBV S379A ( n = 11 ) or EBV S457A / T465V EBV ( n = 9 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O

(B) Survival curve of humanized mice infected with 105 GRU EBV wt (n = 13), EBV S379A (n = 11) or EBV S457A/T465V EBV (n = 9).


( E ) Development of viral loads in blood of infected mice over a period of five . Error bars indicate mean ± . Number of biological replicates per group : EBNA2 WT EBV week 2 - 5 : n = 8 ; EBNA2 S379A EBV week 2 - 5 : n = 7 ; EBNA2 S457A T465V EBV week 2 : n = 9 , week 3 : n = 8 , week 4 week 5 : n =

O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(E) Development of viral loads in blood of infected mice over a period of five weeks. Error bars indicate mean ± SEM. Number of biological replicates per group: EBNA2 WT EBV week 2 - 5: n = 8; EBNA2 S379A EBV week 2 - 5: n = 7; EBNA2 S457A T465V EBV week 2: n = 9, week 3: n = 8, week 4 and week 5: n = 7


( F ) Flow - cytometric analyses of CD8 + / CD4 + T ratios in blood of infected mice over a period of five . : EBNA2 WT EBV week 0 , 2 , 4 , 5 : n = 13 , week 3 : n = 8 ; EBNA2 S379A week 0 , 2 , 4 , 5 : n = 11 , week 3 : n = 7 ; EBNA2 S457A T465V week 0 , 2 : n = 12 , week 3 : n = 7 , week 4 : n = 10 , week 5 : n = 9 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O B-ORGANISM O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Flow-cytometric analyses of CD8+/CD4+ T cell ratios in the blood of infected mice over a period of five weeks. : EBNA2 WT EBV week 0, 2, 4, 5: n = 13, week 3: n = 8 ; EBNA2 S379A week 0, 2, 4, 5: n = 11, week 3: n = 7 ; EBNA2 S457A T465V week 0, 2: n = 12, week 3: n = 7, week 4: n = 10, week 5: n = 9.


Gel shift of biotin - labeled mascRNA 5S rRNA with cytosol ( Cyto ) postnuclear lysate ( Mem ) .

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O

Gel shift assay of biotin-labeled mascRNA and 5S rRNA with the cytosol (Cyto) and the postnuclear membrane lysate (Mem).


Northern blotting of mascRNA ( top panel ) western blotting of β - Tubulin Mortalin ( bottom two panels ) in fractions postnuclear fractions of HEK293 HepG2 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE O

Northern blotting of mascRNA (top panel) and western blotting of β-Tubulin and Mortalin (bottom two panels) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 cells.


SDS - PAGE coomassie of in pulldown samples . Biotin - labeled mascRNA ( S ) antisense ( AS ) were used to pull down - complexes . Representive data of mass spectrometry of pulldown samples . most enriched in mascRNA ( S ) pulldown sample are listed .

B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O

SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.   Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.


Western blots of in mascRNA S AS pulldown samples ( top 4 panels ) , mascRNA menRNA pulldown samples ( Bottom 2 panels ) . β - tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blots of the proteins in mascRNA S and AS pulldown samples (top 4 panels), and mascRNA and menRNA pulldown samples (Bottom 2 panels). β-tubulin was used as a loading control.


RT - PCR of RNAs extracted from ARS immunoprecipitation ( RIP ) samples ( erro bars : mean ± SD ; biological replicates n = 3 ) . Eight - forming ARSs ( DARS , EPRS , IARS , KARS , LARS , MARS , QARS RARS ) GARS tagged with FLAG were overexpressed in HEK . ARS - complexes were imminoprecipitated with anti - FLAG Magnetic Beads . constructed with PQCXIP vector ( PQ ) was used as a negative control . GAPDH was used as an internal control for qRT - PCR .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O B-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

RT-PCR analysis of RNAs extracted from ARS immunoprecipitation (RIP) samples (erro bars: mean ± SD; biological replicates n = 3). Eight complex-forming ARSs (DARS, EPRS, IARS, KARS, LARS, MARS, QARS and RARS) and GARS tagged with FLAG were overexpressed in HEK cells. ARS-RNA complexes were imminoprecipitated with anti-FLAG Magnetic Beads. The cell line constructed with PQCXIP vector (PQ) was used as a negative control. GAPDH was used as an internal control for qRT-PCR.


Northern blot validation of mascRNA in RIP samples . 5S rRNA from RIP samples was used as an internal control .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O

Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control.


Northern blot ( top 2 panels ) western blot ( bottom 3 panels ) of ARS immunoprecipitation samples ( RIP ) . RIP was performed using EPRS , QARS GARS antibodies .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O

Northern blot (top 2 panels) and western blot (bottom 3 panels) analysis of ARS immunoprecipitation samples (RIP). RIP was performed using EPRS, QARS and GARS antibodies.


Gel shift of biotin - labeled mascRNA with cytosol in presence or absence of a QARS antibody .

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O

Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.


Biotin - labeled mascRNA ( sense ( S ) antisense ( AS ) ) acceptor mutants ( Mut1 - 8 ) were used to pulldown - complexes . Top panel : western blotting of QARS in pulldown samples . β - Tubulin was used as an input control for pulldown . Bottom panel : Biotin detection of biotin - labeled mascRNA mutants . Biotin - labeled mascRNA ( sense ( S ) antisense ( AS ) ) anticodon - loop mutants ( Anti - Mut1 - 9 ) were used to pulldown - complexes . Top panel : western blotting of QARS in pulldown samples . β - Tubulin was used as an input control for pulldown . Bottom panel : Biotin detection of biotin - labeled mascRNA mutants .

B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD 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-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O

Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the acceptor stem mutants (Mut1-8) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.   Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the anticodon stem-loop mutants (Anti-Mut1-9) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.


Gel shift of biotin - labeled mascRNA or mutants ( Anti - Mut1 Mut1 ) with cytosol .

