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 .

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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 )

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


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

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


Western blot detection of ARS 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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


Western blot detection of endogenous 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 .

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


Detection of 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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


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

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Northern 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 .

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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 .

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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 ) .

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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 ) .

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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 ) .

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


Immunoblot detection 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 =

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


PDGFR - β 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

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


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

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


Tissue clearing with Scale 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 .

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


Representative Ki67 immunofluorescence 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 .

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


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 .

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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 .

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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 .

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


PAS 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 .

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


Immunhistological 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 .

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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 .

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


Histological stainings 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 .

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


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

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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

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


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

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


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

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


Creatinine clearance was measured in mice of an 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

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


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

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


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

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


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

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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 .

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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 .

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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 .

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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 .

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


Five days after 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

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


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

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Angiotensin 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 ) .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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

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

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


22 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 ) .

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


Gene expression arrays were performed in 6 week old Foxd1Cre : : Pdgfrb + / J mice compared to wt mice . Listed are genes with log2 fold changes higher or lower than 0 . 6 . Out of these 46 genes , 17 are IFN - regulated genes , 5 are involved in autophagy 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 .

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


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

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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 .

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


( b ) RT - PCR 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 ( - ) .

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


( a - e ) T1 - weighted imaging shows hyperintensity of 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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( E ) Oligomeric state of Dyn1ΔΔ at room temperature .

(E) Oligomeric state of Dyn1ΔΔ at room temperature.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( A , B ) Transferrin receptor ( TfnR ) uptake , a measure of CME , shown as % uptake of total steady - state surface TfnR for control H1299 cells , and H1299 cells stabily expressing low amounts of siRNA - resistant Dyn2WT - EGFP ( A ) or Dyn2S619L - EGFP ( B ) with or without treatment of siRNA - directed towards endogenous Dyn2 .

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(A,B) Transferrin receptor (TfnR) uptake, a measure of CME, shown as % uptake of total steady-state surface TfnR for control H1299 cells, and H1299 cells stabily expressing low amounts of siRNA-resistant Dyn2WT-EGFP (A) or Dyn2S619L-EGFP (B) with or without treatment of siRNA-directed towards endogenous Dyn2.


( C ) Fixed cell TIRF - M images of Dyn2WT - EGFP or Dyn2S619L - EGFP expressing H1299 cells with or without Dyn2 - siRNA treatment showing co - localization with clathrin light chain immunostained rabbit anti - CLC and Alexa 547 - conjugated secondary antibodies .

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(C) Fixed cell TIRF-M images of Dyn2WT-EGFP or Dyn2S619L-EGFP expressing H1299 cells with or without Dyn2-siRNA treatment showing co-localization with clathrin light chain immunostained rabbit anti-CLC and Alexa 547-conjugated secondary antibodies.


( D ) Percentage of Dyn2 - EGFP positive clathrin coated pits detected by live cell TIRF - M and automated master / slave image analysis ( Aguet et al , 2013 ) .

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(D) Percentage of Dyn2-EGFP positive clathrin coated pits detected by live cell TIRF-M and automated master/slave image analysis (Aguet et al, 2013).


( E ) Lifetime analysis of CCPs which are positive for EGFP - Dyn2WT or EGFP - Dyn2S619L with or without siRNA - mediated specific knockdown of endogenous Dyn2 .

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(E) Lifetime analysis of CCPs which are positive for EGFP-Dyn2WT or EGFP-Dyn2S619L with or without siRNA-mediated specific knockdown of endogenous Dyn2.


B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and / or Chloramphenicol ( Chl ) . After fixation , AF647 - azide ( magenta ) was used for click reaction ( see A ) , and cells were immunostained for TOM20 as mitochondrial marker ( green ) . Representative epifluorescent microscopy images are shown . Magenta images shown in the presence of only Chl and in the absence of both inhibitors ( none ) are presented at lower brightness level than the other panels , duo to their very strong intensities . An inset of Harr panel is zoomed in the panel below for better visualization . Met , methionine . Scale bars , 50 µm and 10 µm for the zoom panel .

