G , H When tumors reached 100 mm3 , mice were injected subcutaneously with decitabine ( 0 . 4 mg / kg ) or PBS and / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . ( G ) Tumor volumes were measured twice a week . The statistical significance of the differences obtained between the control PBS - group and treated ( DAC + net1 - mAb ) group was determined by two - way ANOVA and post - hoc Tukey - test . * * * P < 0 . 0001 . ( H ) Tumor weights were measured 3 days after the end of the experiment . The statistical significance of the differences obtained between the groups was determined by one - way ANOVA , * * * P < 0 . 0001 . Error bars = s . e . m .

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

G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.


A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors , compared with the control PBS - group . The percentage of mean DNA methylation of the 11 DAPK1 - CpGs was 94 % ( 550 amplicons analyzed ) , while that of the 7 NTN1 - CpGs was 64 % ( 213 amplicons analyzed ) . Two - way ANOVA and post - hoc Tukey - test .

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

A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.


B Expression of DAPK1 , UNC5B , and NTN1 was measured by Q - RT - PCR in PDX tumors after 7 days of in vivo DAC treatment ( 0 . 4 mg / kg ) . The level of PBGD expression was used as an internal control . Data are expressed as mean ± s . e . m . for at least 3 grafts per group . * * * * P < 0 . 0001 , , two - way ANOVA and post - hoc Tukey - test .

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

B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.


C Levels of DAPK1 , UNC5B , and netrin - 1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry . Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS - group . * * * * P < 0 . 0001 , one - way ANOVA . Error bars = s . e . m .

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

C Levels of DAPK1, UNC5B, and netrin-1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry. Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS-group. **** P<0.0001, one-way ANOVA. Error bars = s.e.m.


D Cleaved caspase - 3 and DNA fragmentation ( TUNEL ) median number of cells per mm2 were measured in treated xenografts . Data represent mean ± s . e . m . from at least 3 tumors per group . * * * * P < 0 . 0001 , one - way ANOVA . E Representative sections of the xenograft PDX analyzed in ( D ) . Scale bars = 50 μm . Arrows indicate positive staining .

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

D Cleaved caspase-3 and DNA fragmentation (TUNEL) median number of cells per mm2 were measured in treated xenografts. Data represent mean ± s.e.m. from at least 3 tumors per group. **** P<0.0001, one-way ANOVA.E Representative sections of the xenograft PDX analyzed in (D). Scale bars = 50 μm. Arrows indicate positive staining.


F The combination of net1 - mAb and DAC reduces humanbreasttumor growth in immuno - compromised mice . After anesthesia , mice were engrafted in the interscapular area with a ≈ 60 mm3 patient - derived tumor . When the tumors reached 120 - 150 mm3 , mice were injected subcutaneously with decitabine ( 0 . 4 mg / kg ) or PBS and / or intraperitoneally with net1 - mAb ( 10 mg / kg ) or with a humanIgG1 control isotype antibody ( Ctrl IgG1 , 10 mg / kg ) from day 1 to day 21 . Tumor volumes were measured twice a week ; n = 7 mice per group . The statistical significance of the differences obtained between the DAC + Ctrl IgG1 - group and the DAC + net1 - mAb - group was determined by two - way ANOVA2 and a post - hoc Tukey - test , * * * * P < 0 . 0001 . Error bars = s . e . m .

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

F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.


A , D Stably transfected MDA - MB - 231 cells bearing a control ( A ) , DAPK1 ( B ) , UNC5B ( C ) , or NTN1 shRNA ( D ) were injected into the mammary fat pad of immuno - compromised mice . When tumors reached 100 - 120 mm3 , mice were injected subcutaneously with DAC ( 0 . 4 mg / kg ) or PBS and / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . Tumor volumes were measured twice a week , n = 8 mice per group . The statistical significance of the differences obtained between DAC - group and DAC + net1 - mAb - group for shControl , DAC - group and DAC + net1 - mAb - group for shDAPK1 and shUNC5B , and PBS - group and DAC - group for shNTN1 , respectively , were determined by two - way ANOVA and a post - hoc Tukey - test . * * * * P < 0 . 0001 , , ns = not significant . Error bars = s . e . m .

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

A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.


A Representative fluorescent images of centrosomes in RPE1 WT , CEP250 KO and CROCC KO cells , without and with MT depolymerization using Nocodazole . PCNT was used to mark centrosomes and DNA was stained with DAPI . The boxes in the right - hand corner show enlargement of the centrosome signals of the cell in the main panel . White scale bars , 5 μm . Yellow scale bars , 1 μm . B Quantification of cells with separated centrosomes ( centrosome distance > 2 μm ) from RPE1 samples ( A ) . N = 50 cells per experiment , n = 3 independent experiments . For WT with Nocodazole , N = 50 cells , n = 2 independent experiments . Bar and error represent mean and SD . Unpaired t - tests . n . s . : not significant p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A Representative fluorescent images of centrosomes in RPE1 WT, CEP250 KO and CROCC KO cells, without and with MT depolymerization using Nocodazole. PCNT was used to mark centrosomes and DNA was stained with DAPI. The boxes in the right-hand corner show enlargement of the centrosome signals of the cell in the main panel. White scale bars, 5 μm. Yellow scale bars, 1 μm. B Quantification of cells with separated centrosomes (centrosome distance > 2 μm) from RPE1 samples (A). N = 50 cells per experiment, n = 3 independent experiments. For WT with Nocodazole, N = 50 cells, n = 2 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.