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O

Gel shift assay of biotin-labeled mascRNA or the mutants (Anti-Mut1 and Mut1) with the cytosol.


Northern blot of mascRNA in mascRNA overexpressing HEK ( mascRNA ) expressing scrambled ( Scramble ) . 5S rRNA was used as an internal control .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O

Northern blot analysis of mascRNA in mascRNA overexpressing HEK cells (mascRNA) and the cells expressing the scrambled RNA (Scramble). 5S rRNA was used as an internal control.


qRT - PCR of MALAT1 ( total RNAs used as templates ) in mascRNA overexpressing ( mascRNA ) scrambled expressing ( Scramble ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

qRT-PCR analysis of MALAT1 (total RNAs used as templates) in mascRNA overexpressing (mascRNA) and the scrambled RNA expressing (Scramble) cells.


Detection of newly synthesized in HEK stably expressing mascRNA ( mascRNA ) or scrambled ( Scramble ) . Left : biotin detection of labeled nascent ; right : coomassie of total . of of labeled nascent in Panel C .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel C .


Northern blot of mascRNA in HEK transiently transfected with in vitro synthesized eGFP fragment or mascRNA . 5S rRNA was used as an internal control .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O

Northern blot analysis of mascRNA in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. 5S rRNA was used as an internal control.


Detection of newly synthesized in HEK transiently transfected with in vitro synthesized eGFP fragment or mascRNA . Left : biotin detection of labeled nascent ; right : coomassie of total . of of labeled nascent in Panel F .

O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

Detection of the newly synthesized proteins in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel F.


Northern blot of mascRNA in mascRNA overexpressing ( mascRNA ) , mutants ( Anti - Mut1 Mut1 ) or scrambled ( Scramble ) expressing HEK . 5S rRNA was used as an internal control .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD I-GENEPROD O O O O O O O

Northern blot analysis of mascRNA in mascRNA overexpressing (mascRNA), and the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble) expressing HEK cells. 5S rRNA was used as an internal control.


Detection of newly synthesized in HEK stably expressing mascRNA ( mascRNA ) , mutants ( Anti - Mut1 Mut1 ) or scrambled ( Scramble ) . Left : biotin detection of labeled nascent ; right : coomassie of total . of of labeled nascent in Panel I .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel I.


Decay of Biotin - labeled in HEK stably expressing mascRNA ( mascRNA ) or scrambed ( Scramble )

B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O

Decay of the pulse Biotin-labeled proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambed RNA (Scramble)


Western blot detection of ARS in mascRNA overexpressing ( mascRNA ) or scrambled expressing ( Scramble ) . β - Tubulin was used as a loading control . of in Panel A .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells. β-Tubulin was used as a loading control.   Quantification of the protein levels in Panel A.


Western blot detection of ARS in HEK stably expressing mascRNA ( mascRNA ) , mutants ( Anti - Mut1 Mut1 ) or scrambled ( Scramble ) . β - Tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control.


Western blot detection of ARS in HEK stably expressing mascRNA ( mascRNA ) , mutants ( Anti - Mut1 Mut1 ) or scrambled ( Scramble ) . β - Tubulin was used as a loading control . of in Panel C .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control. Quantification of the protein levels in Panel C.


qRT - PCR of MALAT1 in MALAT1 knockdown control .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O

qRT-PCR analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells.


Northern blot of mascRNA in MALAT1 knockdown control . 5S rRNA was used as an internal control .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O

Northern blot analysis of mascRNA in MALAT1 knockdown and the control cells. 5S rRNA was used as an internal control.


Western blot detection of ARS in MALAT1 knockdown control . of in Panel G .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O

Western blot detection of ARS proteins in MALAT1 knockdown and the control cells.   Quantification of the protein levels in Panel G.


qRT - PCR of GARS QARS mRNAs in mascRNA overexpressing ( mascRNA ) or scrambled expressing ( Scramble ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O

qRT-PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells.


Western blot detection of ARS in mascRNA overexpressing ( mascRNA ) or scrambled expressing ( Scramble ) , before after emetine cycloheximide treatment . β - Tubulin was used as a loading control . of relative QARS in Panel J . of relative GARS in Panel J .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells, before and after emetine and cycloheximide treatment. β-Tubulin was used as a loading control.   Quantification of the relative QARS protein levels in Panel J.   Quantification of the relative GARS protein levels in Panel J.


Western blot detection of endogenous FLAG - tagged ARS in HEK transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blot detection of endogenous and FLAG-tagged ARS proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of newly synthesized in HEK transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of labeled nascent ; right , coomassie of total . of labeled nascent in Panel B .

O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Detection of the newly synthesized proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel B.


Western blot detection of FLAG - tagged ARS in mascRNA overexpressing transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blot detection of FLAG-tagged ARS proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of newly synthesized in mascRNA overexpressing transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of labeled nascent ; right , coomassie of total . of labeled nascent in Panel E .

O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Detection of the newly synthesized proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel E.


Western blot detection of FLAG - tagged ARS in mascRNA anticodon - loop mutant Anti - Mut1 expressing transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blot detection of FLAG-tagged ARS proteins in mascRNA anticodon stem-loop mutant Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of newly synthesized in mascRNA Anti - Mut1 expressing transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of labeled nascent ; right , coomassie of total .

O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O

Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.


Detection of newly synthesized in mascRNA Anti - Mut1 expressing transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of labeled nascent ; right , coomassie of total . of labeled nascent in Panel H .

O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel H.


Western blot detection of FLAG - tagged ARS in MALAT1 knockdown with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . β - Tubulin was used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Western blot detection of FLAG-tagged ARS proteins in MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). β-Tubulin was used as a loading control.


