F - J B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD20 antibody . The calcium fluxes were measured by FACScan .

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 and pGSK3β at Ser9 ) and total GSK3α ; and ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 and MAPK1 at Thr183 / Tyr185 ) and total MAPK . Right , Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - test with Benjamini - Hochberg correction .

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(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.


( E ) Kinases with altered activity between CTL and micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 and ZT6 . Graph shows the z - score value of each kinase assigned by RoKAI . Kinases above and below the dotted line represent prediction of up - and downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) .

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(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).


( F Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) and PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - test with Benjamini - Hochberg correction .

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(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.


G ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - test with Benjamini - Hochberg correction .

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G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


( 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 .

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(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 .

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(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 .

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(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 ) .

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(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).


Mitochondrial 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 .

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Mitochondrial 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 .

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(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 )

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(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 .

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(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 ) .

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(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 ) .

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(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).


( 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 .

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(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 ) .

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(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 ) .

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(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).


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

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


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

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(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 .

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(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 .

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(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 .

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


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

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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


( A ) Representative 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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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).


Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before and on the day of LPS administration , using routes and doses specified in the methods . C57bl / 6 female mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in concentration between drug and vehicle - treated pre - and post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . Percent survival was compared using a log rank Mantel Cox test

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Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


A Representative images of alkaline phosphatase ( ALP ) , Alizarin Red S ( ARS ) and Van Gieson ' s staining of human

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A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs


B , C Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 6 ; * * : p < 0 . 01 and * * * : p < 0 . 001 by t test

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B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test


D , E Quantitative RT - PCR and western blot analyses of the expression of osteogenic markers . Cells were cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t

D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


F H & E staining of the MSCs - mediated ectopic bone formation . Scale bar , 50 μm

F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm


G Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 6 . * : p < 0 . 05 by t

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G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test


A Scatter plot of RNA - seq expression analysis . 508 genes were upregulated and 391 genes were downregulated . Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days . Two biological replicates per group

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A Scatter plot of RNA-seq expression analysis. 508 genes were upregulated and 391 genes were downregulated. Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days. Two biological replicates per group


C Heatmap of representative genes associated with BMP2 signaling pathway


D Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * * * : p < 0 . 001 by t

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D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t test


E Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were starved overnight and then stimulated with 100 ng / ml BMP2 for 24

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E Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


F Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Cells were depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by t

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F Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t test


G Representative images of ALP and ARS staining . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 10 and 21 days ,

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G Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectively


H , I Quantitative analyses of the ALP activity ( n = 4 ) and calcium mineralization ( n = 5 ) . Results are shown as mean ± SEM ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t

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H, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


J , K Representative microCT images and quantification of newly formed bone in response to BMP2 . Results are shown as mean ± SEM ; n = 8 . * : p < 0 . 05 by t test . Scale bar , 2500 μm

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J, K Representative microCT images and quantification of newly formed bone in response to BMP2. Results are shown as mean ± SEM; n=8. *: p<0.05 by t test. Scale bar, 2500 μm


L H & E staining of BMP2 - induced newly formed bone . Scale bar , 50

L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μm


M Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

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M Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


N H & E staining of calvarial organ cultures stimulated with or without 100 ng / ml BMP2 . Red dotted lines indicate the interface between original and newly formed bone . Scale bar , 50

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N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μm


O Quantitative analysis of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

O Quantitative analysis of newly formed bone volume versus original bone volume (%). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


A Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged Smad1 in 293T

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A Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged Smad1 in 293T cells


B Immunoblot analysis showing decreased FLAG - Smad1 after USP34 knockdown

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B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown


C Immunoblot of Smad1 - linked polyUb . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 10 µM MG132 for 4 hours before collection

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C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection


D Immunoblot analysis of the degradation of FLAG - Smad1 protein . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 50 µg / ml cycloheximide ( CHX ) before collection at designated time points . Right : relative quantification of FLAG - Smad1 protein levels at different time

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D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time points


E Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged RUNX2 in 293T

E Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cells


F Co - immunoprecipitation of USP34 with endogenous RUNX2 in mouse

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F Co-immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCs


G Immunoblot of RUNX2 - linked polyUb . Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection . Prx1 - Cre ( + ) indicates MSCs isolated from Prx1 - Cre ; Usp34f / f mice , and Prx1 - Cre ( - ) indicates those from Usp34f / f control