C Representative fluorescent images of centrosomes in HCT116 WT , CEP250 KO , and CROCC KO , without and with MT depolymerization using Nocodazole . γ - tubulin was used to mark centrosomes and DNA was stained with DAPI . The boxes in the right - hand corner show enlargements of the centrosome signals of the cell in the main panel . White scale bars , 10 μm . Yellow scale bars , 2 μm . D Quantification of cells with separated centrosomes ( centrosome distance > 1 μm ) from HCT116 samples ( C ) . N = 50 cells per experiment , n = 3 independent experiments . Bar and error represent mean and SD . Unpaired t - tests . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

C Representative fluorescent images of centrosomes in HCT116 WT, CEP250 KO, and CROCC KO, without and with MT depolymerization using Nocodazole. γ-tubulin was used to mark centrosomes and DNA was stained with DAPI. The boxes in the right-hand corner show enlargements of the centrosome signals of the cell in the main panel. White scale bars, 10 μm. Yellow scale bars, 2 μm. D Quantification of cells with separated centrosomes (centrosome distance > 1 μm) from HCT116 samples (C). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001.


E Representative fluorescent images of RPE1 and HCT116 cells . Rootletin and CEP68 were stained , PCNT was used as centrosome marker and DNA was stained with DAPI . The boxes on top show the centrosome signals of the cell in the main panel . Scale bars in white : 5 μm . Scale bars in yellow : 1 μm . F Quantification of relative Rootletin and CEP68 intensity at the centrosome in RPE1 and HCT116 cells . Mean value of relative intensity at the centrosome from N = 50 in each replicate , n = 4 are shown . Bar and error represent mean and SD . Unpaired t - test . * p < 0 . 05 , * * * p < 0 . 001 .

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

E Representative fluorescent images of RPE1 and HCT116 cells. Rootletin and CEP68 were stained, PCNT was used as centrosome marker and DNA was stained with DAPI. The boxes on top show the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. F Quantification of relative Rootletin and CEP68 intensity at the centrosome in RPE1 and HCT116 cells. Mean value of relative intensity at the centrosome from N = 50 in each replicate, n = 4 are shown. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, *** p < 0.001.


G Abundance of Rootletin and CEP68 in RPE1 and HCT116 cells was examined by immunoblotting . Tubulin was used as loading control . Representative blot is shown . The original HCT116 protein sample was diluted 1 : 10 . The relative abundance of Rootletin and CEP68 was normalized to the corresponding tubulin signal in each condition .

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

G Abundance of Rootletin and CEP68 in RPE1 and HCT116 cells was examined by immunoblotting. Tubulin was used as loading control. Representative blot is shown. The original HCT116 protein sample was diluted 1:10. The relative abundance of Rootletin and CEP68 was normalized to the corresponding tubulin signal in each condition.


A - H ( A , C , E , G ) Time - lapse images ( 4 min interval ) of RPE1 WT , CEP250 KO , NIN KO and CROCC KO cells expressing γ - tubulin - mRuby2 ( red ) as marker for the centrosomes with and without Nocodazole treatment . Representative still images ( top rows ) and kymographs of centrosomes ( bottom rows ) are shown . The DNA was marked with SPY - DNA650 ( grey ) . White scale bars , 5 μm . Yellow scale bars , 2 μm . ( B , D , F , H ) Intercentrosomal distances ( μm ) from ( A ) , ( C ) , ( E ) and ( G ) were plotted over time ( n = 15 cells ) .

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

A-H (A, C, E, G) Time-lapse images (4 min interval) of RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells expressing γ-tubulin-mRuby2 (red) as marker for the centrosomes with and without Nocodazole treatment. Representative still images (top rows) and kymographs of centrosomes (bottom rows) are shown. The DNA was marked with SPY-DNA650 (grey). White scale bars, 5 μm. Yellow scale bars, 2 μm. (B, D, F, H) Intercentrosomal distances (μm) from (A), (C), (E) and (G) were plotted over time (n = 15 cells).


A Indirect immunofluorescence analysis of Ninein at the centrosomes in RPE1 WT , CEP250 KO and CEP128 KO cells . CEP164 marks the mother centriole , γ - tubulin was used as centrosome marker . The model panel illustrates the position of the centrioles and centriole appendages . Scale bars : 1 μm . B STED super resolution image and model of Ninein at the centrosome in RPE1 WT , CEP250 KO and CEP128 KO cells . CEP164 , C - Nap1 and GT335 were used to mark the mother centriole , the proximal end of centrioles , and the centriole wall , respectively . The model panel illustrates the position of the centrioles and appendages ( colours as indicated ) . For WT and CEP250 KO a blue centriole model was used for better illustration of the different positions . Scale bars : 1 μm .

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

A Indirect immunofluorescence analysis of Ninein at the centrosomes in RPE1 WT, CEP250 KO and CEP128 KO cells. CEP164 marks the mother centriole, γ-tubulin was used as centrosome marker. The model panel illustrates the position of the centrioles and centriole appendages. Scale bars: 1 μm. B STED super resolution image and model of Ninein at the centrosome in RPE1 WT, CEP250 KO and CEP128 KO cells. CEP164, C-Nap1 and GT335 were used to mark the mother centriole, the proximal end of centrioles, and the centriole wall, respectively. The model panel illustrates the position of the centrioles and appendages (colours as indicated). For WT and CEP250 KO a blue centriole model was used for better illustration of the different positions. Scale bars: 1 μm.