Detection of newly synthesized in wild - type HEK ( WT ) MALAT1 knockdown ( sh - MALAT1 ) , or MALAT1 knockdown with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . Left , biotin detection of labeled nascent ; right , coomassie of total .

O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O

Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.


Detection of newly synthesized in wild - type HEK ( WT ) MALAT1 knockdown ( sh - MALAT1 ) , or MALAT1 knockdown with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . Left , biotin detection of labeled nascent ; right , coomassie of total . of labeled nascent in Panel K .

O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel K.


Detection of newly synthesized in QARS ( shQARS ) or GARS ( shGARS ) knockdown control stably overexpressing mascRNA ( mascRNA ) or expressing scrambled ( Scramble ) . Left , biotin detection of labeled nascent ; right , coomassie of total . of labeled nascent in Panel M .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Detection of the newly synthesized proteins in QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the scrambled RNA (Scramble). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel M.


Northern blotting of mascRNA 5S rRNA in lysates of HEK before after FBS starvation .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O B-TISSUE O O

Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation.


Northern blotting of mascRNA 5S rRNA in lysates of human embryonic on different days of differentiation .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O

Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation.


Soft agar colony formation of HEK , HeLa or MCF - 7 overexpressing mascRNA or expressing scrambled . of stained colones in Panel C .

Soft agar colony formation assay of HEK, HeLa or MCF-7 cells overexpressing mascRNA or expressing the the scrambled RNA.   Quantification of stained colones in Panel C.


Soft agar colony formation of HEK overexpressing mascRNA , or expressing mutants ( Anti - Mut1 Mut1 ) or scrambled ( Scramble ) .

O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O

Soft agar colony formation assay of HEK cells overexpressing mascRNA, or expressing the mutants (Anti-Mut1 and Mut1) or the the scrambled RNA (Scramble).


Soft agar colony formation of QARS ( shQARS ) or GARS ( shGARS ) knockdown control stably overexpressing mascRNA ( mascRNA ) or expressing scrambled ( Scramble ) .

O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

Soft agar colony formation assay of QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the the scrambled RNA (Scramble).


Soft agar colony formation of MALAT1 knockdown ( shMALAT1 ) control ( Control ) with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) .

O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O

Soft agar colony formation assay of MALAT1 knockdown cells (shMALAT1) and the control cells (Control) with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ).


Immunoblot detection its of PDGFR - β in kidney cortex lysates of healthy diseased human kidneys shows twice as much PDGFR - β abundance in diseased tissue . A stronger signal for phosphorylated PDGFR - β indicates active status of receptor . high α - SMA content in diseased kidneys shows that they are affected by fibrosis . Bar graphs show means ± SD , healthy n = 5 , diseased n =

B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O B-DISEASE O O O O O O O O O O O O O O O O

Immunoblot detection and its quantification of PDGFR-β in kidney cortex lysates of healthy and diseased human kidneys shows twice as much PDGFR-β abundance in diseased tissue. A stronger signal for phosphorylated PDGFR-β indicates the active status of the receptor. The high α-SMA content in the diseased kidneys shows that they are affected by fibrosis. Bar graphs show means ± SD, healthy n = 5, diseased n = 6


PDGFR - β is detectable in mesangium of healthy ( a ) diseased ( b - d ) glomeruli of human kidneys . PDGFR - β positive mesangial expand in mesangioproliferating scarring glomerular changes . Similarly , population of PDGFR - β positive interstitial is discrete in healthy kidney cortex ( e ) but expands in fibrosis ( f - h ) . Arrows point to selected areas of positive PDGFR - β . Scale bar = 50

B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O B-TISSUE O B-ORGANISM B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O O O O O B-DISEASE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

PDGFR-β staining is detectable in mesangium of healthy (a) and diseased (b-d) glomeruli of human kidneys. PDGFR-β positive mesangial cells expand in mesangioproliferating and scarring glomerular changes. Similarly, the population of PDGFR-β positive interstitial cells is discrete in healthy kidney cortex (e) but expands in fibrosis (f-h). Arrows point to selected areas of positive PDGFR-β staining. Scale bar = 50 µm


GFP expression under Pdgfrb - promoter in mice is present in mesangial of glomeruli interstitial of cortex medulla , but not in tubular . Nuclei are stained with DAPI ( blue ) . Scale bar = 50

B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE O B-TISSUE B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O

GFP expression under Pdgfrb-promoter in mice is present in mesangial cells of glomeruli and interstitial cells of cortex and medulla, but not in tubular cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm


Tissue clearing with Scale 3D reconstruction of Pdgfrb - GFP reporter mice in a healthy fibrotic kidney ( UUO day 5 ) shows expansion of Pdgfrb expressing in fibrosis .

O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-DISEASE I-DISEASE O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O

Tissue clearing with Scale and 3D reconstruction of Pdgfrb-GFP reporter mice in a healthy and fibrotic kidney (UUO day 5) shows the expansion of Pdgfrb expressing cells in fibrosis.


Representative Ki67 immunofluorescence ( green ) in wt Foxd1Cre : : Pdgfrb + / J mice , showing increased proliferation in transgenic mice . Glomeruli are outlined with circles , arrowheads point to Ki67 positive interstitial . Nuclei are stained with DAPI ( blue ) . Scale bar = 50 µm .

O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-EXP_ASSAY O O B-ORGANISM O B-TISSUE O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

Representative Ki67 immunofluorescence staining (green) in wt and Foxd1Cre::Pdgfrb+/J mice, showing increased proliferation in the transgenic mice. Glomeruli are outlined with circles, arrowheads point to Ki67 positive interstitial cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm.


of proliferating Ki67 - positive specifically in glomeruli ( C ) , interstitium ( D ) tubules ( E ) in Foxd1Cre : : Pdgfrb + / J mice ( black bars ) wt mice ( bars ) of 6 , 14 , 25 35 of . Foxd1Cre : : Pdgfrb + / J mice exhibited increased proliferation of glomerular interstitial , whereas tubular epithelial proliferation was not altered . Data in C , D E are shown as means ± SD of n = 3 per group .