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G Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control mice


H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were stimulated with BMP2 , treated with 50 µg / ml CHX , and collected at designated time points . Right : relative quantification of RUNX2 protein levels at different time

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H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time points


I Representative images of ALP and ARS staining . Usp34f / f and Prx1 - Cre ; Usp34f / f MSCs were transduced with GFP , Smad1 , Runx2 or Smad1 + Runx2 adenoviruses , and then cultured in osteogenic medium for 5 and 10 days ,

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I Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectively


J , K Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

J, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


L , M H & E staining and quantitative analysis of MSCs - mediated ectopic bone formation . Scale bar , 50 μm . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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L, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


A Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * : p < 0 . 05 by ANOVA with Tukey ' s post hoc

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A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc test


B Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , starved overnight and then stimulated with 100 ng / ml BMP2 for 24

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B Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


C Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , and depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


D ALP staining and quantitative measurement of ALP activity . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 5 days . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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D ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test


E Representative images of ARS staining and quantification . Cells were cultured with normal medium supplemented with 100 ng / ml BMP2 for 14 days . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test

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E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test


F Representative images of ALP and ARS staining . Mouse MSCs were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 and 10 days ,

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F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectively


G , H Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc test

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G, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


I , J Quantitative RT - PC analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


western blot analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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western blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


B Phospholipid transport / synthesis , as measured by 3H - serine incorporation , in human fibroblasts ( mean ± SE ; n = 9 , with 5 replicates / experiment ) and in primary mouse hippocampal neurons ( mean ± SE ; n = 3 , with 4 or 5 replicates / experiment ) . * , p < 0 . 05 vs E3 .

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B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.


C Comparison of 3H - serine incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).


D Duramycin sensitivity in ApoE ACM - treated fibroblasts . Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4 - treated cells , which was blocked upon the addition of 15 μM exogenous PtdEtn ( mean ± SD ; n = 3 , with 3 replicates / experiment ) .

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D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).


B Fractions from the discontinuous gradient were blotted with anti - ApoE ( WUE - 4 ) antibody ; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [ 16 ] .

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B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].


C An ApoE - containing fraction ( fraction 5 [ + ] ) , an ApoE - negative fraction ( fraction 3 [ - ] ) , and recombinant lipid - free ApoE protein were applied to human fibroblasts . Note increase in phospholipid synthesis using an ApoE4 - containing lipoprotein fraction , but not with either an ApoE4 - negative fraction or with lipoprotein - free recombinant ApoE3 or ApoE4 protein ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).


A Cholesteryl ester synthesis , as measured by 3H - oleate incorporation . Note increased CE in ApoE4 - treated cells , whereas there was essentially no difference in triglyceride ( TAG ) production ( mean ± SE ; n = 3 . with 3 replicates / experiment ) . † , p = 0 . 06 vs E3 .

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A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.


B Comparison of 3H - oleate incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 5 replicates / experiment ) .

B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).


C ApoE ACM - treated fibroblasts were stained with LipidTox green neutral lipid stain , and lipid droplets were visualized ( example at left ) and counted ( right ) . Note significantly more lipid droplets in ApoE4 - treated cells ( mean ± SD , n = 3 ) .

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C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).


HeLa cells were transfected with plasmids to visualize ER ( in green ) and mitochondria ( in red ) , and then were treated with ApoE3 - and ApoE4 - ACM for 24 hours . Shown is a box - and - whisker plot comparing co - localization in ApoE3 ACM ( n = 15 images analyzed [ gray circles , average of co - localization in ~ 5 cells in each image field ] ) compared to that in ApoE4 ACM ( n = 18 images ) .

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HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).


A Expression of transcripts coding for PI3K subunit genes in pDCs ( n = 12 biological replicates ) . In the boxplot , the middle , the bottom , and the top of the box represent the median , the 1st quantile and the 3rd quantile , respectively ; the height of the box is given by the difference between the 3rd and the 1st quantiles ( IQR , interquartile range ) ; the whiskers length is 1 . 5 times the interquartile range . Data points beyond the whiskers represent values more extreme than 1 . 5 times the interquartile range above or below the 3rd or the 1st quantile , respectively .

