( E ) Graph shows the percentage of GFP + cells in the VZ / SVZ , IZ and CP . The number of GFP + cells in the VZ / SVZ region increased significantly , and the number of GFP + in the CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


( I ) Representative images of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT and KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of the percentage of GFP + cells in the VZ / SVZ , IZ and CP . Overexpression of NKX2 - 2 restored the distribution of GFP + cells to the normal state . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


J , Ridge graph comparisons of cyclin expression across the U5 - hNSC defined cell cycle phases . The x - axis is relative expression of the cyclin , and the y - axis is counts of cells per phase . The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot .

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J, Ridge graph comparisons of cyclin expression across the U5-hNSC defined cell cycle phases. The x-axis is relative expression of the cyclin, and the y-axis is counts of cells per phase. The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot.


C , Number of putative stem - like cells in each ccAF cell cycle phase , and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem - like cells in Bhaduri et al . , 2020 .

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C, Number of putative stem-like cells in each ccAF cell cycle phase, and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem-like cells in Bhaduri et al., 2020.


D , Putative stem - like cells and expression of the genes used to identify the putative stem - like cells projected onto Neftel et al . , 2019 . Expression of genes are overlaid as the color of each cell ( white = 0 , dark blue = max expression ) . Quadrants are broken up by dashed grey lines : OPC = upper left ; NPC = upper right ; AC = lower left ; and MES = lower right . Percentages of gene expression greater than 0 are shown in the corners of the plot .

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D, Putative stem-like cells and expression of the genes used to identify the putative stem-like cells projected onto Neftel et al., 2019. Expression of genes are overlaid as the color of each cell (white = 0, dark blue = max expression). Quadrants are broken up by dashed grey lines: OPC = upper left; NPC = upper right; AC = lower left; and MES = lower right. Percentages of gene expression greater than 0 are shown in the corners of the plot.


C , Violin plot showing PLP1 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of PLP1 in single cells is much higher in oligodendrocytes ( Oligo ) than in Neural G0 or oligodendrocyte progenitor cells ( OPC ) . D , Violin plot showing GFAP expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types . E , Violin plot showing AQP4 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit .

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C, Violin plot showing PLP1 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of PLP1 in single cells is much higher in oligodendrocytes (Oligo) than in Neural G0 or oligodendrocyte progenitor cells (OPC). D, Violin plot showing GFAP expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types. E, Violin plot showing AQP4 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit.


B , Representative contour maps of flow cytometry for FUCCI following the loss of NF2 , PTPN14 , TAOK1 , CREBBP , and TP53 .

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B, Representative contour maps of flow cytometry for FUCCI following the loss of NF2, PTPN14, TAOK1, CREBBP, and TP53.


D , G0 / G1 and S / G2 / M transit times using time - lapse microscopy and FUCCI . Differences in G0 / G1 are statistically significant with p < 0 . 001 for targeted U5 - NSCs and p = 0 . 0006 for GSC - 131 compared to NTC .

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D, G0/G1 and S/G2/M transit times using time-lapse microscopy and FUCCI. Differences in G0/G1 are statistically significant with p<0.001 for targeted U5-NSCs and p=0.0006 for GSC-131 compared to NTC.


( A ) After 4 h of starvation in 1 % K ‐ acetate medium in the presence of PMSF , autophagic vesicles can be visualized in vacuoles of wild ‐ type and pep4prb1 cells using Normaski optics . In aut2Δ and aut7Δ cells no autophagic vesicles can be seen . Bar represents 20 μm .

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(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.


( B ) Total protein breakdown of cells starved on nitrogen ‐ free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild ‐ type cells . pep4Δ ( pra1Δ ) cells are almost completely impaired in starvation ‐ induced vacuolar protein breakdown . Cells were pulse ‐ labeled with [ 35S ] methionine and chased on non ‐ radioactive starvation medium . Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al . ( 1997 ) .

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(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).


( C ) Survival during starvation . Like pep4Δ ( pra1Δ ) cells , which are almost completely impaired in vacuolar proteolysis , aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen ‐ free medium as compared with wild ‐ type cells . Aliquots of cells incubated in 1 % K ‐ acetate were plated and growing colonies were determined according to Straub et al . ( 1997 ) .

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(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).


Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) and aut7Δ ( C ) cells . Further details are outlined in the text . Crude extracts were prepared from cells starved 4 h on 1 % K ‐ acetate .

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Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.


( A ) Detection of protein - protein interactions using the two ‐ hybrid system . Intense staining indicates an interaction between Aut2p and Aut7p , Aut2p and Tub1p , and Aut2p and Tub2p . AUT7 , TUB1 and TUB2 are cloned in ‐ frame with the GAL4 ‐ activator domain . AUT2 and AUT7 were fused to the GAL4 ‐ binding domain ( see Materials and methods ) .

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(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods).


( B ) Affinity chromatography confirms protein - protein interaction between Aut2p and Aut7p . A fusion protein of Aut2p with the 26 kDa glutathione ‐ S ‐ transferase ( GST ) domain from Schistosoma japonicum ( see Materials and methods ) was bound to glutathione - Sepharose 4B . This GST - Aut2p column was incubated with a crude extract from aut7Δ cen Met25 : : AUT7 - GFP cells . The non ‐ bound supernatant ( S ) fraction ( lane 1 ) was taken away . Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non ‐ specifically bound proteins . After the final 500 mM NaCl wash ( W , lane 2 ) , bound proteins were eluted with Laemmli buffer ( E , lane 3 ) . Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP . Using Aut2p - GST , a clear band corresponding to Aut7p - GFP is seen in the eluate fraction ( lane 3 , arrowhead ) . As a control , GST alone was attached to the column and no band was detectable ( lane 6 ) .

