Figure 5 ( 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 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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.(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.(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.


Figure 1Electron 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 . 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 ± SEMQuantitative 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 . 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 .

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

Figure 1Electron 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.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 ±SEMQuantitative 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.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.


Figure 2Western 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 . 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 ) . 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 .

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

Figure 2Western 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.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).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.


Figure 3Western 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 . 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 . 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 .

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

Figure 3Western 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.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.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.


Figure 4Heatmaps 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 " 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 .

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

Figure 4Heatmaps 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"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.


Figure 5The 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 . 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 . 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 ±

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

Figure 5The 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.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.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


Figure 6Western 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 . 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 . 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 .

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

Figure 6Western 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.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.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.


Figure 7Construction 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 μmWestern 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 . 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 . 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 .

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

Figure 7Construction 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 μmWestern 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.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.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.


Figure 1 . ( 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 .

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

Figure 1.(A) 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.


Figure 2 . ( 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 . ( 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 .

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

Figure 2.(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.(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.


Figure 3 . ( 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 . ( 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 .

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

Figure 3.(A) 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.(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.


Figure 4 . ( 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 .

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

Figure 4.(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.


Figure 5 . ( 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 . ( 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 .

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

Figure 5.(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.(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.


Figure 6 . ( 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 . ( C ) YFP - TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - Fis1 and anti - Cytochrome c antibodies . Scale bars , 20 μm . ( D ) YFP or YFP - Fis1 was co - overexpressed with HA - TBC1D15 or HA - TBC1D17 in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies . ( E ) YFP , YFP - TBC1D15 , or YFP - TBC1D17 was co - overexpressed with HA - TBC1D15 ( upper panel ) or HA - TBC1D17 ( lower panel ) in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP and anti - HA antibodies . ( F ) HA - TBC1D15 ( Δ221 - 250 ) together with Fis1 and YFP - TBC1D15 WT or YFP - TBC1D17 WT were transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - HA and anti - Fis1 antibodies . Images of HA and Fis1 staining were merged in the right panels . Scale bars , 20 μm .

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

Figure 6.(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.(C) YFP-TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-Fis1 and anti-Cytochrome c antibodies. Scale bars, 20 μm.(D) YFP or YFP-Fis1 was co-overexpressed with HA-TBC1D15 or HA-TBC1D17 in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.(E) YFP, YFP-TBC1D15, or YFP-TBC1D17 was co-overexpressed with HA-TBC1D15 (upper panel) or HA-TBC1D17 (lower panel) in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP and anti-HA antibodies.(F) HA-TBC1D15 (Δ221-250) together with Fis1 and YFP-TBC1D15 WT or YFP-TBC1D17 WT were transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Images of HA and Fis1 staining were merged in the right panels. Scale bars, 20 μm.


Figure 7 . ( A ) The indicated cells transiently expressing YFP - GABARAPL1 ( green ) and mCherry - Parkin ( red ) were treated with valinomycin for 3 hr . Scale bars , 10 μm . ( B ) YFP - TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST - fused proteins ( GABA , L1 , and L2 represent GABARAP , GABARAPL1 , and GABARAPL2 , respectively ) . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) . Coomassie brilliant blue ( CBB ) staining shows GST - fusion proteins in bound fractions ( lower panel ) . ( C ) Binding assay carried out as in ( B ) with GST - GABARAPL1 WT ( GST - WT ) or its Y49A / L50A mutant ( GST - YL ) . Immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) are shown . ( D ) Cell extracts from HEK293 overexpressed HA - TBC1D15 and YFP , YFP - GABARAPL1 ( YFP - WT ) , or its Y49AL50A mutant ( YFP - YL ) were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies . ( E - H ) The indicated YFP - tagged TBC1D15 full - length , truncated , or point - mutant protein or YFP - Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST - GABARAPL1 . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) . ( J ) LC3 - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( K ) The YFP - LC3 morphology of cells in ( J ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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

Figure 7.(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm.(B) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown.(D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.(E-H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel).(J) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


Figure 8 . ( A ) Total cell lysates from HCT116 treated with control ( NTC ) or RAB7A siRNA were analyzed by immunoblotting . ( B ) The indicated cells stably expressing YFP - LC3 ( green ) and mCherry - Parkin were treated with control ( NTC ) or Rab7 _ # 5 siRNA . After 3 hr valinomycin treatment , cells were analyzed by immunofluorescence microscopy with anti - TOMM20 antibody ( red ) . Z - stacks of confocal images are shown . Magnified images are also shown . Scale bars , 10 μm . ( C ) YFP - LC3 morphologies of cells in ( B ) were quantified . Percentages of cells harboring diffuse , punctuate or accumulated / tubulated YFP - LC3 are shown . Data and error bars were obtained from at least 50 cells in each of three independent replicates . ( D ) The indicated cells stably expressing YFP - LC3 ( green ) , mCherry - Parkin , and 2HA - Rab7 ( Red ) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti - HA antibody . Magnified images are also shown . Scale bars , 10 μm . ( E ) YFP - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( F ) The YFP - LC3 morphology of cells in ( E ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each replicate .