E - H ( E , G ) Fluorescent images of centrosomes in RPE1 CEP250 KO and CEP128 KO cells with the indicated siRNA depletions , without and with MT depolymerization using Nocodazole . PCNT or γ - tubulin was used to mark centrosomes and DNA was stained with DAPI . The box in the right - hand corner shows the centrosome signals of the cell in the main panel . Scale bars in white : 5 μm . Scale bars in yellow : 1 μm . ( F , H ) Quantification of cells with separated centrosomes ( centrosome distance > 2 μm ) for RPE1 CEP250 KO ( E ) and CEP128 KO ( G ) samples . N = 50 cells per experiment , n = 3 independent experiments . Bar and error represent mean and SD . Unpaired t - test . n . s . : not significant p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E-H (E, G) Fluorescent images of centrosomes in RPE1 CEP250 KO and CEP128 KO cells with the indicated siRNA depletions, without and with MT depolymerization using Nocodazole. PCNT or γ-tubulin was used to mark centrosomes and DNA was stained with DAPI. The box in the right-hand corner shows the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. (F, H) Quantification of cells with separated centrosomes (centrosome distance > 2 μm) for RPE1 CEP250 KO (E) and CEP128 KO (G) samples. N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.


A Representative fluorescent images of MT asters nucleated from the centrosome ( γ - tubulin ) after in vivo MT regrowth assay . The enlargements on the right show the centrosome signal of the cell in the main panel . Scale bars in white : 10 μm ; scale bars in yellow : 1 μm .

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

A Representative fluorescent images of MT asters nucleated from the centrosome (γ-tubulin) after in vivo MT regrowth assay. The enlargements on the right show the centrosome signal of the cell in the main panel. Scale bars in white: 10 μm; scale bars in yellow: 1 μm.


B Quantification of the average number ( left ) and length ( right ) of MTs re - nucleated from the centrosomes in ( A ) . N = 50 cells per experiment , n ≥ 5 independent experiments . Bar and error represent mean and SD . Unpaired t - test . n . s . : not significant p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the average number (left) and length (right) of MTs re-nucleated from the centrosomes in (A). N = 50 cells per experiment, n ≥ 5 independent experiments. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.


C Representative fluorescent images of RPE1 CEP250 KO and CEP250 KO expressing C - Nap1 - HA - mNeonGreen ( CEP250 KO + CEP250 ) . The indicated antibodies were used for indirect fluorescent analysis . Scale bars in white : 5 μm ; scale bars in yellow : 1 μm . D Quantification of centrosome separation from ( C ) . N = 50 cells per experiment , n = 3 independent experiments . Bar and error represent mean and SD Unpaired t - test . n . s . : not significant p > 0 . 05 , * * * * p < 0 . 0001 . E Quantification of the average number ( left ) and length ( right ) of MTs re - nucleated from the centrosomes in ( A ) from RPE1 WT and CEP250 KO cells with PLK4 OE . N = 50 cells per experiment , n = 3 independent experiments . Bar and error represent mean and SD Unpaired t - test . * * p < 0 . 01 .

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

C Representative fluorescent images of RPE1 CEP250 KO and CEP250 KO expressing C-Nap1-HA-mNeonGreen (CEP250 KO + CEP250). The indicated antibodies were used for indirect fluorescent analysis. Scale bars in white: 5 μm; scale bars in yellow: 1 μm. D Quantification of centrosome separation from (C). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD Unpaired t-test. n.s.: not significant p > 0.05, **** p < 0.0001. E Quantification of the average number (left) and length (right) of MTs re-nucleated from the centrosomes in (A) from RPE1 WT and CEP250 KO cells with PLK4 OE. N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD Unpaired t-test. ** p < 0.01.


B Fluorescent images of centrosomes in Dox - inducible Flag - PLK4 RPE1 WT , CEP250 KO , NIN KO and CROCC KO cells , with and without Dox induction . Flag - PLK4 was stained with Flag antibodies , PCNT was used as centrosome marker and DNA was stained with DAPI . The boxes on top show enlargement of the centrosome signals of the cell in the main panel . Scale bars in white : 5 μm ; scale bars in yellow : 1 μm .

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

B Fluorescent images of centrosomes in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells, with and without Dox induction. Flag-PLK4 was stained with Flag antibodies, PCNT was used as centrosome marker and DNA was stained with DAPI. The boxes on top show enlargement of the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm; scale bars in yellow: 1 μm.


D Fluorescent images of centrosomes in Dox - inducible Flag - PLK4 RPE1 WT , CEP250 KO , NIN KO and CROCC KO cells upon 48 h Dox induction , without and with MT depolymerization using Nocodazole . DNA was stained with DAPI . Scale bars : 5 μm . E Quantification of the diameter of amplified centrosomes from ( D ) as outlined in ( A ) . A representative dataset from six replicates ( three replicates per experiment ) is shown . N = 50 cells . Dot represents the diameter of amplified centrosomes in each cell with amplified centrosomes . The red lines represent the median . Unpaired t - test . n . s . : not significant p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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

D Fluorescent images of centrosomes in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells upon 48 h Dox induction, without and with MT depolymerization using Nocodazole. DNA was stained with DAPI. Scale bars: 5 μm. E Quantification of the diameter of amplified centrosomes from (D) as outlined in (A). A representative dataset from six replicates (three replicates per experiment) is shown. N = 50 cells. Dot represents the diameter of amplified centrosomes in each cell with amplified centrosomes. The red lines represent the median. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.