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B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and/or Chloramphenicol (Chl). After fixation, AF647-azide (magenta) was used for click reaction (see A), and cells were immunostained for TOM20 as mitochondrial marker (green). Representative epifluorescent microscopy images are shown. Magenta images shown in the presence of only Chl and in the absence of both inhibitors (none) are presented at lower brightness level than the other panels, duo to their very strong intensities. An inset of Harr panel is zoomed in the panel below for better visualization. Met, methionine. Scale bars, 50 µm and 10 µm for the zoom panel.


A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths ( heat map ) and cells immunostained for TOM20 ( gray ) . Representative confocal images are shown for each time point . The same 5 - minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row . Scale bars , 10 µm . Mitochondrial protein synthesis was quantified over time . 30 cells from N = 3 independent experiments were analyzed for each time point ( Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values . Error bars , + / - SEM ) . A hyperbolic graph was fitted to the data . R - squared , 0 . 97 .

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A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97.


B As in A , Mitochondrial translation products were labeled for increasing time ( 10 and 20 minutes ) in HeLa cells . The number of analyzed cells was increased in the plots from 25 to 300 per time point . 150 or 300 random cells from N = 3 independent experiments were analyzed . Kruskal - Wallis followed by Fisher ' s test . * * * * p < 0 . 0001 . Error bars , + / - SEM .

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B As in A, Mitochondrial translation products were labeled for increasing time (10 and 20 minutes) in HeLa cells. The number of analyzed cells was increased in the plots from 25 to 300 per time point. 150 or 300 random cells from N = 3 independent experiments were analyzed. Kruskal-Wallis followed by Fisher's test. ****p < 0.0001. Error bars, +/- SEM.


C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 ( magenta ) and cells immunostained for TOM20 ( green ) in fibroblasts , iPSCs - derived cardiomyocytes , and hippocampal neurons . Representative epifluorescent microscopy images are shown . The correlation of HPG and TOM20 channels is represented in fire lookup table , after calculating the Pearson Correlation values over each pixel of the images . Scale bars , 25 µm .

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C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.


A Mitochondrial translation products were labeled using a FUNCAT approach and [ 35S ] Met labeling in control and TSFM patient fibroblasts . Representative epifluorescent microscopy and the autoradiography images are shown . Scale bars , 50 µm . Changes in mitochondrial protein synthesis were quantified for both approaches . For microscopy image analysis , > 300 cells from N = 3 independent experiments were analyzed for each cell type . Mann - Whitney U test , * * * * p < 0 . 0001 . For radiolabeling analyses , N = 3 independent samples were quantified for each cell type . Student t test , * * p < 0 . 01 . Error bars , + / - SEM . B As in A using control and COA6 KO HEK cells . Scale bars , 25 µm . For microscopy image analysis , * * * * p < 0 . 0001 , and for radiolabeling experiments , * p < 0 . 05 were calculated . Error bars , + / - SEM .

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A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM. B As in A using control and COA6 KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM.


A Mitochondrial translation products were labeled with HPG in hippocampal neurons , immunostained for ANK - G ( initial axon segment ) , and plasma membrane was stained with DiO . Representative epifluorescent images and zoomed insets of neural branches are shown . In both axon ( ANK - G positive , arrow ) and dendrite ( star ) , HPG incorporation revealing mitochondrial protein synthesis was detected . Scale bars , 20 µm .

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A Mitochondrial translation products were labeled with HPG in hippocampal neurons, immunostained for ANK-G (initial axon segment), and plasma membrane was stained with DiO. Representative epifluorescent images and zoomed insets of neural branches are shown. In both axon (ANK-G positive, arrow) and dendrite (star), HPG incorporation revealing mitochondrial protein synthesis was detected. Scale bars, 20 µm.


B Representative confocal images of a dendrite ( top ) and an axon ( bottom ) in which mitochondrial translation products were labeled with HPG ( magenta ) . HOMER1 and VGLUT1 were used as post - and presynapse markers , respectively ( green ) and DiO stained randomly neurites ( gray ) . In each neurite , an exemplary region was selected and the DiO was outlined to mark the outer edge of the region . As a control , cells were treated with Chloramphenicol ( Chl ) to inhibit mitochondrial translation . Scale bars , 5 µm .

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B Representative confocal images of a dendrite (top) and an axon (bottom) in which mitochondrial translation products were labeled with HPG (magenta). HOMER1 and VGLUT1 were used as post- and presynapse markers, respectively (green) and DiO stained randomly neurites (gray). In each neurite, an exemplary region was selected and the DiO was outlined to mark the outer edge of the region. As a control, cells were treated with Chloramphenicol (Chl) to inhibit mitochondrial translation. Scale bars, 5 µm.