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(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).


( C ) As described in ( B ) , bovine tubulin binds to GST - Aut2p glutathione - Sepharose 4B ( lane 12 ) , but not to GST glutathione - Sepharose 4B ( lane 9 ) . Protein staining with Coomassie is shown in lanes 1 - 6 ; in lanes 7 - 12 tubulin was detected using monoclonal antibodies ( Boehringer Mannheim ) . Further details are given in Materials and methods .

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(C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods.


( D ) Similar to ( C ) , bovine tubulin does not bind to GST - Aut7p glutathione - Sepharose 4B ( lane 12 ) and to GST glutathione - Sepharose 4B ( lane 9 ) . Lanes 1 - 6 , protein staining with Coomassie ; lanes 7 - 12 probed with antibodies directed against tubulin .

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(D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.


Benomyl sensitivity of wild ‐ type cells overexpressing AUT2 and AUT7 , and of aut2Δ and aut7Δ cells is not altered . Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg / ml benomyl . Plates were incubated at 30 ° C .

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Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.


( A ) Autophagosome ‐ like vesicles ( arrowhead ) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1 % K ‐ acetate in the presence of PMSF . Bar represents 100 nm .

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(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.


( B ) Electron micrographs unravels the accumulation of double membrane ‐ layered autophagosome ‐ like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen ‐ free medium in the presence of PMSF . Arrowheads indicate the clearly distinguishable two membrane layers . Bar represents 100 nm .

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(B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.


( C and D ) Cytosol containing autophagosome ‐ like vesicles accumulating in the cytosol of aut2Δ cells ( D ) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction . In wild ‐ type cells ( C ) , no P100 is detectable . Fatty acid synthase ( Fas ) was used as a cytosolic protein . Further details are given in the text .

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(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.


Human placental syncytiotrophoblast cells express markers of cellular senescence , p16 Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - staining for senescence regulators ( A ) p16 Green label : the syncytiotrophoblast marker βHCG , red label : p16 Blue label : DAPI nuclear stain . Arrowheads indicate positively stained syncytiotrophoblast cells βHCG + / p16 + ( A ) Scale bar , 50 µm .

Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 Green label: the syncytiotrophoblast marker βHCG, red label: p16 Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A) Scale bar, 50 µm.


Human placental syncytiotrophoblast cells express markers of cellular senescence , p21 . Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - staining for senescence regulators ( B ) p21 . Green label : the syncytiotrophoblast marker βHCG , red label : p21 . Blue label : DAPI nuclear stain . Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG + / p21 + ( B ) . Scale bar , 50 µm .

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Human placental syncytiotrophoblast cells express markers of cellular senescence, p21. Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (B) p21. Green label: the syncytiotrophoblast marker βHCG, red label: p21. Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG+/p21+ (B). Scale bar, 50 µm.


( C - F ) Sections of human normal third - trimester placentas post - partum ( n = 4 normal placentas ) and IUGR - complicated placentas of the same gestational age ( n = 4 IUGR placentas ) were stained for senescence - related markers of p15 ( C ) , p16 ( D ) , p21 ( E ) , and p53 ( F ) in the syncytiotrophoblast ( n = 3 sections , derived from each normal and IUGR placenta ) . Percentages of positively stained syncytia were quantified ( n = at least 12 fields of view from all sections ) . Scale bar , 100 µm . Arrowheads indicate syncytiotrophoblast cells . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

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(C-F) Sections of human normal third-trimester placentas post-partum (n=4 normal placentas) and IUGR-complicated placentas of the same gestational age (n=4 IUGR placentas) were stained for senescence-related markers of p15 (C), p16 (D), p21 (E), and p53 (F) in the syncytiotrophoblast (n=3 sections, derived from each normal and IUGR placenta). Percentages of positively stained syncytia were quantified (n= at least 12 fields of view from all sections). Scale bar, 100 µm. Arrowheads indicate syncytiotrophoblast cells. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


( G ) Immunoblot analysis of the protein content of p16 , p21 , p53 and DCR in normal ( n = 4 placentas ) and in IUGR - complicated placentas ( n = 4 placentas ) . Each lane represents one independent placenta .

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(G) Immunoblot analysis of the protein content of p16, p21, p53 and DCR in normal (n=4 placentas) and in IUGR-complicated placentas (n=4 placentas). Each lane represents one independent placenta.


( H ) Quantitative RT - PCR analysis of p16 , p21 and DCR2 expression in IUGR - complicated and in normal placentas . Results were obtained from four human normal and four IUGR placentas . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

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(H) Quantitative RT-PCR analysis of p16, p21 and DCR2 expression in IUGR-complicated and in normal placentas. Results were obtained from four human normal and four IUGR placentas. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( A ) Representative T1 - weighted image of a pregnant WT mouse and a selection of four different placentas ( outlined by colored lines ) .

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DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines).


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( B ) Representative SI dynamics of a single WT placenta ( marked in A ) , following biotin - BSA - GdDTPA administered for 60 min . SI parameters of Initial Enhancement and Recovery are illustrated .

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DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( C ) Representative SI dynamics of a single WT placenta ( marked in A ) over time . Scale bar , 2 . 5 mm .