G The mean of I50 values from ( F ) . N ≥ 22 cells per replicate , n = 3 biological replicates . Bar and error represent mean and SD . Unpaired t - test . n . s . : not significant p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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G The mean of I50 values from (F). N ≥ 22 cells per replicate, n=3 biological replicates. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


A Representative images of live cell imaging analysis of RPE1 WT , CEP250 KO , WT PLK4 OE and CEP250 KO PLK4 OE cells expressing LaminB1 - mNeonGreen ( magenta ) and γ - tubulin - mRuby2 ( green ) as markers for the NE and centrosomes , respectively . Yellow arrows indicate first NE fold . White arrows indicate first NE discontinuation . The inlet boxes show enlargement of first NE discontinuation . Scale bars : 5 μm .

A Representative images of live cell imaging analysis of RPE1 WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells expressing LaminB1-mNeonGreen (magenta) and γ-tubulin-mRuby2 (green) as markers for the NE and centrosomes, respectively. Yellow arrows indicate first NE fold. White arrows indicate first NE discontinuation. The inlet boxes show enlargement of first NE discontinuation. Scale bars: 5 μm.


C Quantification of NEBD preparation time . WT : n = 54 cells from 6 independent experiments , CEP250 KO : n = 56 cells from 6 independent experiments , WT PLK4 OE : n = 48 cells from 7 independent experiments , CEP250 KO PLK4 OE : n = 47 cells from 8 independent experiments . D Quantification of NEBD duration . WT : n = 54 cells from 6 independent experiments , CEP250 KO : n = 56 cells from 6 independent experiments , WT PLK4 OE : n = 48 cells from 7 independent experiments , CEP250 KO PLK4 OE : n = 47 cells from 8 independent experiments .

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C Quantification of NEBD preparation time. WT: n = 54 cells from 6 independent experiments, CEP250 KO: n = 56 cells from 6 independent experiments, WT PLK4 OE: n = 48 cells from 7 independent experiments, CEP250 KO PLK4 OE: n = 47 cells from 8 independent experiments. D Quantification of NEBD duration. WT: n = 54 cells from 6 independent experiments, CEP250 KO: n = 56 cells from 6 independent experiments, WT PLK4 OE: n = 48 cells from 7 independent experiments, CEP250 KO PLK4 OE: n = 47 cells from 8 independent experiments.


H Representative images from live cell imaging analysis of WT , CEP250 KO , WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ - tubulin - mRuby2 ( red ) as a marker for the centrosomes . The DNA was marked by SPY - DNA650 dye ( cyan ) . Gray arrows highlight chromosome segregation errors . Scale bars : 5 μm .

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H Representative images from live cell imaging analysis of WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ-tubulin-mRuby2 (red) as a marker for the centrosomes. The DNA was marked by SPY-DNA650 dye (cyan). Gray arrows highlight chromosome segregation errors. Scale bars: 5 μm.


I Fluorescent images of RPE1 WT , CEP250 KO , WT PLK4 OE and CEP250 KO PLK4 OE cells . BUB1 and MAD1 were stained to analyse the signal distribution or accumulation / persistence . CENP - C was used as centromere marker , α - tubulin to visualize the mitotic spindle and DNA was stained with DAPI . Scale bars : 5 μm .

I Fluorescent images of RPE1 WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells. BUB1 and MAD1 were stained to analyse the signal distribution or accumulation/persistence. CENP-C was used as centromere marker, α-tubulin to visualize the mitotic spindle and DNA was stained with DAPI. Scale bars: 5 μm.


A Fluorescent image of mitotic spindles in Dox - inducible Flag - PLK4 RPE1 WT , CEP250 KO , NIN KO and CROCC KO cells upon 48 h Dox induction ( PLK4 OE ) . CW069 was used to partially inhibit HSET . DMSO is solvent control . White scale bars : 5 μm . B Quantification of mitotic cells with multipolar spindle from ( A ) . N = 50 cells per experiment , n = 3 independent experiments . Bar and error represent mean and SD . Unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Quantification of multipolar spindle / chromosome mis - segregation of Dox - inducible Flag - PLK4 RPE1 WT and CEP250 KO cells upon 48 h Dox induction expressing γ - tubulin - mScarlet - I ( red ) as markers for the centrosomes with or without CW069 treatment . The DNA was marked with SPY - DNA650 ( grey ) . WT PLK4 OE : nDMSO = 28 cells , nCW069 = 40 cells , CEP250 KO PLK4 OE : nDMSO = 26 cells , nCW069 = 31 cells ; from 2 independent live cell experiments . Bar represents mean . Unpaired t - test . * p < 0 . 05 .

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A Fluorescent image of mitotic spindles in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells upon 48 h Dox induction (PLK4 OE). CW069 was used to partially inhibit HSET. DMSO is solvent control. White scale bars: 5 μm. B Quantification of mitotic cells with multipolar spindle from (A). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. C Quantification of multipolar spindle/chromosome mis-segregation of Dox-inducible Flag-PLK4 RPE1 WT and CEP250 KO cells upon 48 h Dox induction expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes with or without CW069 treatment. The DNA was marked with SPY-DNA650 (grey). WT PLK4 OE: nDMSO = 28 cells, nCW069 = 40 cells, CEP250 KO PLK4 OE: nDMSO = 26 cells, nCW069 = 31 cells; from 2 independent live cell experiments. Bar represents mean. Unpaired t-test. * p < 0.05.