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A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.


C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone ( Ctrl ) or IFN - γ after 1 − h pretreatment with PI3K inhibitors .

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C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors.


D Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF - β , in the presence or absence ( Ctrl , control ) of catalytic PI3K inhibitors , normalized to the expression of Gapdh and presented relative to results in untreated cells ( dotted line , 1 - fold ) .

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D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold).


E In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs conditioned in vitro with TGF - β ( or medium ) for 24 h in the presence or absence ( Ctrl ) of catalytic inhibitors as in C . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) .

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E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


F Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α - , β - , δ - specific , or negative control ( nc ) siRNA and stimulated for 18 h with TGF - β , normalized to the expression of Gapdh and presented relative to results in unstimulated cells ( dotted line , 1 - fold ) .

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F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold).


G In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs transfected with siRNA as in ( F ) and conditioned in vitro with TGF - β ( or medium ) for 24 h . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) .

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G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


C Pull - down ( PD ) assay of lysates from P1 . HTR cells with unphosphorylated ( YENM ) and tyrosine - phosphorylated IDO1 peptides ( pYENM , pITIM1 , and pITIM2 ) , analyzed by sequential immunoblotting with anti − p85 , − SHP - 1 , and - SHP - 2 antibodies .

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C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.


D Immunoblotting with anti - IDO1 and - p85 antibodies of IDO1 immunoprecipitates obtained from P1 . HTR cells transfected with IDO1 . WT or IDO1 . Y149F . Mock - transfected P1 . HTR cells were used as a control .

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D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.


E Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from lysates of P1 . HTR transfectants as in D .

E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.


F Kyn release over 24 h by P1 . HTR transfectants as in D .

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F Kyn release over 24 h by P1.HTR transfectants as in D.


A Confocal immunofluorescence microscopy images of P1 . HTR cells either untransfected ( Untr ) or transfected with IDO1 . WT ( WT ) or vector alone ( Mock ) and stained with antibodies recognizing IDO1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 µm .

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A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.


B Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT , co - stained with antibodies recognizing IDO1 ( green ) and markers of intracellular organelles and structures ( as indicated ; red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


C Immunoblotting analysis of EE purified from P1 . IDO1 . WT cells . Mock - transfected cells as in A were used as control .

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C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.


D Distribution of the IDO1 . WT protein expressed by P1 . HTR transfected cells in isopycnic sucrose gradients . Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated .

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D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.


A Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT or IDO1 . Y149F and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


B Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR cells transfected as in A , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 24 cells from three independent experiments ) . Statistical analysis was performed using two - tailed unpaired Student ' s t - test . * * * P < 0 . 001 .

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B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001.


C Distribution of IDO1 . WT and the IDO1 . Y149F mutants in homogenates of P1 . HTR cells along the isopycnic sucrose gradient as in Fig 3D . Fractions were immunoblotted using antibodies specific for IDO1 .

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C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1.


D The distribution of IDO1 . WT and IDO1 . Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction ( mean ± SD of three experiments ) .

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D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).


A Pull - down assay of lysates from untransfected P1 . HTR cells with unphosphorylated ( YENM ) and tyrosine - phosphorylated ( pYENM ) IDO1 peptides , analyzed by immunoblotting with anti - p85 , - p110α , - p110β , - p110γ , and - p110δ antibodies . Whole cell lysates ( WCL ) were used as control of lysate amount .

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A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount.


B Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT incubated with class IA PI3K p110 isoform inhibitors ( indicated ) or medium alone ( Ctrl ) for 24 h and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


C Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR cells transfected and treated as in ( B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 cells from two independent experiments ) .

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C Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected and treated as in (B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from two independent experiments).


D Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from P1 . HTR cells transfected with IDO1 . WT cells and treated with PI3K catalytic inhibitors ( indicated ) or medium alone ( ctrl ) . Data represent the mean ± SD ( n = 3 technical replicates ) .

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D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).


A Confocal immunofluorescence microscopy images of freshly purified pDCs co - stained with antibodies recognizing IDO1 ( green ) and markers of intracellular organelles and structures ( indicated ; red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 5 µm .

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A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.


B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF - β or IFN - γ and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 5 µm . C Pearson ' s coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF - β or IFN - γ ( as in B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 cells from three independent experiments ) .