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a and Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) and placental enhancement was monitored at 9 . 4T MRI for 60 min . ( D − G ) Representative SI plots of Cdkn1a - / - ( D ) , p53 - / - ( E ) , Cdkn2a - / - ( F ) , and Cdkn2a - / - ; p53 - / - ( G ) placentas , compared to their WT or HET littermates . ( H − I ) Quantification of the SI Initial Enhancement ( H ) and SI Recovery ( I ) in WT , Cdkn1a - / - , p53 - / - , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas . MRI experiments were repeated at least three times for each murine genotype ( n > = 3 placental SI measurements from each genotype ) . Values are means + SEM ; Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

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

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates. (H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , and Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( A − E ) The labyrinth zone of the WT ( A ) differs from those of Cdkn2a - / - ( B ) and Cdkn2a - / - ; p53 - / - ( C ) placentas . Black arrows indicate regular - sized nuclei in the WT placenta and polyploidy in the Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas . Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas . Labyrinths of p53 - / - ( D ) and Cdkn1a - / - ( E ) placentas did not differ from that of the WT placenta . L , labyrinth . Staining for each placenta was performed in triplicates .

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

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (A−E) The labyrinth zone of the WT (A) differs from those of Cdkn2a-/- (B) and Cdkn2a-/-;p53-/- (C) placentas. Black arrows indicate regular-sized nuclei in the WT placenta and polyploidy in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Labyrinths of p53-/- (D) and Cdkn1a-/- (E) placentas did not differ from that of the WT placenta. L, labyrinth. Staining for each placenta was performed in triplicates.


Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , and Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( F − G ) Ki67 immunostaining in the labyrinth zones of murine WT , p53 - / - , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas , on day E14 . 5 ( n = 3 placentas from each genotype ) . ( F ) Representative images of Ki67 staining in the labyrinth zone are shown . Positive Ki67 trophoblast cells are indicated with arrows . L , labyrinth . ( G ) Proliferation scores ( n = 3 scores , derived from three placentas of each genotype ) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a - / - and Cdkn2a - / - ; p53 - / - placentas relative to WT . Values are means + SEM of three scores for each genotype . * P < 0 . 05 by a one - tailed unpaired Student ' s t - test . Data information : Scale bars , 50 µm .

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (F−G) Ki67 immunostaining in the labyrinth zones of murine WT , p53-/- ,Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas, on day E14.5 (n=3 placentas from each genotype). (F) Representative images of Ki67 staining in the labyrinth zone are shown. Positive Ki67 trophoblast cells are indicated with arrows. L, labyrinth. (G) Proliferation scores (n=3 scores, derived from three placentas of each genotype) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas relative to WT. Values are means +SEM of three scores for each genotype. *P < 0.05 by a one-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.


Histological evaluation of H & E - stained sections in the labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( H ) Representative images of SA - β - gal staining in the labyrinth zones of WT , Cdkn2a - / - , and Cdkn2a - / - ; p53 - / - placentas on day E14 . 5 . ( I ) SA - β - gal activity , quantified by ImageJ sofware in Cdkn2a - / - and Cdkn2a - / - ; p53 - / - labyrinths , is significantly reduced relative to those in WT labyrinth ( n > = 7 fields of view from each genotype ) . Values are means + SEM . ( * * * ) p < 0 . 001 by a two - tailed unpaired Student ' s t - test . Data information : Scale bars , 50 µm .

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

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5) placentas on day E14.5, compared to WT placentas (n=6). (H) Representative images of SA-β-gal staining in the labyrinth zones of WT, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas on day E14.5. (I) SA-β-gal activity, quantified by ImageJ sofware in Cdkn2a-/- and Cdkn2a-/-;p53-/- labyrinths, is significantly reduced relative to those in WT labyrinth (n>=7 fields of view from each genotype). Values are means + SEM. (***) p < 0.001 by a two-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . On days 1 and 5 after seeding , cell fusion was monitored by staining for phalloidin ( F - actin ) ( green ) and DAPI ( blue ) . Scale bar , 50 μm .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( B ) Trophoblast cell fusion was quantified on days 1 , 3 , and 5 post - seeding . Values are means + SEM , derived independently from human placentas of three pregnancies .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (B) Trophoblast cell fusion was quantified on days 1, 3, and 5 post-seeding. Values are means + SEM, derived independently from human placentas of three pregnancies.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( C ) Quantification of the human βhCG hormone , normally secreted by functional syncytiotrophoblast cells , in the medium of a representative trophoblast culture on post - delivery days 2 , 3 and 5 .

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (C) Quantification of the human βhCG hormone, normally secreted by functional syncytiotrophoblast cells, in the medium of a representative trophoblast culture on post-delivery days 2, 3 and 5.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( D ) Gene set enrichment analysis ( GSEA ) of published datasets from human primary trophoblast cultures on days 5 and 2 ( n = 4 trophoblast culture of day 5 and day 2 ) . Gene sets related to pregnancy , aging , immune response , cytokine activity , MAPK , JAK - STAT , NF - κB and metalloproteinase activity were found to be enriched in day 5 cultures , whereas on the same day gene sets related to cell cycle , E2F targets , mitosis and DNA replication were downregulated . Statistical significance was determined by permutation testing with normalized enrichment score ( NES )

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (D) Gene set enrichment analysis (GSEA) of published datasets from human primary trophoblast cultures on days 5 and 2 (n=4 trophoblast culture of day 5 and day 2). Gene sets related to pregnancy, aging, immune response, cytokine activity, MAPK, JAK-STAT, NF-κB and metalloproteinase activity were found to be enriched in day 5 cultures, whereas on the same day gene sets related to cell cycle, E2F targets, mitosis and DNA replication were downregulated. Statistical significance was determined by permutation testing with normalized enrichment score (NES)