Figure 2 . SPOP localizes to nuclear speckles or other nuclear bodies . Confocal microscopy images of fixed NIH 3T3 cells transiently expressing ( A ) full length HA - SPOP , ( B ) GFP - Gli31455 , and ( C ) HA - SPOP + GFP - Gli31 - 455 are shown . DAPI was used to stain the nucleus , SPOP localization was identified using a anti - HA antibody , nuclear speckle localization was identified using a anti - SC - 35 antibody , and Gli31 - 455 localization was identified via GFP fluorescence . SPOP co - localizes with a marker for nuclear speckles or with the substrate Gli31455 . The areas with overlapping HA - and GFP signal contain 75 % of the punctate HA signal , and 100 % of the punctate GFP signal .

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Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies. Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full length HA-SPOP, (B) GFP-Gli31455, and (C) HA-SPOP+GFP-Gli31-455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli31-455 localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli31455. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.


( A - B ) NIH 3T3 cells were ( A ) co - transfected with constructs expressing full length SPOP and GFP - Gli31455 or ( B ) transfected with a construct expressing SC - 35 - GFP , and GFP fluorescence was monitored in live cells . ( A ) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching ( at 0 s ) , recovery , and nuclear body fusion events . The arrow in each panel points to the body that was photobleached and subsequently fuses with another body . These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery . ( B ) Snapshots at indicated timepoints show a nuclear speckle fusion event . Additional images are shown in Fig EV1 .

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(A-B) NIH 3T3 cells were (A) co-transfected with constructs expressing full length SPOP and GFP-Gli31455 or (B) transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1.


( C ) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec . Data from 45 individual cells and FRAP events were corrected for background , normalized to the minimum and maximum intensity , and the mean is shown with error bars representing the standard error of the mean . The mean characteristic recovery time is indicated ± SEM .

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(C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.


( D - E ) Nuclear bodies are close to spherical . ( D ) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA - SPOP ( 151 cells ) ( D ) , or GFP - Gli31 - 455 and HA - SPOP ( 155 cells ) ( E ) were calculated as described previously ( Brangwynne et al , 2011 ) . Representative individual images of cells are shown as insets in each panel .

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(D-E) Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) (D), or GFP-Gli31-455 and HA-SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.


( A ) SEC chromatograms of given loading concentrations of SPOP28 - 359 are shown . The concentrations were normalized to the monomer molecular weight , i . e . , identical concentrations of dimeric SPOP ΔBACK ( see Fig EV2 ) and oligomeric SPOP28 - 359 contain identical numbers of protomers . ( B ) SEC chromatograms for SPOP constructs defective in self - association in one or both oligomerization domains are shown . Proteins were injected at the same loading concentration ( 533 µM ) and the elution volume of globular molecular weight standards is noted above the graph . ( C ) SEC chromatograms of SPOP28 - 359 ( 200 µM ) , SPOP mutBACK ( 718 µM ) , and mixtures of the two ( ratios given in the Figure , SPOP at 200 µM ) are shown . The elution volume of globular molecular weight standards is noted above the graph .

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(A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph.


( D ) Constructs for expressing full - length HA - SPOP or HA - SPOP mutants capable of oligomerization through only one ( or neither ) of the self - association domains were transfected into NIH 3T3 cells . DAPI was used to stain the nucleus and SPOP localization was identified using an anti - HA antibody . Experiments were performed at least twice on four biological samples . Multiple cells were examined and representative cells are shown . For additional images see Appendix Fig S3 . Scale bar represents 5 µm .

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(D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.


( A ) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide . All SPOP proteins bound substrate with an affinity similar to that of SPOP28 - 359 ( 4 - 8 µM , see Table 2 ) . Experimental data are shown as circles ; the solid lines are fits to eq . 2 ( Roehrl et al , 2004 ) .

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(A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004).


( B ) In vitro crosslinking assays were performed for SPOP28 - 359 and each mutant at 30 μM protein with the amide - specific BS3 crosslinker . Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non - specific crosslinking of protein species . ( C ) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP , SPOP mutBACK , SPOP mutBTB , or SPOP mutBTB - BACK . SPOP1 , SPOP2 and SPOPn identify SPOP monomers , dimers and larger species , respectively . For loading levels see Appendix Fig S4 .