O B-EXP_ASSAY B-GENEPROD O O O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

Quantification of proliferating Ki67-positive cells specifically in glomeruli (C), interstitium (D) and tubules (E) in Foxd1Cre::Pdgfrb+/J mice (black bars) and wt mice (white bars) of 6, 14, 25 and 35 weeks of age. Foxd1Cre::Pdgfrb+/J mice exhibited increased proliferation of glomerular and interstitial cells, whereas tubular epithelial cell proliferation was not altered. Data in C, D and E are shown as means ± SD of n = 3 animals per group.


FoxD1 - reporter mice ( Foxd1 : : tdTomato ) confirmed expansion of mesenchymal in 14 old Foxd1Cre : : Pdgfrb + / J mice both in glomeruli interstitium . Circles outline glomeruli . Scale bar = 50 µm . of FoxD1 - Tomato positive in cortical interstitium in 14 old Foxd1Cre : : Pdgfrb + / J wt mice confirmed significantly increased expansion of mesenchymal by 42 % in Foxd1Cre : : Pdgfrb + / J mice . were counted in six viewfields at 40x magnification . Bar graphs show means ± SD of n = 3 mice per group .

B-GENEPROD O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O

FoxD1-reporter mice (Foxd1::tdTomato) confirmed the expansion of mesenchymal cells in 14 weeks old Foxd1Cre::Pdgfrb+/J mice both in glomeruli and interstitium. Circles outline glomeruli. Scale bar = 50 µm. Quantification of FoxD1-Tomato positive cells in the cortical interstitium in 14 weeks old Foxd1Cre::Pdgfrb+/J and wt mice confirmed the significantly increased expansion of mesenchymal cells by 42% in Foxd1Cre::Pdgfrb+/J mice. Cells were counted in six viewfields at 40x magnification. Bar graphs show means ± SD of n = 3 mice per group.


Isolated fibroblasts mesangial from Foxd1Cre : : Pdgfrb + / J mice have higher proliferation rates assessed by bromodeoxyuridine ( BrdU ) incorporation compared to from wt mice . Bar graphs show means ± SD , n = 4 per group , * : p ≤ 0 . 05 compared to wt of same time point .

O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Isolated primary fibroblasts and mesangial cells from Foxd1Cre::Pdgfrb+/J mice have higher proliferation rates assessed by bromodeoxyuridine (BrdU) incorporation assay compared to cells from wt mice. Bar graphs show means ± SD, n = 4 per group, *: p≤0.05 compared to wt of same time point.


PAS of glomeruli of 25 old wt of 6 35 old Foxd1Cre : : Pdgfrb + / J mice shows pathological changes of glomeruli in Foxd1Cre : : Pdgfrb + / J mice . Glomerular tuft of Foxd1Cre : : Pdgfrb + / J mice was significantly increased compared to wt mice during time course .

B-SMALL_MOLECULE O B-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O B-TISSUE O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O

PAS staining of glomeruli of 25 weeks old wt and of 6 and 35 weeks old Foxd1Cre::Pdgfrb+/J mice shows pathological changes of the glomeruli in Foxd1Cre::Pdgfrb+/J mice. Glomerular tuft size of Foxd1Cre::Pdgfrb+/J mice was significantly increased compared to wt mice during the time course.


Immunhistological of α - SMA its histomorphometric ( D ) reveals expansion of activated mesangial in glomerular tuft of in transgenic mice in time course .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O

Immunhistological staining of α-SMA and its histomorphometric quantification (D) reveals expansion of activated mesangial cells in the glomerular tuft of in the transgenic mice in the time course.


Immunhistological of collagen I its histomorphometric ( F ) shows that collagen I deposits in an increasing manner in Foxd1Cre : : Pdgfrb + / J mice during time course .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O

Immunhistological staining of collagen I and its histomorphometric quantification (F) shows that collagen I deposits in an increasing manner in Foxd1Cre::Pdgfrb+/J mice during the time course.


Histological stainings its histomorphometric ( B ) of kidney cortex for α - SMA of wt ( 25 ) Foxd1Cre : : Pdgfrb + / J mice of an of 6 35 show expansion of myofibroblasts in interstitium of transgenic mice .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-CELL_TYPE O B-TISSUE O O B-ORGANISM O

Histological stainings and its histomorphometric quantification (B) of kidney cortex for α-SMA of wt (25 weeks) and Foxd1Cre::Pdgfrb+/J mice of an age of 6 and 35 weeks show expansion of myofibroblasts in the interstitium of the transgenic mice.


Collagen I its histomorphometric ( D ) show progression of interstitial fibrosis in time course .

B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O B-DISEASE O O O O

Collagen I staining and its histomorphometric quantification (D) show the progression of interstitial fibrosis in the time course.


tubular stress marker Lipocalin - 2 ( LCN2 ) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent , as seen on histological

B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O B-DISEASE O O O O O O O B-EXP_ASSAY

The tubular stress marker Lipocalin-2 (LCN2) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent, as seen on histological staining


tubular stress marker Lipocalin - 2 ( LCN2 ) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent mRNA

B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O B-DISEASE O O O B-EXP_ASSAY

The tubular stress marker Lipocalin-2 (LCN2) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent and mRNA level


A slight , but significant increase in interstitial ErHr3 positive stained macrophages was observed , particularly at later stages ( G ) . For evaluation , ErHr3 positive were counted on 20 view fields on 40x magnification ( H ) .

O O O O O O O B-TISSUE O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

A slight, but significant increase in interstitial ErHr3 positive stained macrophages was observed, particularly at later stages (G). For evaluation, ErHr3 positive cells were counted on 20 view fields on 40x magnification (H).