D Time - lapse analysis of RPE1 WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ - tubulin - mScarlet - I ( red ) as markers for the centrosomes followed by 30 h siHSET . The DNA was marked with SPY - DNA650 ( grey ) . ( D ) shows representative still images of live cell imaging analysis . Scale bars : 10 μm . E Quantification of multipolar spindle / chromosome mis - segregation from ( D ) in RPE1 WT PLK4 OE and CEP250 KO PLK4 OE with siControl and siHSET for 30 h . WT PLK4 OE : nsiControl = 59 cells , nsiHSET = 59 cells , CEP250 KO PLK4 OE : nsiControl = 45 cells , nsiHSET = 32 cells ; from 3 independent experiments . Bar and error represent mean and SD . Unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 .

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D Time-lapse analysis of RPE1 WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes followed by 30 h siHSET. The DNA was marked with SPY-DNA650 (grey). (D) shows representative still images of live cell imaging analysis. Scale bars: 10 μm. E Quantification of multipolar spindle/chromosome mis-segregation from (D) in RPE1 WT PLK4 OE and CEP250 KO PLK4 OE with siControl and siHSET for 30 h. WT PLK4 OE: nsiControl = 59 cells, nsiHSET = 59 cells, CEP250 KO PLK4 OE: nsiControl = 45 cells, nsiHSET = 32 cells; from 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01.


( A ) Mitochondrial morphology in HeLa cells depleted of Sept2 , Sept7 and Sept9 . Mitochondria were labeled with mitotracker ( green ) . Scale bar : 10 µm . The inset represents a twofold enlargement . ( B ) Quantification of mean mitochondrial length in mock treated cells and in cells depleted of Sept2 , Sept7 and Sept9 . n > 250 individual mitochondria from three independent experiments .

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(A) Mitochondrial morphology in HeLa cells depleted of Sept2, Sept7 and Sept9. Mitochondria were labeled with mitotracker (green). Scale bar: 10 µm. The inset represents a twofold enlargement. (B) Quantification of mean mitochondrial length in mock treated cells and in cells depleted of Sept2, Sept7 and Sept9. n>250 individual mitochondria from three independent experiments.


( C ) Mitochondrial morphology in mock treated cells or HeLa cells depleted of Sept2 and then transfected with empty vector , siRNA - resistant HA - tagged Sept2 ( Sept2 - rescue ) or Sept7 ( Sept7 - rescue ) . Mitochondria were labeled with mitotracker ( shown in green ) , asterisks mark transfected cells . Scale bar : 10 µm , insets are twofold enlargements . ( D ) Quantification of mean mitochondrial length in mock treated cells , in cells depleted of Sept2 or in cells depleted of Sept2 and transfected with siRNA - resistant Sept2 ( Sept2 - rescue ) or Sept7 ( Sept7 - rescue ) . n > 200 individual mitochondria from two independent experiments .

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(C) Mitochondrial morphology in mock treated cells or HeLa cells depleted of Sept2 and then transfected with empty vector, siRNA-resistant HA-tagged Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). Mitochondria were labeled with mitotracker (shown in green), asterisks mark transfected cells. Scale bar: 10 µm, insets are twofold enlargements. (D) Quantification of mean mitochondrial length in mock treated cells, in cells depleted of Sept2 or in cells depleted of Sept2 and transfected with siRNA-resistant Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). n>200 individual mitochondria from two independent experiments.


( E ) Mock treated or Sept2 depleted U2OS cells stained for Sept2 ( green ) and the Golgi apparatus ( GM130 , red ) , the ER ( Sec61b - GFP , displayed in red ) or peroxisomes ( PMP70 , red ) . ( F ) Quantification of the mean Golgi area ( n = 40 cells , three independent experiments ) ( G ) Quantification showing the percentage of ER traversing well - resolved mitochondrial tubules ( n = 15 cells from three independent experiments ) . ( H ) Quantification of average peroxisome counts per cell ( n = 40 cells from three independent experiments ) . Scale bar : 10 µm , insets are enlarged twofold .

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(E) Mock treated or Sept2 depleted U2OS cells stained for Sept2 (green) and the Golgi apparatus (GM130, red), the ER (Sec61b-GFP, displayed in red) or peroxisomes (PMP70, red). (F) Quantification of the mean Golgi area (n=40 cells, three independent experiments) (G) Quantification showing the percentage of ER traversing well-resolved mitochondrial tubules (n=15 cells from three independent experiments). (H) Quantification of average peroxisome counts per cell (n=40 cells from three independent experiments). Scale bar: 10 µm, insets are enlarged twofold.


( A ) Live cell imaging of mock treated or Sept2 depleted U2OS cells analyzed with the mito - PA - GFP mitochondrial fusion assay . Mito - PA - GFP is photoactivated in the indicated ROI and the decrease in fluorescence is followed in the same ROI , correcting for cell movement ( B ) Quantification of mitochondrial fusion rates in mock treated or Sept2 depleted cells expressed as a means of three independent experiments SEM . ( C ) Quantification of fission rates in mock and Sept2 depleted cells . n = 116 fission events for Sept2 siRNA , n = 83 fission events for mock , two independent experiments .