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the cell cycle - related genes p16 , p21 and CCNE1 Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the cell cycle-related genes p16, p21 and CCNE1 Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the syncytiotrophoblast markers GCM1 , CGA and CGB ( F ) . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the syncytiotrophoblast markers GCM1, CGA and CGB (F). Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . ( G ) Immunoblot analysis of the indicated proteins in human trophoblast cells , derived independently from two placentas , on days 2 and 5 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (G) Immunoblot analysis of the indicated proteins in human trophoblast cells, derived independently from two placentas, on days 2 and 5.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the SASP components CCL2 , CCL5 , CCL8 , IL6 , IL1β , TGF - β and PDG - FC ( H ) in human primary trophoblast cultures on days 5 and 2 . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the SASP components CCL2, CCL5, CCL8, IL6, IL1β, TGF-β and PDG-FC (H) in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected and cytotrophoblast cells were extracted and seeded . Quantitative RT - PCR analysis of expression of the metalloproteases MMP2 , MMP3 and MMP9 ( I ) , in human primary trophoblast cultures on days 5 and 2 . Data information : Quantitative RT - PCR values are expressed as means + SEM . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . All RT - PCR experiments were performed in triplicates and repeated at least three times . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the metalloproteases MMP2, MMP3 and MMP9 (I), in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


( A ) Immunoblot analysis of the indicated proteins in WT and Cdkn2a - / - ; p53 - / - placentas , on day E14 . 5 ( two independent placentas out of four from each genotype are shown ) .

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

(A) Immunoblot analysis of the indicated proteins in WT and Cdkn2a-/-;p53-/- placentas, on day E14.5 (two independent placentas out of four from each genotype are shown).


RT - PCR analysis of expression of Mmp2 in murine Cdkn2a - / - ; p53 - / - and in WT placentas on day E14 . 5 ( n > = 4 fields of view for each genotype ) . Values are means + SEM of at least three experimental repeats . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

RT-PCR analysis of expression of Mmp2 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


RT - PCR analysis of expression of Mmp9 in murine Cdkn2a - / - ; p53 - / - and in WT placentas on day E14 . 5 ( n > = 4 fields of view for each genotype ) . Values are means + SEM of at least three experimental repeats . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

RT-PCR analysis of expression of Mmp9 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( D , E ) Representative images ( D ) and quantification ( E ) of in - situ gelatin zymography of the labyrinth zone of murine WT and Cdkn2a - / - ; p53 - / - placentas on day E14 . 5 . Scale bar , 50 μm . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

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

(D, E) Representative images (D) and quantification (E) of in-situ gelatin zymography of the labyrinth zone of murine WT and Cdkn2a-/-;p53-/- placentas on day E14.5. Scale bar, 50 μm. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( F , G ) Representative images ( F ) and quantification ( G ) of in - situ zymography of human normal third trimester post - partum placentas and placentas from IUGR - complicated pregnancies of the same gestational age ( n > = 9 fields of view of normal and IUGR placentas ) . Scale bar , 100 µm . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

(F, G) Representative images (F) and quantification (G) of in-situ zymography of human normal third trimester post-partum placentas and placentas from IUGR-complicated pregnancies of the same gestational age (n>=9 fields of view of normal and IUGR placentas). Scale bar, 100 µm. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( H ) Immunoblot analysis of the protein content of p - p65 , p65 , p - STAT3 and STAT3 in normal placentas ( n = 4 ) and placentas from IUGR - complicated pregnancies ( n = 4 ) . Each lane represents one independent placenta .

(H) Immunoblot analysis of the protein content of p-p65, p65, p-STAT3 and STAT3 in normal placentas (n=4) and placentas from IUGR-complicated pregnancies (n=4). Each lane represents one independent placenta.


( I ) Quantitative RT - PCR analysis of expression of MMP2 , MMP9 , IL6 , and the JAK - STAT targets IFNAR1 and IFNAR2 , in normal placentas and placentas from IUGR - complicated pregnancies . Results are from four human normal placentas and four placentas from IUGR - complicated pregnancies . Data information : Values are means + SEM of at least three experimental repeats . Statistical significance was determined by unpaired two - tailed Student ' s t - test ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

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

(I) Quantitative RT-PCR analysis of expression of MMP2, MMP9, IL6, and the JAK-STAT targets IFNAR1 and IFNAR2, in normal placentas and placentas from IUGR-complicated pregnancies. Results are from four human normal placentas and four placentas from IUGR-complicated pregnancies. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


Electron microscopy revealed a number of demyelinated or degenerated axons in PLP - 150Q mice at the age of 5 months . LAQ ( 5 mg / kg ) treatment at 3 months of age for 2 months improved myelination in PLP - 150Q mice . Scale bars : 2μm ( Low magnification ) and 0 . 5 μm ( High magnification ) .

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

Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification).


G - ratios , which were calculated and plotted against axon diameter with linear regression , were shown beneath the micrographs and were significantly decreased in PLP - 150Q mice after LAQ treatment ( G = 0 . 6418 ± 0 . 0191 ) , compared with age - matched vehicle group ( G = 0 . 7727 ± 0 . 0203 ) . One - way ANOVA with Tukey ' s test . * P = 0 . 0166 . At least 182 axons per genotype were examined from 3 mice in each group .

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

G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group.