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

Figure 8.(A) Total cell lysates from HCT116 treated with control (NTC) or RAB7A siRNA were analyzed by immunoblotting.(B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates.(D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm.(E) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.


Figure 1Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib , Nilotinib , Bosutinib and DMSO ( N = 7 ) . [ Correction added after publication on 4 July 2013 : The y axis of the graph shown in Fig . 1A has been corrected from " concentration ( mM ) " to " concentration ( nM ) " ] WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Abl ( top blot ) T412 Abl ( 2nd blot ) , soluble parkin level ( 3rd blot ) , and LC3 ( 4th blot ) relative to MAP - 2 ( N = 9 ) . Quantitative ELISA showing soluble ( STEN extract ) and insoluble ( 4 M urea extract ) mouseparkin in Tg - APP ( N = 9 ) . Parkin − / − brains were used as a specificity control . ( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin antibody ( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin antibody , and ( E ) control IgG in parallel with immunoprecipitates . ( F ) immunoprecipitatedBeclin - 1 probed with parkin and the reverse experiment in post - mortem human AD cortex analyzed on 4 - 12 % SDS - NuPAGE gel . In situ proximity ligation assay ( PLA ) shows endogenous parkin - Beclin - 1 complexes in ( G ) WT C57BL / 6mice ( N = 5 ) and ( H ) parkin − / − as control . PLA in Tg - APPmice IP injected once daily for 3 weeks with ( I ) DMSO ( J ) 5 mg / kg Bosutinib and ( K ) 10 mg / kg Nilotinib ( N = 5 ) . PLA in human post - mortem brains in the ( L ) cortex of a normal subject and ( M ) cortex of an AD patient ; the hippocampus of ( N ) a normal subject and ( O ) an AD patient ; the caudate of ( P ) a normal subject and ( Q ) an AD patient . Graph represents human Aβ1 - 42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1 - 42 ( or LacZ ) or Beclin - 1 shRNA for 24 hr , and then treated with 1 μM Bosutinib for an additional 24 hr ( N = 12 ) . * Significantly different to control or as indicated , Mean ± SEM , ANOVA with NeumannKeuls multiple comparison .

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

Figure 1Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from "concentration (mM)" to "concentration (nM)"]WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9).Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouseparkin in Tg-APP (N = 9). Parkin−/−brains were used as a specificity control.(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates.(F) immunoprecipitatedBeclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4-12% SDS-NuPAGE gel.In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient.Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.


Figure 2Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different ( A , B ) Tg - APP + DMSO and ( C ) thioflavin - S staining in Tg - APP treated with DMSO ( N = 7 ) . ( D , E ) Tg - APP treated with 5 mg / kg Bosutinib for 3 - weeks and ( F ) thioflavin - s staining . Staining of 20 μm thick brain sections shows ( G ) parkin , ( H ) Aβ1 - 42 and ( I ) merged figure in cortex of Tg - APPmice after 3 weeks of DMSO treatment , and ( J ) parkin , ( K ) Aβ1 - 42 and ( L ) merged figure in cortex of Tg - APPmice after 3 weeks of 5 mg / kg Bosutinib treatment . ( M ) Parkin , ( N ) Aβ1 - 42 and ( O ) merged figure in hippocampus of Tg - APPmice after 3 weeks of DMSO treatment , and ( P ) parkin , ( Q ) Aβ1 - 42 and ( R ) merged figure in cortex of Tg - APPmice after 3 weeks of Bosutinib treatment ( N = 7 ) . ( S ) Graphs represent quantification of amyloidplaques in Tg - APP with and without Bosutinib . WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( T ) levels of BACE1 ( 1st blot ) , ADAM - 10 ( 2nd blot ) and presinilin - 1 ( 3rd blot ) relative to actinWB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( U ) total APP ( top blot ) , CTFs ( 2nd blot ) and phospho - tyrosine ( 3rd blot ) relative to actin ( N = 9 ) .

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

Figure 2Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1-42 and (I) merged figure in cortex of Tg-APPmice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1-42 and (L) merged figure in cortex of Tg-APPmice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1-42 and (O) merged figure in hippocampus of Tg-APPmice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1-42 and (R) merged figure in cortex of Tg-APPmice after 3 weeks of Bosutinib treatment (N = 7).(S) Graphs represent quantification of amyloidplaques in Tg-APP with and without Bosutinib.WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (T) levels of BACE1 (1st blot), ADAM-10 (2nd blot) and presinilin-1 (3rd blot) relative to actinWB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (U) total APP (top blot), CTFs (2nd blot) and phospho-tyrosine (3rd blot) relative to actin (N = 9).