(A) Live cell imaging of mock treated or Sept2 depleted U2OS cells analyzed with the mito-PA-GFP mitochondrial fusion assay. Mito-PA-GFP is photoactivated in the indicated ROI and the decrease in fluorescence is followed in the same ROI, correcting for cell movement (B) Quantification of mitochondrial fusion rates in mock treated or Sept2 depleted cells expressed as a means of three independent experiments SEM. (C) Quantification of fission rates in mock and Sept2 depleted cells. n=116 fission events for Sept2 siRNA, n=83 fission events for mock, two independent experiments.


( D ) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane ( OM - GFP ) , showing delayed FCCP - induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells . White arrowheads point at fission examples , yellow arrowheads to looping . Scale bar : 10 µm , insets are enlarged twofold . ( E ) Quantification of mitochondrial elongation ( long / short axis ) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells , mean of four independent experiments SEM .

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(D) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane (OM-GFP), showing delayed FCCP-induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells. White arrowheads point at fission examples, yellow arrowheads to looping. Scale bar: 10 µm, insets are enlarged twofold. (E) Quantification of mitochondrial elongation (long /short axis) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells, mean of four independent experiments SEM.


( A ) Immunofluorescence microscopy analysis of endogenous Sept2 ( green ) and mitochondria ( Tom20 , red ) in HeLa cells . The insets show a twofold enlargement with arrowheads highlighting the presence of small Sept2 structures on mitochondria . Scale bar 10 µm . ( B ) 3D rendering of confocal microscopy sections showing Sept2 at mitochondrial constrictions ( white arrowheads ) and constricted mitochondria that are not marked by Sept2 ( yellow arrowhead ) Scale bar 2 µm .

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(A) Immunofluorescence microscopy analysis of endogenous Sept2 (green) and mitochondria (Tom20, red) in HeLa cells. The insets show a twofold enlargement with arrowheads highlighting the presence of small Sept2 structures on mitochondria. Scale bar 10 µm. (B) 3D rendering of confocal microscopy sections showing Sept2 at mitochondrial constrictions (white arrowheads) and constricted mitochondria that are not marked by Sept2 (yellow arrowhead) Scale bar 2 µm.


( C ) CoDiM super - resolution imaging of endogenous Sept2 . Arrowheads highlight the presence of Sept2 at mitochondrial constrictions . Insets show a threefold enlargement Scale bar 10 μm .

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(C) CoDiM super-resolution imaging of endogenous Sept2. Arrowheads highlight the presence of Sept2 at mitochondrial constrictions. Insets show a threefold enlargement Scale bar 10 μm.


( D ) Still images from a live cell imaging experiment showing Sept2 ( green ) accumulation during two mitochondrial fission events ( mitotracker orange , red ) , highlighted by yellow ( first fission ) or green arrowheads ( second fission ) . Scale bar 2 µm .

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(D) Still images from a live cell imaging experiment showing Sept2 (green) accumulation during two mitochondrial fission events (mitotracker orange, red), highlighted by yellow (first fission) or green arrowheads (second fission). Scale bar 2 µm.


( A ) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co - immunoprecipitates with Sept2 . The asterisks mark the IgG heavy chains .

(A) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co-immunoprecipitates with Sept2. The asterisks mark the IgG heavy chains.


( B ) Elyra SIM image of U2OS cells stained for Sept2 ( green ) , mitochondria ( GFP targeted to the mitochondrial outer membrane ( OM - GFP ) , shown in red ) , Drp1 ( blue ) . Yellow arrows point at tetraspeck beads used for image alignment . Insets represent sevenfold enlargements . White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria . Scale bar : 10 µm , insets are enlarged fourfold .

(B) Elyra SIM image of U2OS cells stained for Sept2 (green), mitochondria (GFP targeted to the mitochondrial outer membrane (OM-GFP), shown in red), Drp1 (blue). Yellow arrows point at tetraspeck beads used for image alignment. Insets represent sevenfold enlargements. White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria. Scale bar: 10 µm, insets are enlarged fourfold.


( C ) GST pulldown of recombinant Sept2 with GST - Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1 .

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(C) GST pulldown of recombinant Sept2 with GST-Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1.


( D ) Sept2 immunoprecipitation from mock or CCCP - treated HeLa cells probed for Drp1 and Sept2 . ( E ) Quantification of two independent experiments showing stimulated Drp1 co - immunoprecipitation with Sept2 in FCCP - treated cells .

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(D) Sept2 immunoprecipitation from mock or CCCP-treated HeLa cells probed for Drp1 and Sept2. (E) Quantification of two independent experiments showing stimulated Drp1 co-immunoprecipitation with Sept2 in FCCP-treated cells.


( F ) Sept2 immunoprecipitation from mock or Mdivi - 1 - treated HeLa cells probed for Drp1 and Sept2 . ( G ) Quantification of two independent experiments showing decreased Drp1 co - immunoprecipitation with Sept2 upon Mdivi - 1 treatment .

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(F) Sept2 immunoprecipitation from mock or Mdivi-1-treated HeLa cells probed for Drp1 and Sept2. (G) Quantification of two independent experiments showing decreased Drp1 co-immunoprecipitation with Sept2 upon Mdivi-1 treatment.