Creatinine clearance was measured in mice of an of 10 , 14 , 25 35 decreased continuously in Foxd1Cre : : Pdgfrb + / J mice , with a predicted complete loss of renal function 62 in transgenic mice using linear regression analyses as described previously ( Steiger et al , 2018 ) . 10 n = 5 wt n = 4 Foxd1Cre : : Pdgfrb + / J , 14 n = 2 per group , 25 n = 8 wt n = 8 Foxd1Cre : : Pdgfrb + / J , 35 n = 3 per group

B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

Creatinine clearance was measured in mice of an age of 10, 14, 25 and 35 weeks and decreased continuously in Foxd1Cre::Pdgfrb+/J mice, with a predicted complete loss of renal function 62 weeks in transgenic mice using linear regression analyses as described previously (Steiger et al, 2018). 10 weeks n = 5 wt and n = 4 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 2 per group, 25 weeks n = 8 wt and n = 8 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group


blood urea nitrogen ( BUN ) concentrations were higher in Foxd1Cre : : Pdgfrb + / J mice compared to wt mice , apart from 25 old group . Bar graphs show means ± SD , 10 n = 7 wt n = 5 Foxd1Cre : : Pdgfrb + / J , 14 n = 3 per group , 25 n = 11 wt n = 12 Foxd1Cre : : Pdgfrb + / J , 35 n = 3 per

B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O

The blood urea nitrogen (BUN) concentrations were higher in Foxd1Cre::Pdgfrb+/J mice compared to wt mice, apart from 25 weeks old group. Bar graphs show means ± SD, 10 weeks n = 7 wt and n = 5 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 3 per group, 25 weeks n = 11 wt and n = 12 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group


Erythrocyte numbers in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point .

B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Erythrocyte numbers in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.


hematocrit in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point .

B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hematocrit in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.


hemoglobin content ( C ) decrease in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point .

B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hemoglobin content (C) decrease in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.


EPO shown as delta from respective wt . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point .

B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EPO levels shown as delta from respective wt littermates. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.


in - situ hybridization ( RNAScope ) of Epo ( green ) Pdgfrb ( purple ) in renal kidney cortex of 35 week old mice shows that EPO - producing co - express Pdgfrb . Foxd1Cre : : Pdgfrb + / J mice have less EPO - producing ( arrows ) than wt mice .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O B-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O O O B-ORGANISM O

RNA in-situ hybridization (RNAScope) of Epo (green) and Pdgfrb (purple) in renal kidney cortex of 35 week old mice shows that EPO-producing cells co-express Pdgfrb. Foxd1Cre::Pdgfrb+/J mice have less EPO-producing cells (arrows) than wt mice.


After induction of anemia by blood taking , erythrocyte numbers recovered after one week in wt mice , whereas Foxd1Cre : : Pdgfrb + / J mice were not able to recover their erythrocytes numbers at . Data are means ± SD of n = 5 per group .

O O O B-DISEASE O B-TISSUE O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-ORGANISM O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O

After induction of anemia by blood taking, the erythrocyte numbers recovered after one week in wt mice, whereas Foxd1Cre::Pdgfrb+/J mice were not able to recover their erythrocytes numbers at all. Data are means ± SD of n = 5 per group.


Five days after induction of unilateral ureteral obstruction ( UUO ) , a model of obstructive nephropathy interstitial fibrosis , we assessed fibrosis ( collagen I , α - SMA ) inflammation ( F4 / 80 ) , classical read - out parameters in this model . Compared to wt mice with UUO , Foxd1Cre : : Pdgfrb + / J mice developed significantly stronger fibrosis inflammation ( A ) . Representative pictures of histological stainings are shown in ( B ) . Scale bar = 50 µm data in A B show means ± SD , wt n = 4 , Foxd1Cre : : Pdgfrb + / J n = 3 . * : p ≤ 0 . 05 compared to

O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O B-DISEASE O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-DISEASE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE I-DISEASE O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O

Five days after the induction of unilateral ureteral obstruction (UUO), a model of obstructive nephropathy and interstitial fibrosis, we assessed fibrosis (collagen I, α-SMA) and inflammation (F4/80), the classical read-out parameters in this model. Compared to wt mice with UUO, Foxd1Cre::Pdgfrb+/J mice developed significantly stronger fibrosis and inflammation (A). Representative pictures of the histological stainings are shown in (B). Scale bar = 50 µm All data in A and B show means ±SD, wt n = 4, Foxd1Cre::Pdgfrb+/J n = 3. *: p≤0.05 compared to wt


Angiotensin II infusion via an osmotic pump induced hypertension similarly in wt Foxd1Cre : : Pdgfrb + / J mice ( C ) .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-DISEASE O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O

Angiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre::Pdgfrb+/J mice (C).


In this model of hypertensive injury , kidney function even histopathology was largely unaffected in wt mice , whereas Foxd1Cre : : Pdgfrb + / J mice showed a significantly increased BUN values prominent glomerulopathy after 28 days compared to starting values ( day 0 ) , to wt after 28 days to - matched Foxd1Cre : : Pdgfrb + / J mice without Angiotensin II infusion ( D ) .

O O O O B-DISEASE O O B-TISSUE O O B-EXP_ASSAY O O O O O B-ORGANISM O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

In this model of hypertensive injury, the kidney function and even the histopathology was largely unaffected in wt mice, whereas the Foxd1Cre::Pdgfrb+/J mice showed a significantly increased BUN values and prominent glomerulopathy after 28 days compared to the starting values (day 0), to wt animals after 28 days and to age-matched Foxd1Cre::Pdgfrb+/J mice without Angiotensin II infusion (D).


Consistently creatinine clearance per g bodyweight decreased in hypertensive Foxd1Cre : : Pdgfrb + / J mice ( E ) .