Western blotting showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months up - regulates multiple myelin proteins ( MBP , MOBP and MOG ) in the corpus callosum in PLP - 150Q mice , which were treated from 3 months of age . Ratios of MBP , MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right . One - way ANNOVA followed with Tukey ' s test . MBP : * * * P = 0 . 0009 ; MOG : * * * P = 0 . 0003 ; MOBP : * * P = 0 . 0067 . Data are mean ±

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

Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM


Quantitative PCR of the transcript expression of myelin associated genes ( MBP and MOG ) in PLP - 150Q mouse corpus callosum at 5 months of age after LAQ ( 5 or 25 mg / kg ) treatment for 2 months . Student ' s t - test . MBP : * * * P = 0 . 0007 ; MOG : * * * P = 0 . 0009 . Data are mean ± SEM ( n = 3 ) .

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Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3).


The MBP DNA promoter was inserted into the pGL4 . 1 luciferase report vector and was co - expressed with N - terminal MYRF ( nMYRF ) and mHTT to assess its transcription activity via the luciferase assay . MYRF markedly enhanced the MBP promoter activity . N - terminal mutant HTT significantly inhibited the reporter activity , * * * P < 0 . 001 ; which was reversed by LAQ ( 5 μM ) treatment , * * P < 0 . 01 . The ratios were obtained from 3 independent experiments . One - way ANNOVA followed with Tukey ' s test . Data are mean ± SEM .

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The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM.


Immunoprecipitation of transgenic mHTT from 3 - month - old PLP - 150Q mouse brains revealed that 5 or 25 mg / kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF . fMYRF : full - length MYRF ; nMYRF : N - terminal MYRF . The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right . One - way ANNOVA followed with Tukey ' s test . * * P = 0 . 009 . Data are mean ± SEM .

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Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.


Western blotting of the phosphorylated - NF - kB , - Akt , - JNK in PLP - 150Q mouse corpus callosum at 3 months of age showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months could dephosphorylate these proteins . Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right . One - way ANNOVA with Tukey ' s test . NF - kB : * * * P = 0 . 00067 ( left ) and 0 . 00086 ( right ) ; JNK : * * * P = 0 . 00013 ( left ) and 0 . 00047 ( right ) ; Akt : * * P = 0 . 0014 and * * * P = 0 . 00012 . Data are mean ± SEM .

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Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM.


In vitro phosphorylation assay of N - terminal MYRF ( nMYRF ) . GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP - 23Q or PLP - 150Q mice that were treated with vehicle or 5 mg / kg LAQ ( left panel ) . GST fusion proteins containing wild type N - terminal MYRF , S259A , or S261A were incubated with PLP - 150Q mouse tissue lysates ( right panel ) . The beads were then centrifuged and analyzed by western blotting with anti - GST ( lower panels ) and anti - phosphor - serine ( upper panels ) . Note that LAQ treatment could eliminate mutant HTT ( PLP - 150Q ) - mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation .

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In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation.


Co - immunoprecipitation of nMYRF and N - terminal HTT ( 1 - 212 aa ) containing 150Q in HEK293 cells . Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co - immunoprecipitated HTT . Λ - PPase served as the positive control for the dephosphorylation of MYRF . The ratios of the pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots , which were obtained from 3 independent western blotting experiments . One - way ANNOVA with Tukey ' s test . * * * P = 6 . 51x10 - 5 . Data are mean ± SEM .

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Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM.


Co - transfection of nMYRF with N - terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ ( 5 μM ) treatment . Coomassie blue staining confirms the presence of immunoprecipitated N - terminal MYRF bands ( arrow ) .

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Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow).


Transcriptional activity of wide type N - terminal MYRF , S259A , or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay . Compared to wide type MYRF or S261A , S259A ( non - phosphorylated ) remained transcriptional activity that was not inhibited by N - terminal mutant HTT . The ratios were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


Co - transfection of wide type N - terminal MYRF , S259A , or S261A with N - terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF . Compared to wide type MYRF and S261A , less S259A ( non - phosphorylated ) was precipitated with mutant HTT . The ratios of the pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P = 2 . 30x10 - 5 . Data are mean ± SEM .

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Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.


Western blotting analysis of the corpus callosum in PLP - 23Q or PLP - 150Q mice with anti - pS259 and an antibody to total MYRF . Note that LAQ ( 5 mg / kg ) treatment for 2 months decreased the phosphorylation in both full length ( fMYRF ) and N - terminal MYRF ( nMYRF ) in PLP - 150Q mice .

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Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice.


Ratios of phosphor - S259 - MYRF ( pMYRF ) and total MYRF to GAPDH . The ratios were obtained from 3 independent western blotting experiments . Student ' s t - test ; * * * P = 0 . 0007 . Data are mean ± SEM .

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Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM.


Anti - Ser259 - MYRF immunohistochemical staining of the corpus callosum ( CC , indicated between two dotted lines ) of PLP - 23Q and PLP - 150Q mice showing decreased expression of Ser259 - MYRF by LAQ ( 5 mg / kg ) treatment for 2 months . Micrographs at 10X ( upper ) and 40X ( lower ) are shown . Scale bars : 40 μm .

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Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm.


Quantitative statistics of the cells stained by anti - Ser259 MYRF in each filed was presented . Twenty random fields ( 40X ) of each section were examined to count anti - Ser259 positive cells , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test . * * * P = 0 . 0003 ; * * P = 0 . 0039 . Data are mean ± SEM .

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Quantitative statistics of the cells stained by anti-Ser259 MYRF in each filed was presented. Twenty random fields (40X) of each section were examined to count anti-Ser259 positive cells, n=3 mice in each group. One-way ANNOVA with Tukey's test. ***P=0.0003; **P=0.0039. Data are mean ±SEM.