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B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).


( C ) The Hippo WW domain - containing proteins show binding specificity to the known Hippo PY motif - containing proteins . TAP - MS analysis of a series of WW domain - containing proteins were performed and their binding with the indicated Hippo PY motif - containing proteins was summarized in a heatmap .

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(C) The Hippo WW domain-containing proteins show binding specificity to the known Hippo PY motif-containing proteins. TAP-MS analysis of a series of WW domain-containing proteins were performed and their binding with the indicated Hippo PY motif-containing proteins was summarized in a heatmap.


( E ) Validation of the binding specificity for the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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(E) Validation of the binding specificity for the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( F ) Validation of the binding specificity for the derived WW domains from the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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(F) Validation of the binding specificity for the derived WW domains from the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( C - G ) Validation of the identified 9 - amino acid sequence in determining the Hippo WW domain binding specificity . The requirement of the identified 9 - amino acid sequence for AMOT association was respectively examined for TAZ ( C ) , TAZ - WW domain ( D ) , KIBRA ( E ) , YAP ( F ) and SAV1 ( G ) . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( B ) STXBP4 forms a complex with several Hippo PY motif - containing proteins . Immunoprecipitation was performed with STXBP4 antibody .

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(B) STXBP4 forms a complex with several Hippo PY motif-containing proteins. Immunoprecipitation was performed with STXBP4 antibody.


( C ) The identified 9 - amino acid sequence is required for the association between STXBP4 and AMOT . HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay .

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(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.


( E ) The YAP - WW1 / SMAD7 - PY and STXBP4 - WW / SMAD7 - PY complexes show similar cumulative average trend and average binding free energy ( ∆ G ) within standard deviation ( the shaded region ) of one another .

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(E) The YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes show similar cumulative average trend and average binding free energy (∆G) within standard deviation (the shaded region) of one another.


( F ) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation . Western blotting was performed with the indicated antibodies .

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(F) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation. Western blotting was performed with the indicated antibodies.


Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP nuclear translocation ( G )

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Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP nuclear translocation (G)


Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP downstream gene transcription ( mean ± s . d . , n = 3 biological replicates ) ( H ) . Scale bar , 20 μm . * * * p < 0 . 001 ( Student ' s t - test ) .

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Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP downstream gene transcription (mean ± s.d., n=3 biological replicates) (H). Scale bar, 20 μm.*** p < 0.001 (Student's t-test).


( I ) WW domain is required for the STXBP4 - mediated YAP cytoplasmic translocation . STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed . HA - positive cells ( arrows ) from ~ 30 different views ( ~ 200 cells in total ) were randomly selected and quantified for YAP localization . Percentage of HA - positive cells with nuclear YAP enrichment is shown . Scale bar , 20 μm .

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(I) WW domain is required for the STXBP4-mediated YAP cytoplasmic translocation. STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed. HA-positive cells (arrows) from ~30 different views (~200 cells in total) were randomly selected and quantified for YAP localization. Percentage of HA-positive cells with nuclear YAP enrichment is shown. Scale bar, 20 μm.


( J ) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition . The indicated cells were subjected to serum starvation ( treatment with no - serum medium for 12 hours ) , glucose starvation ( treatment with no - glucose medium for 6 hours ) or actin inhibition ( treatment with 0 . 5 μg / mL latrunculin B or 5 μM blebbistatin for 30 min ) . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


( B ) STXBP4 forms a protein complex with α - catenin and a group of Hippo pathway proteins .

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(B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins.


( C ) STXBP4 promotes the association of α - catenin with AMOT and LATS1 . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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(C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( D ) Loss of STXBP4 diminishes the association of α - catenin with AMOT , LATS1 and YAP . HEK293A and STXBP4 KO cells were transfected with the SFB - tagged α - catenin construct and subjected to the pulldown assay .

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(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.


( E ) STXBP4 induces the co - localization between α - catenin and AMOT as well as LATS1 and YAP . HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed . Scale bar , 20 μm .

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(E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm.


Identification of several STXBP4 missense mutations that disrupt its interaction with α - catenin and AMOT . The identified missense mutations respectively disrupted the STXBP4 - α - catenin ( G ) and STXBP4 - AMOT ( H ) complex formation .