( H ) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 ( green , Sept2 - YFP ) , Drp1 ( red , Drp1 - mCherry ) and mitochondria ( blue , mitotracker DeepRed ) during mitochondrial fission . Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission ( yellow arrowheads ) . Scale bar : 2 µm .

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(H) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 (green, Sept2-YFP), Drp1 (red, Drp1-mCherry) and mitochondria (blue, mitotracker DeepRed) during mitochondrial fission. Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission (yellow arrowheads). Scale bar: 2 µm.


( A ) Immunofluorescence microscopy of Sept2 ( blue ) , mitochondria ( Tom20 , red ) and Drp1 ( green ) in mock treated and Sept2 depleted cells . Scale bar : 10 µm , insets are enlarged fourfold . ( B ) Quantification of mitochondria - associated Drp1 in mock treated and Sept2 depleted cells ( mean of 3 independent experiments , n = 44 - 51 cells ) ( C ) Average distance between mitochondria - associated Drp1 oligomers ( representative experiment with n > 25 individual mitochondria ) .

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(A) Immunofluorescence microscopy of Sept2 (blue), mitochondria (Tom20, red) and Drp1 (green) in mock treated and Sept2 depleted cells. Scale bar: 10 µm, insets are enlarged fourfold. (B) Quantification of mitochondria-associated Drp1 in mock treated and Sept2 depleted cells (mean of 3 independent experiments, n=44-51 cells) (C) Average distance between mitochondria-associated Drp1 oligomers (representative experiment with n>25 individual mitochondria).


( D ) HeLa cytosol and crude mitochondrial fractions prepared from mock and Sept2 siRNA treated cells . The samples were analysed by western blotting with indicated antibodies , and show a decrease in mitochondria - associated Drp1 in Sept2 depleted cells .

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(D) HeLa cytosol and crude mitochondrial fractions prepared from mock and Sept2 siRNA treated cells. The samples were analysed by western blotting with indicated antibodies, and show a decrease in mitochondria-associated Drp1 in Sept2 depleted cells.


( A ) Immunofluorescence microscopy analysis of P - MLC ( green ) recruitment to mitochondria ( red , Tom20 ) in mock treated and Sept2 depleted HeLa cells . Insets show twofold enlargements . Scale bar : 10 µm . ( B ) Colocalization of P - MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells .

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(A) Immunofluorescence microscopy analysis of P-MLC (green) recruitment to mitochondria (red, Tom20) in mock treated and Sept2 depleted HeLa cells. Insets show twofold enlargements. Scale bar: 10 µm. (B) Colocalization of P-MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells.


( C ) Mock treated or Sept2 depleted Drp1 - / - MEF cells were labeled with mitotracker orange ( red ) , treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin ( green ) . Insets are enlarged twofold .

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(C) Mock treated or Sept2 depleted Drp1 -/- MEF cells were labeled with mitotracker orange (red), treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin (green). Insets are enlarged twofold.


( D ) Immunofluorescence microscopy analysis of the actin binding proteins Arp3 and cofilin ( green ) , colocalized with cytocrome c ( red ) and cortactin ( green ) , colocalized with Hsp60 ( red ) in mock treated or Sept2 depleted Drp1 - / - MEF cells . Scale bars : 10 µm .

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(D) Immunofluorescence microscopy analysis of the actin binding proteins Arp3 and cofilin (green), colocalized with cytocrome c (red) and cortactin (green), colocalized with Hsp60 (red) in mock treated or Sept2 depleted Drp1 -/- MEF cells. Scale bars: 10 µm.


( E ) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 - / - MEF cells and analyzed by western blotting with the indicated antibodies .

(E) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 -/- MEF cells and analyzed by western blotting with the indicated antibodies.


( F ) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation . Immunoprecipitates were analyzed by western blot for Drp1 and Sept2 , showing that Drp1 co - immunoprecipitation with Sept2 is not affected by cytochalasin D treatment .

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(F) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation. Immunoprecipitates were analyzed by western blot for Drp1 and Sept2, showing that Drp1 co-immunoprecipitation with Sept2 is not affected by cytochalasin D treatment.


( A ) Live imaging of C . elegans expressing TOM70 : : GFP in body wall muscle cells . Depletion of the UNC - 59 or UNC - 61 led to an increase in mitochondrial network connectivity as shown by quantification in ( B ) . Insets represent a twofold magnification . Scale bar : 10 µm .

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(A) Live imaging of C. elegans expressing TOM70::GFP in body wall muscle cells. Depletion of the UNC-59 or UNC-61 led to an increase in mitochondrial network connectivity as shown by quantification in (B). Insets represent a twofold magnification. Scale bar: 10 µm.


( C ) Live imaging of C . elegans body wall muscle cells expressing TOM70 : : GFP . Depletion of DRP - 1 or UNC - 61 caused an increase in mitochondrial elongation and network connectivity , which did not increase upon combination of DRP - 1 and UNC - 61 . Insets represent a twofold magnification . Scale bar : 10 µm .

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(C) Live imaging of C. elegans body wall muscle cells expressing TOM70::GFP. Depletion of DRP-1 or UNC-61 caused an increase in mitochondrial elongation and network connectivity, which did not increase upon combination of DRP-1 and UNC-61. Insets represent a twofold magnification. Scale bar: 10 µm.