Figure 3Graphs represent ELISA of human Aβ1 - 42 in ( A ) brain levels and ( B ) bloodAβ1 - 42 levels in 8 - month old Tg - APPmice injected I . P . every other day for 6 weeks with Bosutinib or Nilotinib ( N = 10 ) . ELISA of human soluble and insoluble brain ( C ) Aβ1 - 42 and ( D ) Aβ1 - 40 levels in 8 - month old Tg - APPmice injected IP daily for 3 weeks with 5 mg / kg Bosutinib ( N = 9 ) . ELISA of ( E ) mouse p - Tau levels in 8 - month old Tg - APP mice injected IP daily for 3 weeks with 5 mg / kg Bosutinib ( N = 9 ) . WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Tau ( 1st blot ) , serine 396 p - Tau ( 2nd blot ) , threonine 231 ( AT180 , 3rd blot ) and serine 199 p - Tau ( AT8 , 4th blot ) relative to actin ( N = 7 ) .

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

Figure 3Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).


Figure 4Staining of 20μm brain sections shows intracellular Aβ1 - 42 within the ( A ) hippocampus of lentiviralAβ1 - 42 injected WT mice , and ( B ) Bosutinib clearance of intracellular Aβ1 - 42 . Staining of 20μm brain sections shows intracellular Aβ1 - 42 within the ( C ) cortex of WT mice with the lentiviralAβ1 - 42 , and ( D ) Bosutinib clearance of intracellular Aβ1 - 42 ( N = 7 ) . ELISA of human soluble and insoluble Aβ1 - 42 at 6 weeks post - injection of lentiviralAβ1 - 42 and daily treatment with 5 mg / kg Bosutinib ( N = 9 ) for 3 weeks . * Significantly different to control or as indicated , Mean ± SEM , ANOVA with Neumann Keuls multiple comparison . WB on 4 - 12 % NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin − / − mice treated with 5 mg / kg Bosutinib for 3 weeks on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing parkin ( 1st blot ) total Abl ( 2nd blot ) , T412 Abl ( 3rd blot ) , Beclin - 1 ( 4th blot ) and LC3 ( 5th blot ) relative to actin ( N = 7 ) . Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post - injection with ( G ) lentiviralAβ1 - 42 + 3 weeks DMSO treatment and ( H ) IP injection with 5 mg / kg Bosutinib 3 weeks post - lentiviral expression ( 3 weeks treatment ) clears plaques in WT mice ( N = 7 ) . ( I ) Lentiviral Aβ1 - 42 + DMSO and ( J ) IP injection with 5 mg / kg Bosutinib 3 weeks post - lentiviral expression ( 3 weeks treatment ) in parkin − / − mice ( N = 7 ) .

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

Figure 4Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7).ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).


Figure 5Graphs represent ELISA ( N = 5 ) in autophagic vacuoles ( AVs ) in the brain of 4 and 8 months old Tg - APPmice treated with 10 mg / kg Nilotinib or 5 mg / kg Bosutinib ( N = 5 ) for 3 weeks showing ( A ) humanAβ1 - 42 ( Insert is WB analysis of AVs showing LC3 - B in AV10 and AV20 ( 1st blot ) and LAMP2a in Lys ( 2nd blot ) fraction ) and ( B ) Aβ1 - 40 , ( C ) p - Tau and ( D ) parkin . ELISA in autophagic vacuoles of 1 year old WT and parkin − / − mice ( N = 5 ) injected with lentiviralAβ1 - 42 for 3 weeks and treated with 10 mg / kg Nilotinib and 5 mg / kg Bosutinib for 3 additional weeks showing ( E ) humanAβ1 - 42 and ( F ) p - Tau levels .

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

Figure 5Graphs represent ELISA (N = 5) in autophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APPmice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)humanAβ1-42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.ELISA in autophagic vacuoles of 1 year old WT and parkin−/−mice (N = 5) injected with lentiviralAβ1-42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) humanAβ1-42 and (F) p-Tau levels.


Figure 6 ( A ) Represents results of Morris water maze test in lentiviralAβ1 - 42 - injected ± Bosutinib WT ( N = 12 ) and parkin − / − ( N = 10 ) mice , and ( B ) heat maps for each group . Graphs represent total number of entry into platform area and distance travelled . Represents results of Morris water maze test in Tg - APP ± Bosutinib ( N = 12 ) mice . Graphs represent total number of entry into platform area and distance travelled .

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

Figure 6(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled.Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.