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Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation.


( I ) Inhibition of actin cytoskeleton promotes the STXBP4 - associated protein complex formation . HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre - immune serum and anti - STXBP4 serum under the indicated treatments .

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(I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments.


( J ) The missense mutations of STXBP4 ( F ) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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(J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


STXBP4 is downregulated in human kidney cancer . The mRNA level of STXBP4 is analyzed in the Firebrowse web database ( A ) , where 14 , 729 tumor sample data generated by TCGA were included . The first quartile , median and third quartile values were indicated as the boxplots . Outliers were plotted as individual points . Error bars indicated the standard deviation above and below the mean of the data .

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STXBP4 is downregulated in human kidney cancer. The mRNA level of STXBP4 is analyzed in the Firebrowse web database (A), where 14,729 tumor sample data generated by TCGA were included. The first quartile, median and third quartile values were indicated as the boxplots. Outliers were plotted as individual points. Error bars indicated the standard deviation above and below the mean of the data.


STXBP4 is downregulated in human kidney cancer . The expression of STXBP4 was also examined using kidney tissue microarray , where percentage of the indicated tissue samples with downregulated STXBP4 was shown ( B ) . The p value was calculated by using the paired Student ' s t - test .

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STXBP4 is downregulated in human kidney cancer. The expression of STXBP4 was also examined using kidney tissue microarray, where percentage of the indicated tissue samples with downregulated STXBP4 was shown (B). The p value was calculated by using the paired Student's t-test.


( C ) Kaplan - Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level . Clinical data of STXBP4 were analyzed in TCGA - KIRC project containing total 611 patient samples . The p value was calculated by using the Log - rank ( Mantel - Cox ) test .

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(C) Kaplan-Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level. Clinical data of STXBP4 were analyzed in TCGA-KIRC project containing total 611 patient samples. The p value was calculated by using the Log-rank (Mantel-Cox) test.


( D ) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray , where the indicated regions in the box were shown three times enlarged . Brown staining indicates positive immunoreactivity . Scale bar , 100 μm .

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(D) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray, where the indicated regions in the box were shown three times enlarged. Brown staining indicates positive immunoreactivity. Scale bar, 100 μm.


( E ) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables . Statistical significance was determined by chi - square test . R , correlation coefficient . N , nuclear localization . C , cytoplasmic localization .

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(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.


( F ) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting .

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(F) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting.


( G and H ) Both the association with α - catenin and the functional WW domain are required for the STXBP4 ' s tumor suppressive function in 786 - O cells . Overexpression of STXBP4 , but not the indicated STXBP4 missense mutants , significantly suppressed the 786 - O cell xenograft tumor formation . Xenograft tumors are shown in ( G ) , and the tumor weight is quantified in ( H ) ( n = 5 mice , mean ± s . d . ) . * * p < 0 . 01 ( Student ' s t - test ) . Scale bar , 1 cm .

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(G and H) Both the association with α-catenin and the functional WW domain are required for the STXBP4's tumor suppressive function in 786-O cells. Overexpression of STXBP4, but not the indicated STXBP4 missense mutants, significantly suppressed the 786-O cell xenograft tumor formation. Xenograft tumors are shown in (G), and the tumor weight is quantified in (H) (n = 5 mice, mean ± s.d.). ** p < 0.01 (Student's t-test). Scale bar, 1 cm.


A , RT - qPCR ( n = 3 - 12 biological replicates ) analyses of CGN expression in different mouse organs at P21 .

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A, RT-qPCR (n = 3-12 biological replicates) analyses of CGN expression in different mouse organs at P21.


B . Western blot analyses of CGN expression in different mouse organs at P21 .

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B. Western blot analyses of CGN expression in different mouse organs at P21.


G . Whole mount immunofluorescence of CGN expression in P3 , P14 and P21 mouse cochlea coimmunostained with cuticular plate marker LMO7 ( G ) . CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals ( n = 130 - 193 hair cells from 3 - 4 cochleae ) . Data information : Data are presented as mean ± SEM ; one - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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G. Whole mount immunofluorescence of CGN expression in P3, P14 and P21 mouse cochlea coimmunostained with cuticular plate marker LMO7 (G). CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals (n = 130-193 hair cells from 3-4 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


B . MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies .