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(B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.


( A ) SV - AUC data for 0 . 2 nM AF488 - SPOP ΔBACK . Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation ( circles ) . The data were subjected to standard c ( s ) analysis in SEDFIT ( Schuck , 2000 ) and the results of this fit are shown ( solid lines , rainbow color scheme ) .

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(A) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme).


( B ) Sedimentation coefficient distributions for a selection of values from the dilution series are shown .

(B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.


( C ) The isotherm of weighted - average s - values vs . concentration ( black circles ) , was fit to a dimer self - association model and reveals a KD of 1 . 11 nM , [ 95 % confidence interval 0 . 88 , 1 . 40 nM ] ( solid line ) .

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(C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line).


( D ) The BACK domain dimerizes with micromolar affinity . Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP mutBTB ( black circles ) was fitted to a self - association model ( see text for details ) for BACK - BACK dimerization ( black line ) yielding a KD of 36 ± 3 µM ( average and standard deviation from 3 independent experiments ) . Fits to the monomer / dimer equilibrium model and to alternative monomer / trimer , tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines , respectively . Residuals for all models are depicted in the lower panel .

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(D) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (Mw) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a KD of 36 ± 3 µM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.


( B ) Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP28 - 359 ( orange circles ) was fitted to an isodesmic self - association model in which SPOP dimers are the self - associating unit ( orange line ) . The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ( ( SPOP2 ) 11 ) . The fits from three independent experiments yielded a KD2 of 2 . 4 ± 0 . 4 µM . Lines for fits of the data to self - association models that assume formation of individual oligomeric species instead of isodesmic self - association are shown for reference ( gray lines ) .

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(B) Experimental weight-average molar mass (Mw) from CG-MALS for SPOP28-359 (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP2)11). The fits from three independent experiments yielded a KD2 of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines).


( C ) Experimental weight - average molar mass ( Mw ) from CG - MALS data ( circles ) and fits ( solid lines ) are depicted for each SPOP construct assayed , showing that a progressive increase in SPOP oligomer size is observed only when both self - association domains are interaction - competent . This Figure is comprised of data shown in Figures 6D , 7B , and Appendix Fig S5A for direct comparison .

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(C) Experimental weight-average molar mass (Mw) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison.


( A ) Native MS spectrum of 30 µM SPOP28 - 359 confirms that SPOP self - associates through addition of dimers . The increase in mass indicates that the dimer is the stable building block within the assemblies .

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(A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies.


( C ) Concentration - dependence of the SPOP oligomer size distribution ( mole fraction ) from isodesmic modeling of CG - MALS data .

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(C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.


Figure 9 . The self - association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo . UAS transgenes were expressed under the control of an epithelial driver C765 - Gal4 in Drosophila melanogaster . ( A ) UAS vector served as a control and yielded a normal wing . Longitudinal veins are denoted by numbers 1 - 5 . Expression of ( B ) SPOP WT resulted in a modest Hh loss - of function phenotype with fusion of LV3 and LV4 ( arrow ) . ( C ) mutBTB acts in a dominant - negative manner , as evidenced by a modest Hh gain - of function phenotype with expansion of the LV3 - LV4 intervein space . Expression of ( D ) mutBACK and ( E ) mutBTB - BACK does not induce a wing patterning defect . ~ 50 animals each were analyzed from two independent crosses .

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Figure 9. The self-association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo. UAS transgenes were expressed under the control of an epithelial driver C765-Gal4 in Drosophila melanogaster. (A) UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of (B) SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). (C) mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of (D) mutBACK and (E) mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.


Representative H & E ( top ) and immunofluorescent ( IF ) images ( bottom ) of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( for secretory cell , white ) , Acetylated tubulin ( Act - Tub , for ciliated cell , green ) , and DAPI ( blue ) . Scale bar , 100μm . Statistical quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells in ( B ) .

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Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm. Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B).


Representative IF images of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( for AT1 cell , white ) , and DAPI ( blue ) . Scale bar , 100μm . Quantification of Scgb1a1 lineage - labeled tdTomato + AQP5 + AT1 cells in ( D ) . Data are presented as mean ± SEM ( n = 5 mice for each group ) .

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Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group).