Figure 2Figure 2 . Rpp30 is required for pre - tRNA processing . Whole fly RNA extracts were used to study tRNA processing by Northern Blot . Top box : the three different probes used are depicted : 5 ' and 3 ' probes ( red and green , respectively ) can detect full pre - tRNAs and tRNAs intermediates , but will not detect mature tRNAs . An Internal Probe ( IP , blue ) corresponding to the anti - codon region was used to detect all non - mature and mature tRNAs . The genotypes used in this experiment are numbered at the bottom ( 1 - 6 ) . Northern Blot panels : tRNA - his 5 ' probe ( top ) , tRNA - his 3 ' probe ( middle ) and tRNA - his IP ( bottom ) . U6 probe was used as loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.


Figure 3A . Rpp3018 . 2 homozygous early oogenesis arrest is partially rescued by p53 mutation . Scale bar , 100μm . B . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is rescued by chk2 mutation ( Rpp3018 . 2 , mnkp6 and Rpp3018 . 2 / Rpp30PE , mnkp6 ) . Scale bar , 100μm . Star : example of one rescued stage 9 egg chamber . Arrow : example of non - rescued egg chambers . C . Germline clones mutant for Rpp3018 . 2 were immunostained for PCNA ( red ) . DAPI is in blue . Magnifications : PCNA signal in a control or mutant nurse cell . Scale bar , 10μm . D . Germline clones mutant for Rpp3018 . 2 were immunostained for Brf ( pol - III ) ( red ) . DAPI is in blue . A Z - projection of Brf staining is shown . The arrow points Brf aggregates in a mutant chamber . Scale bar , 10μm . E . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is partially rescued by claspin mutation ( Rpp3018 . 2 ; claspinEP / TM3 ) . Scale bar , 100μm . G . Top panel . Early oogenesis arrest found in Rpp30182 . / PEtransheterozygous ovaries is rescued by sqd - S - HAoverexpression . Scale bars : 100µm . Low panel . Rpp3018 . 2 / PE ; sqd - S - HA / sqd - S - HA ovaries were dissected and stained for Orb ( green ) , HA ( squid , red ) and DAPI ( blue ) . Scale bar , 10µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,100μm.B. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers.C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm.D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm.E. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm.G. Top panel. Early oogenesis arrest found in Rpp30182./PEtransheterozygous ovaries is rescued by sqd-S-HAoverexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.


Figure 5 . D . Nuage specific markers ( Aub , Ago3 and Mael ) are shown in stage 3 wild - type and germline mutant clones for Rpp3018 . 2 . Scale bar , 10µm . E - H . Double mutant ovaries for Rpp30 and chk2 ( Rpp3018 . 2 , mnkp6 ) rescued the specific and general piRNA populations , nuage morphology and the characteristic ping - pong signal . Scale bar , 10µm .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.


Figure 6D . Testes from control heterozygous , transheterozygous Rpp3018 . 2 / Rpp30PE or aubHN / QC42 were fixed and stained for Stellate crystal formation . Z - projections are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.


Figure 1 ( A ) 3D time ‐ lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron . Neurons were transfected with Mito ‐ DsRed2 , pretreated with the pan ‐ caspase inhibitor zVAD ‐ fmk methyl ester ( 100 μM ) , and exposed to SNOC ( 200 μM ) . Images were 3D iso ‐ surface rendered . Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure ( upper panels ; scale bar , 15 μm ) and closeup views ( lower panels ; scale bar , 3 μm ) . See Supplementary Video 1 . ( B ) Mitochondrial fragmentation within the field of view shown in ( A ) results in increased mitochondrial numbers . ( C ) NO induces dose ‐ dependent mitochondrial fission . Neurons expressing Mito ‐ DsRed2 were exposed to SNOC and fixed at 60 min . The fraction of neurons displaying fragmented mitochondria is shown as the mean ± s . e . m . ( D ) Mito ‐ DsRed2 ‐ expressing corticalneurons were pretreated with 1 mM nitro ‐ L ‐ arginine ( 20 min ) and exposed to 25 μM NMDA . The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean ± s . e . m . of quintuplicate samples from a representative experiment ( † † † significance at P0 . 001 or n . s . † , not significant compared to control ; * * * significance at P0 . 001 as compared to NMDA treatment ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.(B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers.(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).