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B. MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies.


D . RT - qPCR of CGN expression in transfected MDCK cells ( n = 3 biological replicates ) . Data information : Data are presented as mean ± SEM ; unpaired Student ' s t - test was used in ( D ) . ns , not significant , P > 0 . 05 .

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D. RT-qPCR of CGN expression in transfected MDCK cells (n = 3 biological replicates). Data information: Data are presented as mean ± SEM; unpaired Student's t-test was used in (D). ns, not significant, P > 0.05.


A - C . DPOAE thresholds ( A ) , ABR thresholds ( B ) and ABR P1 amplitudes ( C ) of the 2 - month old control ( Cgnfl / fl ) and Cgn - cKO ( Cgnfl / fl ; Pou4f3creER / + ) mice . Control mice ( circle ) , n = 16 mice ( female = 8 , male = 8 ) ; Cgn - cKO mice ( square ) , n = 19 mice ( female = 11 , male = 8 ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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A-C. DPOAE thresholds (A), ABR thresholds (B) and ABR P1 amplitudes (C) of the 2-month old control (Cgnfl/fl) and Cgn-cKO (Cgnfl/fl;Pou4f3creER/+) mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


D - I . Growth curves of ABR P1 amplitudes at different cochlear frequencies ( 5 . 6 - 32 kHz ) of the 2 - month old control and Cgn - cKO mice . Control mice ( circle ) , n = 16 mice ( female = 8 , male = 8 ) ; Cgn - cKO mice ( square ) , n = 19 mice ( female = 11 , male = 8 ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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D-I. Growth curves of ABR P1 amplitudes at different cochlear frequencies (5.6 - 32 kHz) of the 2-month old control and Cgn-cKO mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


J . Whole mount immunofluorescence of the organ of Corti from 2 - month old control and Cgn - cKO mice . Phalloidin ( F - actin , magenta ) ; Myosin7a ( hair cells , green ) . White arrows label the lost OHCs .

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J. Whole mount immunofluorescence of the organ of Corti from 2-month old control and Cgn-cKO mice. Phalloidin (F-actin, magenta); Myosin7a (hair cells, green). White arrows label the lost OHCs.


C . Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse .

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C. Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse.


D - I . DPOAE thresholds ( D , E ) , ABR thresholds ( F , G ) , and ABR P1 amplitudes at 32 kHz ( H , I ) of 2 - month old ( D , F , H ) or 6 - month old ( E , G , I ) CgndelG mice . Control Cgn + / + mice ( circle ) , n = 3 mice ( 2 - month , female = 1 , male = 2 ) or 15 mice ( 6 - month , female = 4 , male = 11 ) ; CgndelG / + mice ( triangle ) , n = 8 mice ( 2 - month , female = 4 , male = 4 ) or 20 mice ( 6 - month , female = 9 , male = 11 ) ; CgndelG / delG mice ( square ) , n = 7 mice ( 2 - month , female = 4 , male = 3 ) or 16 mice ( 6 - month , female = 6 , male = 10 ) . Cgn + / + vs CgndelG / delG , * ( red ) ; Cgn + / + vs CgndelG / + , * ( green ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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D-I. DPOAE thresholds (D, E), ABR thresholds (F, G), and ABR P1 amplitudes at 32 kHz (H, I) of 2-month old (D, F, H) or 6-month old (E, G, I) CgndelG mice. Control Cgn+/+ mice (circle), n = 3 mice (2-month, female = 1, male = 2) or 15 mice (6-month, female = 4, male = 11); CgndelG/+ mice (triangle), n = 8 mice (2-month, female = 4, male = 4) or 20 mice (6-month, female = 9, male = 11); CgndelG/delG mice (square), n = 7 mice (2-month, female = 4, male = 3) or 16 mice (6-month, female = 6, male = 10). Cgn+/+ vs CgndelG/delG, * (red); Cgn+/+ vs CgndelG/+, * (green). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


J . Whole mount immunofluorescence of the organ of Corti from 7 - month old CgndelG mice . White arrows indicate lost OHCs by F - actin labeling ( Phalloidin ) .

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J. Whole mount immunofluorescence of the organ of Corti from 7-month old CgndelG mice. White arrows indicate lost OHCs by F-actin labeling (Phalloidin).