O B-SMALL_MOLECULE O O O O O O B-DISEASE B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O

Consistently the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre::Pdgfrb+/J mice (E).


PAS showed more prominent mesangial expansion intratubular casts indicative of proteinuria only in hypertensive Foxd1Cre : : Pdgfrb + / J mice .

B-SMALL_MOLECULE O O O O O O O O O B-DISEASE O O B-DISEASE B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O

PAS staining showed more prominent mesangial expansion and intratubular protein casts indicative of proteinuria only in hypertensive Foxd1Cre::Pdgfrb+/J mice.


Hypertension did not induce mesangial expansion activation in wt mice , but led to a significant increase in hypertensive Foxd1Cre : : Pdgfrb + / J mice as visualized by α - SMA . ( H ) Histomorphometric of α - SMA positive of glomerular tuft .

B-DISEASE O O O O O O O O B-ORGANISM O O O O O O O O B-DISEASE B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O

Hypertension did not induce mesangial expansion and activation in wt mice, but led to a significant increase in hypertensive Foxd1Cre::Pdgfrb+/J mice as visualized by α-SMA staining. (H) Histomorphometric analysis of α-SMA positive percentage of the glomerular tuft area.


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Histomorphometric quantifications showed a significant reduction of α - SMA abundance in glomerular tufts of imatinib treated Foxd1Cre : : Pdgfrb + / J mice .

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications showed a significant reduction of α-SMA abundance in the glomerular tufts of imatinib treated Foxd1Cre::Pdgfrb+/J mice.


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Glomerular cellularity evaluated by counting total number of per glomerular tuft , normalized to , were significantly reduced in imatinib treated Foxd1Cre : : Pdgfrb + / J mice compared to control mice .

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Glomerular cellularity evaluated by counting the total number of cells per glomerular tuft, normalized to the area, were significantly reduced in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to control mice.


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in glomeruli ( K ) , whereas reduced interstitial fibrosis in imatinib treated Foxd1Cre : : Pdgfrb + / J mice compared to controls was found ( L ) .

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O B-DISEASE I-DISEASE O B-TISSUE O O O O O O B-DISEASE I-DISEASE O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in the glomeruli (K), whereas reduced interstitial fibrosis in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to controls was found (L).


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Hemoglobin serum were significantly improved in imatinib treated mice .

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE B-TISSUE O O O O B-SMALL_MOLECULE O B-ORGANISM O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Hemoglobin serum levels were significantly improved in imatinib treated mice.


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Representative pictures of histological stainings of α - SMA ( left ) collagen I ( middle : glomeruli , right : cortical interstitium ) . Scale bar = 50

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Representative pictures of the histological stainings of α-SMA (left) and collagen I (middle: glomeruli, right: cortical interstitium). Scale bar = 50 µm


22 old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Total PDGFR - β were decreased in Foxd1Cre : : Pdgfrb + / J mice after imatinib treatment , as shown using western blot ( O ) its densitometric evaluation ( P ) .

O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Total PDGFR-β protein levels were decreased in Foxd1Cre::Pdgfrb+/J mice after imatinib treatment, as shown using western blot (O) and its densitometric evaluation (P).


Gene expression arrays were performed in 6 week old Foxd1Cre : : Pdgfrb + / J mice compared to wt mice . Listed are genes with log2 fold changes higher or lower than 0 . 6 . Out of these 46 genes , 17 are IFN - regulated genes , 5 are involved in autophagy 5 in matrix remodeling fibrosis . Same genes were analysed in microdissected glomeruli ( G ) tubulointerstitium ( T ) of with diabetic ( DN , G : n = 14 , T : n = 18 ) , hypertensive ( HT , G : n = 15 , T : n = 21 ) nephropathy living donors ( LD , G : n = 42 , T : n = 42 ) as controls , revealing a similar expression profile .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O 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-DISEASE O O O O O O O B-TISSUE O O O B-TISSUE O O O O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

Gene expression arrays were performed in 6 week old Foxd1Cre::Pdgfrb+/J mice compared to wt mice. Listed are genes with log2 fold changes higher or lower than 0.6. Out of these 46 genes, 17 are IFN-regulated genes, 5 are involved in autophagy and 5 in matrix remodeling and fibrosis. Same genes were analysed in microdissected glomeruli (G) and tubulointerstitium (T) of patients with diabetic (DN, G: n=14, T: n=18), hypertensive (HT, G: n=15, T: n=21) nephropathy and living donors (LD, G: n=42, T: n=42) as controls, revealing a similar expression profile.


depicting pathway activities in Foxd1Cre : : Pdgfrb + / J mice human CKD according to PROGENy .

O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM I-ORGANISM B-DISEASE O O O O

Heatmap depicting pathway activities in Foxd1Cre::Pdgfrb+/J mice and human CKD patients according to PROGENy.


on gene array datasets transcription factor activities were estimated by using DoRothEA . Depicted are most regulated ones regulated both in Foxd1Cre : : Pdgfrb + / J mice in human CKD .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM B-DISEASE O

Based on the gene array datasets transcription factor activities were estimated by using DoRothEA. Depicted are the most regulated ones regulated both in Foxd1Cre::Pdgfrb+/J mice and in human CKD patients.


( b ) RT - PCR using blue primer set ( left ) green primer set ( right ) from a . Whereas control cDNA samples showed a single product corresponding to wild - type allele ( WT ) , an apparently longer product was observed in subjects 1 , 2 5 , indicating that only transcripts from mutant allele were expressed . In subject 3 , both wild - type mutant alleles were expressed . Template without reverse transcriptase was used as a negative control , RT ( - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-ORGANISM O O O 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) RT-PCR analysis using the blue primer set (left) and green primer set (right) from a. Whereas control cDNA samples showed a single product corresponding to the wild-type allele (WT), an apparently longer product was observed in subjects 1, 2 and 5, indicating that only the transcripts from the mutant allele were expressed. In subject 3, both wild-type and mutant alleles were expressed. Template without reverse transcriptase was used as a negative control, RT(-).