Figure 2Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria . Neurons were co ‐ transfected with vectors encoding ( A ) Mito ‐ DsRed2 and ( B ) MyrPalm ‐ mCFP plus ( C ) AIF ‐ GFP and the effects of SNOC were recorded using 3D time ‐ lapse imaging . Projection images zoomed in onto the dendritic arbor of one neuron ( representative of n = 42 ) . Arrows indicate dendritic spines . The inset shows the soma at appropriate grayscale levels . The apparent brightening of AIF ‐ GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF . Scale bars , 10 μm . See also Supplementary Video 2 . ( D ) 3D time ‐ lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition . Scale bar , 10 μm . Five mitochondria were selected and motion tracked throughout the imaging series . ( E ) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion . Each trace color corresponds to the same colored mitochondrion . ( representative of n = 9 ) ( F - G ) Immunocytochemistry for staining anti ‐ AIF antibodies ( red ) anti ‐ NeuN antibodies ( green ) and nuclei labeled with Hoechst 33342 dye ( blue ) after ( F ) aged SNOC or ( G ) SNOC ( 150 μM ; 4 h ) treatment . Volume rendered 3D reconstruction of confocal image stacks are shown above , and a single confocal plane in grayscale below . Grid , 3 . 3 μm for ( F ) and 1 . 9 μm for ( G ) , scale bars , 5 μm . ( H ) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body , outlined by the NeuN staining ( excluding the nucleus ) . The bar diagram summarizes the mean ± s . e . m . of 282 and 262 neurons measured in n = 19 and 20 image stacks in three independent experiments for aged SNOC and SNOC , respectively ( P ∼ 0 . 99 not significant by Student ' s t ‐ test ) . ( I ) DEVDase activity . Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine , as positive control . The rate of zDEVD ‐ AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units . Data show mean ± s . e . m . for four independent experiments ( * significance at P0 . 01 by ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.(E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9)(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).(I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).


Figure 3 ( A ) Elongated mitochondrion ( arrow ) in neuronal process of the unaffected hemisphere ( top ) . Small mitochondrial segments ( arrows ) are in close proximity to each other in a neuronal process of the ischemic brain region ( bottom ) . Images are representative of three analyzed mice . Scale bars , 500 nm . ( B ) Histogram comparing mitochondrial cross ‐ section length of contralateral , control ( red ) and ischemic ( black ) hemispheres . The lower bins of the histogram ( 2000 nm ) were compared with χ2 ‐ test and were not different . The table shows the presence of long mitochondrial cross ‐ sections ( > 2000 nm ) in the control hemisphere and their absence in the ischemic hemisphere ( P0 . 01 ; Fisher ' s exact test ) . ( C ) Proximities of mitochondria comparing the contralateral ( red ) and ischemic ( black ) hemispheres . Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm . The same data set is summarized as a contingency table on the right , ( P0 . 001 ; two ‐ way Fisher ' s exact test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Elongated mitochondrion (arrow) in neuronal process of the unaffected hemisphere (top). Small mitochondrial segments (arrows) are in close proximity to each other in a neuronal process of the ischemic brain region (bottom). Images are representative of three analyzed mice. Scale bars, 500 nm.(B) Histogram comparing mitochondrial cross‐section length of contralateral, control (red) and ischemic (black) hemispheres. The lower bins of the histogram (2000 nm) were compared with χ2‐test and were not different. The table shows the presence of long mitochondrial cross‐sections (>2000 nm) in the control hemisphere and their absence in the ischemic hemisphere (P0.01; Fisher's exact test).(C) Proximities of mitochondria comparing the contralateral (red) and ischemic (black) hemispheres. Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm. The same data set is summarized as a contingency table on the right, (P0.001; two‐way Fisher's exact test).


Figure 4Fission 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Fission 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.


Figure 5 ( 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 length of mitochondria or ( C ) as mitochondrial numbers . ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 length of mitochondria or (C) as mitochondrial numbers.(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 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.


Figure 6 ( 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 ) . ( 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 . 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 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).(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. 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 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).


Figure 7 ( 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 ) 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 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 . 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 ) 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 . 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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) 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 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. 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) 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. 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).


Figure 1A , 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 -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, 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


Figure 2GFP - 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 and endogenous PLK1 .

O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O

Figure 2GFP-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 and endogenous PLK1.