Western blotting analysis of MBP and Ser259 - MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non - HD control individuals . Ratios of MBP to GAPDH , or Ser259 ( pMYRF ) to total MYRF are presented on the right . One - way ANNOVA with Tukey ' s test . MBP : * * * P = 0 . 0004 ; pMYRF : * * P = 0 . 0051 . Data are mean ± SEM .

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Western blotting analysis of MBP and Ser259-MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non-HD control individuals. Ratios of MBP to GAPDH, or Ser259 (pMYRF) to total MYRF are presented on the right. One-way ANNOVA with Tukey's test. MBP: ***P=0.0004; pMYRF: **P=0.0051. Data are mean ±SEM.


Western blotting analysis of the expression of Ser259 ( pMYRF ) and total MYRF ( full - length : fMYRF and N - terminal : nMYRF ) in 13 - months - old HD - KI and WT mouse cortex . Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots . One - way ANNOVA with Tukey ' s test . * * * P = 0 . 00083 . Data are mean ± SEM .

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Western blotting analysis of the expression of Ser259 (pMYRF) and total MYRF (full-length: fMYRF and N-terminal: nMYRF) in 13-months-old HD-KI and WT mouse cortex. Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots. One-way ANNOVA with Tukey's test. ***P=0.00083. Data are mean ±SEM.


Heatmaps of gene expression in PLP - 150Q mouse corpus callosum at 5 months after LAQ ( 5 mg / kg ) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes .

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Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes.


The individual networks of up - regulated gene were heat mapped typically , on the clusters of " REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS " , and " POSITIVE _ REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS "

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The individual networks of up-regulated gene were heat mapped typically, on the clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC _PROCESS"


Western blotting showing down - regulation of PRKG2 in PLP - 150Q mouse corpus callosum at 3 months of age by 5 mg / kg LAQ . Ratios of PRKG2 or phosphorylated VASP ( p - VASP ) to GAPDH obtained from 3 independent experiments were presented on the right . One - way ANNOVA with Tukey ' s test . PRKG2 : * * P = 0 . 0076 ( left ) and * * * P = 0 . 0015 ( right ) ; p - VASP : * * * P = 3 . 86x10 - 5 ( left ) and 1 . 01x10 - 5 ( right ) . Data are mean ± SEM .

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Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM.


Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP - 150Q mouse corpus callosum at 5 months of age after LAQ ( 5 mg / kg ) treatment for 2 months . Only PRKG2 was decreased by LAQ . n = 3 mice in each group . Student ' s t - test ; * * P = 0 . 0073 ; * * * P = 0 . 00041 . Data are mean ± SEM .

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Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.


The different PRKG2 promoter regions were inserted into the pGL4 . 1 luciferase report vector . The core promoter reporter vector of PRKG2 ( - 472 / + 131 ) was determined for transcription activity in cultured N2a cells , obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s test ; * * * P < 0 . 001 . Data are mean ± SEM .

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The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from 3 independent experiments. One-way ANNOVA with Tukey's test; ***P<0.001. Data are mean ±SEM.


The treatment of LAQ ( 5 or 10 μM ) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM.


The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a , which were mediated by AhR , in the PLP - 150Q mouse brain ( corpus callosum ) . Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ ( 5 mg / kg ) . n = 3 mice in each group . Student ' s t - test ; Cyp1a1 : * * * P = 0 . 00042 ; Ugt1a6a ; * * * P = 0 . 00057 . Data are mean ± SEM .

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The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM.


The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ . The values of promoter activity via luciferase report assay were obtained from three independent experiments . One - way ANNOVA with Tukey ' s test . * * * P < 0 . 001 . Data are mean ± SEM .

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The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


The semi - PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP assay . The N2a cells were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP assay . The N2a cells were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . The results were obtained from three independent experiments . One - way ANNOVA with Tukey ' s test . LAQ : * * * P = 0 . 00065 ; 2 , 3 , 7 , 8 - TCDD : * * * P = 0 . 00014 . Data are mean ±

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The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM


Western blotting showing that the phosphorylation of transfected N - terminal MYRF ( pMYRF ) was promoted by treatment with the PKG activator of 8 - Br - cGMP ( 10 or 100 μM ) for 1 hr or co - transfected 150Q - HTT for 24 hr , which was blocked by treatment of the PKG inhibitors of RKRARKE ( 50 μM ) or KT5823 ( 5 μM ) for 12 hr . trans - nMYRF : transfected N - terminal MYRF .

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Western blotting showing that the phosphorylation of transfected N-terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8-Br-cGMP (10 or 100 μM) for 1 hr or co-transfected 150Q-HTT for 24 hr, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 hr. trans-nMYRF: transfected N-terminal MYRF.


Western blotting of N2a cell line that was transfected with AAV - PRKG2 for 48 hr . The blots were probed with anti - Ser259 ( pMYRF ) antibody . Note that overexpression of PRKG2 increased phosphorylation of N - terminal MYRF ( nMYRF ) in a dose - dependent manner .

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Western blotting of N2a cell line that was transfected with AAV-PRKG2 for 48 hr. The blots were probed with anti-Ser259 (pMYRF) antibody. Note that overexpression of PRKG2 increased phosphorylation of N-terminal MYRF (nMYRF) in a dose-dependent manner.