( a - e ) T1 - weighted imaging shows hyperintensity of substantia nigra with a central band of T1 - weighted hypointensity ( arrowheads ) . are shown for subject 1 at 33 years ( a ) , subject 2 at 25 years ( b ) , subject 3 at 39 years ( c ) , subject 4 at 46 years ( d ) subject 5 at 33 years ( e ) . ( f - h ) T2 - weighted imaging shows marked hypointensity of globus pallidus ( arrows ) , suggesting iron deposition . Cerebral atrophy mild cerebellar atrophy are also seen . are shown for subject 1 ( f ) , subject 2 ( g ) subject 3 ( h ) . ( i , j ) fluid attenuated inversion recovery ( FLAIR ) image of subject 1 ( i ) T1 - weighted FLAIR coronal image of subject 2 ( j ) also show cerebral atrophy .

O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O

(a-e) T1-weighted imaging shows hyperintensity of the substantia nigra with a central band of T1-weighted hypointensity (arrowheads). Images are shown for subject 1 at 33 years (a), subject 2 at 25 years (b), subject 3 at 39 years (c), subject 4 at 46 years (d) and subject 5 at 33 years (e). (f-h) T2-weighted imaging shows marked hypointensity of the globus pallidus (arrows), suggesting iron deposition. Cerebral atrophy and mild cerebellar atrophy are also seen. Images are shown for subject 1 (f), subject 2 (g) and subject 3 (h). (i,j) The fluid attenuated inversion recovery (FLAIR) image of subject 1 (i) and the T1-weighted FLAIR coronal image of subject 2 (j) also show cerebral atrophy.


( a ) Immunoblot of WIPI4 ( apparent mobility at ∼ 35 kDa ) in LCLs ( top ) . Truncated forms were not detected ( middle ) . HSP90 was used as a loading control ( bottom ) . An asterisk indicates nonspecific immunoreactive bands .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands.


( b ) were treated with 250 nM Torin1 in presence or absence of 20 μM chloroquine for 2 h . lysates were analyzed by SDS - PAGE immunoblotting using antibodies to LC3 , WIPI4 HSP90 . positions of LC3 - I ( ) LC3 - II ( bound ) are indicated .

O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

(b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated.


( c - e ) were cultured in presence of Torin1 for 2 h . ( c ) Cytospun were fixed analyzed by immunofluorescence microscopy using antibodies to LC3 ATG9A . Abnormal colocalization of LC3 with ATG9A was observed in LCLs of affected subjects . Scale bars , 10 μm 1 μm in inset . ( d , e ) numbers of LC3 + ( d ) LC3 + ATG9A + ( e ) foci were quantified from more than 20 from 3 independent samples ( Online Methods ) . Data are presented as mean ± s . e . m . * P 0 . 05 , ANOVA followed by Bonferroni - Dunn post - hoc .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c-e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.


( A ) Crystal structure of a dynamin monomer ( PDB ID : 3SNH ) corresponding color - coded linear domain architecture of dynamin . PHD is shown in both closed ( 3SNH , solution structure ) open ( 3ZYS , cryo - EM reconstruction of - bound dynamin ) states . approximate locations of terminal residues flanking different functional domains are identified with black arrowheads . positions of L1SP L2SP loops are identified by red arrowheads .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O 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) Crystal structure of a dynamin monomer (PDB ID: 3SNH) and corresponding color-coded linear domain architecture of dynamin. The PHD is shown in both the closed (3SNH, solution structure) and open (3ZYS, cryo-EM reconstruction of membrane-bound dynamin) states. The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads. The positions of the L1SP and L2SP loops are identified by red arrowheads.


Changes in HDX behavior for dynamin analyzed ( B ) in solution presence GMPPCP , ( C ) in solution in presence of GDP • AlF4 - , ( D ) - bound , or ( E ) - bound in presence of GMPPCP mapped onto structures of dynamin monomers . Color coding ( see scale below ) shows extent of solvent exchange relative to apo dynamin in solution . HDX MS data shown represents an average of 3 replicates are color coded from red to blue with warm colors representing increased conformational dynamics ( red being highest D2O uptake ) cool colors representing decreased conformational dynamics ( blue being lowest D2O uptake ) . Grey indicates no statistically significant change between two states . Quantitative data on peptides used to generate this map are provided in Appendix ( Figure S1 - S4 ) .

O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE B-EXP_ASSAY O O 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-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Changes in HDX behavior for dynamin analyzed (B) in solution the presence GMPPCP, (C) in solution in the presence of GDP•AlF4-, (D) membrane-bound, or (E) membrane-bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers. Color coding (see scale below) shows the extent of solvent exchange relative to apo dynamin in solution. All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics (red being the highest D2O uptake) and cool colors representing decreased conformational dynamics (blue being the lowest D2O uptake). Grey indicates no statistically significant change between the two states. Quantitative data on the individual peptides used to generate this map are provided in Appendix (Figure S1-S4).


( A ) Changes in solvent exchange superimposed onto structure ( PDB ID : 2SNH ) of G domain or ( B ) stalk / PHD of dynamin in GMPPCP or lipid bound states , respectively . Key structural elements are identified color - coding shows magnitude of solvent exchange relative to apo state ( see scale below ) .

O O O 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-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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) Changes in solvent exchange superimposed onto the structure (PDB ID: 2SNH) of the G domain or (B) the stalk/PHD of dynamin in the GMPPCP or lipid bound states, respectively. Key structural elements are identified and the color-coding shows the magnitude of solvent exchange relative to the apo state (see scale below).