Figure 1 ( A ) Immuno - precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4 ° C overnight . The control antibody ( ab ) is a rabbit anti - yeast Mad2 . ( B ) Single confocal section of a population of S2 cells . Examples of interphase cells are boxed in blue . Mitotic cells positive for the anti - phosphoHistone3 ( pH3 ) are boxed in red . ( C ) Single confocal section of a S2 cell . Anti - Incenp , anti - Cid mark the centromeric chromatin and the inner KT , respectively . ( D - E ) Maximal projections of S2 cells imaged by STED microscopy . 7 z - sections for a total of 1 . 5 um ( D ) and 4 z - sections for a total of 0 . 5 um ( E ) are shown . Anti - CenpC and anti - Spc105R mark the proximal and the distal region of different portions of the outer KT , respectively . ( F - G ) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29 , nuclear pores ( WGA ) , and CyclinB ( F ) , or Snap29 , Spc105R , and the nuclear lamina ( LaminA ) ( G ) . Note the transient perinuclear enrichment of Snap29 in early prophase . WGA stains also cytoplasmic speckles in hemocyte - derived S2 cells ( Rizki Rizki , 1983 ) . High magnification of the insets in C - G shows localization of Snap29 relative to the indicated markers . ( H - J ) Immuno - gold localization of Snap29 in cryo - sections of S2 cells in pro - metaphase . Pseudo - coloring in red identifies chromosomes ( Ch ) , in yellow membrane organelles and in green MTs . The white arrows in H - I indicate examples of signal in the nucleoplasm that is not associated to membranes , but rather , to electron - dense material . MVB : multivesicular body ; NE : nuclear envelope ; PM : plasma membrane .

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

Figure 1(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2.(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.(C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively.(D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively.(F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers.(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.


Figure 2 ( A ) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs . A cartoon of full length Snap29 is shown above the control panel . ( B ) Quantification of the Snap29 signal as in panel A , considering > 30 KTs per sample . Relative mRNA expression of the downregulated genes versus control , measured by Q - PCR , is shown above the graph . Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs . ( C ) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over - expressed . Cartoons of the expressed Snap29 mutant forms are shown above the panels . Anti - pH3 labels mitotic cells and CenpC marks KTs . ( D ) Quantification of Snap29 as in panel C , considering > 28 KTs per sample . Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells .

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

Figure 2(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel.(B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs.(C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells.


Figure 3 ( A ) Selected frames of time - lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29 . Note that depleted cell does not display a recognizable metaphase plate , or fail to segregate all chromosomes correctly , or form tripolar spindles or micronuclei ( arrowheads ; arrowheads in control point to cells dividing normally ) . ( B ) Western blotting of extract from control cells and cells depleted of Snap29 ( dsSnap29 ) relative to the experiment in a . ( C ) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells , based on time - lapse imaging of an average of > 28 individual cells per sample . P value is determined by two - tailed t - test considering all defects together . ( D ) Selected frames of time - lapse imaging of control U2OS cells and U2OS cells treated for the indicated time . Note that the depleted cell at 48 - 66h display altered metaphase plate and chromosome segregation ( arrowheads ) . ( E ) Western blot analysis of extracts relative to the experiments in panel d . ( F ) Quantification of the time from prophase to entry in anaphase based on analysis of > 20 individual cells / sample by time - lapse microscopy . P values are determined by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . OE : Over - expression .

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

Figure 3(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally).(B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a.(C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together.(D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads).(E) Western blot analysis of extracts relative to the experiments in panel d.(F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.


Figure 4 ( A ) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4 ° C and immunostained to reveal the presence of unattached kinetochores . CENPT labels all KTs , while MAD1 identifies unattached ones . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate . ( B ) Quantification of the number of KTs based on attachment , position and MAD1 positivity . Each point per category represents one experiment in which 10 random KTs / cell of 20 cells / sample were counted . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison test . Note that in control cells most of the KT are attached , MAD1 - negative and aligned to the metaphase plate , while in SNAP29KD cells half of the KTs that are unattached , MAD1 - positive and not aligned to the metaphase plate . ( C ) Single confocal sections of HeLa cells . CREST and CENPT were used to label the centromere region and associated KTs , respectively . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown . ( D ) Average of the inter KT distance based on in each of which 10 chromosomes / cell and 10 cells / sample were counted . P values are obtained by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs . ( E ) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA . ( F ) Averaged spindle pole length based on > 20 cells with bipolar spindles / sample . The P value is obtained by two - tailed paired t - test .

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

Figure 4(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.(C) Single confocal sections of HeLa cells. CREST and CENPT were used to label the centromere region and associated KTs, respectively. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown. (D) Average of the inter KT distance based on in each of which 10 chromosomes/cell and 10 cells/sample were counted. P values are obtained by Kruskal-Wallis test with Dunn's multiple comparison analysis. Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs.(E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.