Figure 3 ( 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 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 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 .

O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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 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.


Figure 4 ( 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 cellular extracts that were produced for luciferase assays were tested for protein expression using Flag - , HA - and GAPDH - specific antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 cellular extracts that were produced for luciferase assays were tested for protein expression using Flag-, HA- and GAPDH-specific antibodies.


Figure 5 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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 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.


Figure 1Gel 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 ) 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 .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Gel 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) 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.


Figure 2SDS - 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 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 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 the RIP samples . 5S rRNA from the RIP samples was used as an internal control . 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 assay of biotin - labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 2SDS-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 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 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 the RIP samples. 5S rRNA from the RIP samples was used as an internal control.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 assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.


Figure 3Biotin - 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 assay of biotin - labeled mascRNA or the mutants ( Anti - Mut1 and Mut1 ) with the cytosol .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 3Biotin-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 assay of biotin-labeled mascRNA or the mutants (Anti-Mut1 and Mut1) with the cytosol.


Figure 4Northern 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 analysis of MALAT1 ( total RNAs used as templates ) in mascRNA overexpressing ( mascRNA ) and the scrambled RNA expressing ( Scramble ) cells . 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 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 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 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 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 the pulse Biotin - labeled proteins in HEK cells stably expressing mascRNA ( mascRNA ) or the scrambed RNA ( Scramble )

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Northern 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 analysis of MALAT1 (total RNAs used as templates) in mascRNA overexpressing (mascRNA) and the scrambled RNA expressing (Scramble) cells.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 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 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 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 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 the pulse Biotin-labeled proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambed RNA (Scramble)


Figure 5Western 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 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 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 analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells . 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 proteins in MALAT1 knockdown and the control cells . Quantification of the protein levels in Panel G . qRT - PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing ( mascRNA ) or the scrambled RNA expressing ( Scramble ) cells . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western 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 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 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 analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells.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 proteins in MALAT1 knockdown and the control cells.   Quantification of the protein levels in Panel G.   qRT-PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells.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.


Figure 6Western 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 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 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 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 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 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Western 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 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 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 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 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 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 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 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 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 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 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.


Figure 7Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation . Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation . 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 assay of HEK cells overexpressing mascRNA , or expressing the mutants ( Anti - Mut1 and Mut1 ) or the the scrambled RNA ( Scramble ) . 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 assay of MALAT1 knockdown cells ( shMALAT1 ) and the control cells ( Control ) with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation.Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation.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 assay of HEK cells overexpressing mascRNA, or expressing the mutants (Anti-Mut1 and Mut1) or the the scrambled RNA (Scramble).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 assay of MALAT1 knockdown cells (shMALAT1) and the control cells (Control) with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ).


Figure 1Immunoblot 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 = 6PDGFR - 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 µmGFP 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 µmTissue 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunoblot 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 = 6PDGFR-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 µmGFP 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 µmTissue 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.


Figure 2Representative 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative 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.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 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 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.


Figure 3PAS 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3PAS 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 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 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.


Figure 4Histological 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 staining and its histomorphometric quantification ( D ) show the progression of interstitial fibrosis in the time course . 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 stainingThe 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 levelA 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Histological 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 staining and its histomorphometric quantification (D) show the progression of interstitial fibrosis in the time course.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 stainingThe 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 levelA 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).


Figure 5Creatinine 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 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Creatinine 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 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


Figure 6Erythrocyte 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 . 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 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 . 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 , 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Erythrocyte 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.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 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.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, 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.


Figure 7Five 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 wtAngiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre : : Pdgfrb + / J mice ( C ) . 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 the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre : : Pdgfrb + / J mice ( E ) . 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 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 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 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 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 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 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 µm22 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Five 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 wtAngiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre::Pdgfrb+/J mice (C).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 the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre::Pdgfrb+/J mice (E).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 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 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 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 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 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 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 µm22 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).


Figure 8Gene 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 . Heatmap depicting pathway activities in Foxd1Cre : : Pdgfrb + / J mice and human CKD patients according to PROGENy . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Gene 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.Heatmap depicting pathway activities in Foxd1Cre::Pdgfrb+/J mice and human CKD patients according to PROGENy.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.


figf1 ( 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 ( - ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(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(-).


figf2 ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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.