( C ) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP GDP • AlF4 - - bound states . peptides are mapped to secondary structure elements on polypeptide . Differences in kinetics of solvent exchange of select peptides measured ( D ) for dynamin bound to GMPPCP in presence or absence of lipid nanotubes ( E ) for dynamin bound to lipid nanotubes in presence or absence of GMPPCP . HDX data are presented as an average of three independent triplicates color coded as in Figure 1 .

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(C) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP•AlF4--bound states. The peptides are mapped to secondary structure elements on the polypeptide sequence. Differences in kinetics of solvent exchange of select peptides measured (D) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes (E) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP. HDX data are presented as an average of three independent triplicates and color coded as in Figure 1.


( A ) Design of a functional dynamin mutant to probe PHD stalk interactions . Minimal structure of PHD PHD - Stalk interface showing location of important residues discussed in this study . unresolved loop L1NS in stalk hosts IAEDANS label is sensitive to two tryptophans ( W525 W542 ) in PHD . between two introduced cysteines ( S357C native C602 ) used to construct a double cysteine Dyn1 is also shown .

O O O O O O O B-GENEPROD O O O O O O O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O

(A) Design of a functional dynamin mutant to probe PHD stalk interactions. Minimal structure of the PHD and the PHD-Stalk interface showing the location of important residues discussed in this study. The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans (W525 and W542) in the PHD. The distance between two introduced cysteines (S357C and native C602) used to construct a double cysteine Dyn1 is also shown.


( B ) Comparison of emission spectra of various IAEDANS - labeled Dyn1 RCL variants ( D352C , Y354C , S357C , I365C , H367C ) , upon Trp excitation at 295 nm .

O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

(B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm.


( C ) FRET transfer upon excitation is contributed by two neighboring Trps ( W525 W542 ) ; Substitution by Phe resulted in significant loss of FRET .

O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O

(C) FRET transfer upon excitation is contributed by two neighboring Trps (W525 and W542); Substitution by Phe resulted in significant loss of FRET.


( D ) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4 , 5P2 ( ex = 295 ) . arrow indicates addition of lipids .

O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O

(D) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4,5P2 (ex=295). The arrow indicates addition of lipids.


( B ) Histogram shows basal assembly - stimulated GTPase activities of non - crosslinked Dyn1CC vs crosslinked Dyn1Closed Dyn1WT . Inset shows crosslinking of PHD stalk domains in Dyn1Y354C / S607C ( Dyn1CC ) with bifunctional crosslinker , MTS - 2 - MTS , to yield Dyn1Closed , which migrates more slowly on SDS - PAGE , as well as liposome binding of Dyn1CC compared to Dyn1Closed ( Bound , B ; Unbound , UB ) .

O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

(B) Histogram shows basal and assembly-stimulated GTPase activities of non-crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT. Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C/S607C (Dyn1CC) with the bifunctional crosslinker, MTS-2-MTS, to yield Dyn1Closed, which migrates more slowly on SDS-PAGE, as well as liposome binding of Dyn1CC compared to Dyn1Closed (Bound, B; Unbound, UB).


( C ) Ability of Dyn1WT , Dyn1CC Dyn1Closed to catalyze fission vesicle release from SUPER templates .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O

(C) Ability of Dyn1WT, Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates.


( D ) exclusion chromatographic profiles of Dyn1CC , Dyn1Closed at room or after incubation for 10 min at 37 ° C .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

(D) Size exclusion chromatographic profiles of Dyn1CC, Dyn1Closed at room temperature or after incubation for 10 min at 37°C.


( E ) Oligomeric state of Dyn1ΔΔ at room .

(E) Oligomeric state of Dyn1ΔΔ at room temperature.


( F ) Negative - stain electron of Dyn1WT , Dyn1ΔΔ Dyn1Closed assembled onto lipid nanotubes . Scale bar = 200

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O

(F) Negative-stain electron micrographs of Dyn1WT, Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes. Scale bar = 200 nm


( G ) Curvature dependent liposome - stimulated GTPase activity of Dyn1WT Dyn1ΔΔ .

O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O

(G) Curvature dependent liposome-stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ.


( H ) Ability of Dyn1WT Dyn1ΔΔ to catalyze fission vesicle release from SUPER templates .

O O O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O

(H) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates.


( A ) dependence of basal lipid stimulated GTPase activity of Dyn1WT Dyn2S619L .

O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O

(A) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L.


( B ) sensitive fission activity of Dyn1WT , Dyn2WT Dyn2S619L measured using SUPER templates .

O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O

(B) Temperature sensitive membrane fission activity of Dyn1WT, Dyn2WT and Dyn2S619L measured using SUPER templates.


( C ) Detection of Dyn2 PHD - stalk interactions by FRET using IAEDANS - labeled Dyn2L354C .

O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O

(C) Detection of Dyn2 PHD-stalk interactions by FRET using IAEDANS-labeled Dyn2L354C.


( D ) Curvature dependent binding consequent opening of PHD in Dyn1Y354C - IAEDANS Dyn2L354C - IAEDANS .

O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE B-GENEPROD O B-SMALL_MOLECULE O

(D) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C-IAEDANS and Dyn2L354C-IAEDANS.


( E ) Differential dependence of PHD - stalk FRET for WT S619L mutant Dyn2IAEDANS . Data are presented as relative FRET distances assuming a single donor / acceptor pair .

O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O

(E) Differential temperature dependence of PHD-stalk FRET for WT and S619L mutant Dyn2IAEDANS. Data are presented as relative FRET distances assuming a single donor/acceptor pair.


( F ) Differential solvent exchange kinetics of Dyn1S619L at ambient compared to Dyn1WT under identical conditions . Yellow coloring indicates a significant increase in HDX . Quantitative data on peptides used to generate this map are provided in ( Appendix Fig . S5 ) .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(F) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions. Yellow coloring indicates a significant increase in HDX. Quantitative data on the individual peptides used to generate this map are provided in (Appendix Fig. S5).