Stereotaxic injection of AAV - GFP virus into the corpus callosum ( CC , indicated between two dotted lines ) in the mouse brain . Ctx : cortex , Str : striatum . Scale bar : 100 μm . . Immunohistochemical staining with anti - Ser259 ( pMYRF ) showing that AAV - PRKG2 , but not AAV - GFP , promotes MYRF phosphorylation in the corpus callosum . Scale bar : 40 μm .

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Stereotaxic injection of AAV-GFP virus into the corpus callosum (CC, indicated between two dotted lines) in the mouse brain. Ctx: cortex, Str: striatum. Scale bar: 100 μm..   Immunohistochemical staining with anti-Ser259 (pMYRF) showing that AAV-PRKG2, but not AAV-GFP, promotes MYRF phosphorylation in the corpus callosum. Scale bar: 40 μm.


Quantitative analysis of the pMYRF - or total MYRF - positive cells in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 8 . 79x10 - 5 . Data are mean ± SEM .

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Quantitative analysis of the pMYRF- or total MYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=8.79x10-5. Data are mean ±SEM.


Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV - PRKG2 , but not AAV - GFP ( green ) , promotes MYRF phosphorylation ( red ) in the injected corpus callosum . Scale bar : 40 μm .

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Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV-PRKG2, but not AAV-GFP (green), promotes MYRF phosphorylation (red) in the injected corpus callosum. Scale bar: 40 μm.


Western blotting analysis of the mouse corpus callosum tissues using anti - Ser259 ( pMYRF ) , phosphor - PRKG2 ( pPRKG2 ) or phosphor - VASP ( pVASP ) . Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP . fMYRF : full - length MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the mouse corpus callosum tissues using anti-Ser259 (pMYRF), phosphor-PRKG2 (pPRKG2) or phosphor-VASP (pVASP). Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Construction of AAV - U6 - gRNA - CMV - RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter , respectively . AAV - gRNA - PRKG2 was injected into the corpus callosum ( CC , indicated between two dotted lines ) in PLP - 150Q / Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection ( lower panel ) . Ctx : cortex , Str : striatum , LV : lateral ventricle . Scale bar : 100

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Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm


Western blotting analysis of the PLP - 150Q / Cas9 mouse corpus callosum tissues using the antibody to Ser259 ( pMYRF ) , phosphor - PRKG2 or MBP . Note that reduction of mouse PRKG2 by CRISPR / Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP . The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots . Student ' s t - test ; pMYRF : * * * P = 0 . 00026 ; MBP ; * * P = 0 . 0036 . Data are presented as mean ± SEM . fMYRF : full - length MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2 or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots. Student's t-test; pMYRF: ***P=0.00026; MBP; **P=0.0036. Data are presented as mean ±SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP - 150Q mouse . Scale bar : 40 μm .

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Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 μm.


Quantitative analysis of the pMYRF - positive cells in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 0 . 00022 . Data are mean ± SEM .

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Quantitative analysis of the pMYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=0.00022. Data are mean ±SEM.


Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV - PRKG2 - gRNA ( red ) injection increased MBP expression ( green ) as compared with AAV - gRNA - control . Scale bar : 40 μm .

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Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 μm.


Quantitative analysis of the MBP - positive cells in the corpus callosum injected with control - gRNA or PRKG2 - gRNA . Twenty random fields ( 40X ) in each section were examined . n = 3 mice in each group . One - way ANNOVA with Tukey ' s test ; * * * P = 4 . 43x10 - 5 . Data are mean ± SEM .

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Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40X) in each section were examined. n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=4.43x10-5. Data are mean ±SEM.


( A ) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting . For comparison , different amounts of proteins ( 1 : 3 ratio ) were applied . An asterisk indicated non - specific crossreactive bands .

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(A) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting. For comparison, different amounts of proteins (1:3 ratio) were applied. An asterisk indicated non-specific crossreactive bands.


( B ) The indicated cell lines were analyzed by immunofluorescence microscopy using anti - Cytochrome c antibody for mitochondria and anti - PMP70 antibody for peroxisome staining . Images are displayed as z - stacks of 6 confocal slices . Magnified images are also shown for mitochondrial morphologies . Scale bars , 10 μm . ( C ) Quantification of mitochondrial morphologies in ( B ) . Percentages of cells harboring fragmented , tubular , or elongated mitochondria are shown . Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network , respectively . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each of three replicate wells .

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(B) The indicated cell lines were analyzed by immunofluorescence microscopy using anti-Cytochrome c antibody for mitochondria and anti-PMP70 antibody for peroxisome staining. Images are displayed as z-stacks of 6 confocal slices. Magnified images are also shown for mitochondrial morphologies. Scale bars, 10 μm. (C) Quantification of mitochondrial morphologies in (B). Percentages of cells harboring fragmented, tubular, or elongated mitochondria are shown. Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each of three replicate wells.


( A ) The indicated cell lines stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) Quantification of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes . Partial and complete denote that Parkin translocates to some of or all mitochondria , respectively . The error bars represent ± SD from three independent experiments . Over 100 cells were counted in each of three separate wells . ( C ) YFP - LC3 morphologies in ( A ) were quantified . Percentages of cells harboring diffuse , punctate or accumulated YFP - LC3 are shown . The error bars represent ± SD from three independent replicates . Over 100 cells were counted in each replicate . For the criteria of LC3 morphology , see Figure 2 - figure supplement 1 .

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(A) The indicated cell lines stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. The error bars represent ±SD from three independent experiments. Over 100 cells were counted in each of three separate wells. (C) YFP-LC3 morphologies in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. For the criteria of LC3 morphology, see Figure 2-figure supplement 1.


( D ) YFP - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the absence or presence of HA - tagged TBC1D15 WT or Δ221 - 250 mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( E ) The YFP - LC3 morphology of cells in ( D ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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(D) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the absence or presence of HA-tagged TBC1D15 WT or Δ221-250 mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (E) The YFP-LC3 morphology of cells in (D) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F and G ) YFP - LC3 and mCherry - Parkin stably expressing WT ( F ) and TBC1D15 − / − ( G ) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti - GFP antibody . The square in panel a shows enlarged areas in panel b Scale bars , 500 nm .

(F and G) YFP-LC3 and mCherry-Parkin stably expressing WT (F) and TBC1D15−/− (G) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. The square in panel a shows enlarged areas in panel b Scale bars, 500 nm.


( A ) The indicated cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti - Cytochrome c antibody . Confocal images were acquired as z - stacks comprising 6 sequential sections with 0 . 8 μm z - intervals . Scale bars , 10 μm .

(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm.


( B ) TBC1D15 − / − cells prepared as in ( A ) were subjected to immunoelectron microscopy with anti - GFP antibody . White arrowheads indicate YFP - LC3 - labeled tubules . High magnification image is shown in the lower panel . Scale bars , 500 nm .

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(B) TBC1D15−/− cells prepared as in (A) were subjected to immunoelectron microscopy with anti-GFP antibody. White arrowheads indicate YFP-LC3-labeled tubules. High magnification image is shown in the lower panel. Scale bars, 500 nm.


( C ) The indicated cells prepared as in ( A ) were subjected to immunostaining with anti - Tubulin antibody . YFP - LC3 and Tubulin staining are merged in the right panels . Magnified images are shown for TBC1D15 − / − cells . Scale bars , 10 μm .

(C) The indicated cells prepared as in (A) were subjected to immunostaining with anti-Tubulin antibody. YFP-LC3 and Tubulin staining are merged in the right panels. Magnified images are shown for TBC1D15−/− cells. Scale bars, 10 μm.


( D ) The indicated cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr . YFP - LC3 and mCherry - Parkin images are merged in the right panels . Scale bars , 10 μm . ( E ) YFP - LC3 morphologies of cells in ( D ) were quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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(D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A and B ) YFP - Parkin stably expressing cells were treated with valinomycin for indicated times . Total cell lysates were subjected to immunoblotting . I and II denote cytosolic and lipidated LC3B , respectively . An asterisk indicates ubiquitinated TOMM20 . ( C ) Indicated protein amounts as in ( B ) were quantified . The amount of protein without valinomycin treatment was set to 100 % . The error bars represent ± SD from three independent experiments .

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(A and B) YFP-Parkin stably expressing cells were treated with valinomycin for indicated times. Total cell lysates were subjected to immunoblotting. I and II denote cytosolic and lipidated LC3B, respectively. An asterisk indicates ubiquitinated TOMM20. (C) Indicated protein amounts as in (B) were quantified. The amount of protein without valinomycin treatment was set to 100%. The error bars represent ±SD from three independent experiments.


( D ) YFP - Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr . Total cell lysates were analyzed by immunoblotting .

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(D) YFP-Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr. Total cell lysates were analyzed by immunoblotting.


( A ) WT , MFF − / − or DRP1 − / − cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) Quantification of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial and complete denote that Parkin translocates to some of and all mitochondria , respectively . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each replicate . ( C ) YFP - LC3 morphologies of cells in ( A ) were quantified . Percentages of cells harboring diffuse , punctate or accumulated YFP - LC3 are shown . The error bars represent ± SD from three independent replicates . Over 100 cells were counted in each replicate .

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(A) WT, MFF−/− or DRP1−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of and all mitochondria, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate. (C) YFP-LC3 morphologies of cells in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate.


( D ) WT , FIS1 − / − , and TBC1D15 − / − cells stably expressing YFP - LC3 were grown in starvation media . Z - stacks of confocal images are shown . Scale bars , 20 μm . ( E ) The number of YFP - LC3 dots in cells under growth or starvation conditions was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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(D) WT, FIS1−/−, and TBC1D15−/− cells stably expressing YFP-LC3 were grown in starvation media. Z-stacks of confocal images are shown. Scale bars, 20 μm. (E) The number of YFP-LC3 dots in cells under growth or starvation conditions was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F ) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting . LC3 - I and LC3 - II denote cytosolic and lipidated LC3B , respectively .

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(F) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting. LC3-I and LC3-II denote cytosolic and lipidated LC3B, respectively.


( A ) TBC1D15 − / − cells and those stably expressing HA - TBC1D15 WT and HA - TBC1D15 ( Δ221 - 250 ) , and FIS1 − / − cells stably expressing HA - TBC1D15 WT were subjected to immunostaining with anti - TOMM20 and anti - HA antibodies . Scale bars , 10 μm .

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(A) TBC1D15−/− cells and those stably expressing HA-TBC1D15 WT and HA-TBC1D15 (Δ221-250), and FIS1−/− cells stably expressing HA-TBC1D15 WT were subjected to immunostaining with anti-TOMM20 and anti-HA antibodies. Scale bars, 10 μm.


( B ) HA - TBC1D15 WT or HA - TBC1D15 ( Δ221 - 250 ) with or without Fis1 was transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - HA and anti - Fis1 antibodies . Scale bars , 20 μm .

(B) HA-TBC1D15 WT or HA-TBC1D15 (Δ221-250) with or without Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Scale bars, 20 μm.