A Freeze - fracture electron micrograph of wild - type yeast treated with DTT for 3 h . The left panel shows two spherical structures , one in a vacuole membrane invagination and one inside the vacuole . The middle panel shows the boxed area of the left panel at higher magnification . Arrows indicate membrane layers that are free of intramembrane particles . The right panel shows the protoplasmic fracture face ( P face ) of cortical ER with a high density of intramembrane particles . Cy , cytosol ; N , nucleus ; V , vacuole .

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A Freeze-fracture electron micrograph of wild-type yeast treated with DTT for 3 h. The left panel shows two spherical structures, one in a vacuole membrane invagination and one inside the vacuole. The middle panel shows the boxed area of the left panel at higher magnification. Arrows indicate membrane layers that are free of intramembrane particles. The right panel shows the protoplasmic fracture face (P face) of cortical ER with a high density of intramembrane particles. Cy, cytosol; N, nucleus; V, vacuole.


B Fluorescence images of cells expressing GPD promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . The upper and lower panel show vacuole - associated and perinuclear GFP - Pho8 stretches and an intravacuolar GFP - Pho8 ring , respectively . Scale bars : 2 µm .

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B Fluorescence images of cells expressing GPD promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. The upper and lower panel show vacuole-associated and perinuclear GFP-Pho8 stretches and an intravacuolar GFP-Pho8 ring, respectively. Scale bars: 2 µm.


C Correlative light and electron microscopy of cells expressing GPD promoter - driven GFP - Pho8 and Sec63 - mCherry . The lower panel shows the boxed area in the electron micrograph at higher magnification . The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack .

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C Correlative light and electron microscopy of cells expressing GPD promoter-driven GFP-Pho8 and Sec63-mCherry. The lower panel shows the boxed area in the electron micrograph at higher magnification. The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack.


D Fluorescence images of cells expressing Hmg2 - GFP and either additionally expressing Sec63 - mCherry or stained with CMAC . The upper and lower panel show cytosolic and vacuolar Hmg2 - GFP rings , respectively . Scale bars : 2 µm .

D Fluorescence images of cells expressing Hmg2-GFP and either additionally expressing Sec63-mCherry or stained with CMAC. The upper and lower panel show cytosolic and vacuolar Hmg2-GFP rings, respectively. Scale bars: 2 µm.


A Individual frames from time - lapse imaging of a cell expressing GAL promoter - driven GFP - Pho8 and stained with the vacuole membrane dye FM4 - 64 . Numbers indicate the time in minutes after the start of the image sequence . Scale bar : 2 µm .

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A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm.


B Electron micrograph of a cell expressing GPD promoter - driven GFP - Pho8 . Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination . Black arrowheads point to vacuole membrane that encases the bent stack and the whorl .

B Electron micrograph of a cell expressing GPD promoter-driven GFP-Pho8. Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination. Black arrowheads point to vacuole membrane that encases the bent stack and the whorl.


C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks . n = 43 .

C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks. n = 43.


The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl . Scale bar : 2 µm .

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The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.


Fluorescence images of ∆ atg7 and ∆ atg8 cells ( A ) expressing GAL promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . Scale bar : 2 µm .

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Fluorescence images of ∆atg7 and ∆atg8 cells (A) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.


Fluorescence images of ∆ snf7 and ∆ vps4 cells ( B ) expressing GAL promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . Scale bar : 2 µm .

Fluorescence images of ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.


C Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 in tunicamycin - treated cells . The dotted line indicates the activity in ∆ atg7 cells . Mean + SEM , n ≥ 3 .

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C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3.


D , E Relative activity of endogenous Pho8 in untreated cells ( D ) or after tunicamycin treatment ( E ) . Background activity determined in ∆ pho8 mutants was subtracted from the activity in all other strains . Mean + SEM , n = 3 . WT , wild - type .

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D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type.


F Western blot of GFP from cells expressing Sec63 - GFP and treated with tunicamycin ( Tm ) where indicated . The two Atg7 - independent cleavage products of around 55 kDa were used as readout for micro - ER - phagy . Pgk1 served as a loading control . G Quantification of Sec63 - GFP cleavage normalized to the WT from western blots as shown in panel ( F ) . Mean + SEM , n = 4 .

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F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control. G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4.


A Electron micrograph of wild - type yeast treated with DTT for 3 h . Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies ( white arrows ) . Cy , cytosol ; V , vacuole .

A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.


B Relative activity of cyto - Pho8 ∆ 60 after nitrogen starvation , which reports on non - selective macroautophagy of cytosol . Mean + SEM , n ≥ 3 . WT , wild - type .

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B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after nitrogen starvation ( C ) Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after tunicamycin treatment ( D , E ) . Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.


F , G Growth assays of untreated and tunicamycin - treated WT , ∆ atg39 / 40 , ∆ snf7 and ∆ snf7 ∆ atg39 / 40 cells . Numbers indicate growth relative to WT cells based on the areas under the curves . Mean ± SEM , n = 3 .

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F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.


A Fraction of wild - type ( WT ) , ∆ vps23 , ∆ snf7 and ∆ vps4 cells with GFP - Pho8 structures . Mean + SEM , n ≥ 3 .

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A Fraction of wild-type (WT), ∆vps23, ∆snf7 and ∆vps4 cells with GFP-Pho8 structures. Mean + SEM, n ≥ 3.


B Fraction of GFP - Pho8 structures classified as stacks , cytosolic whorls , uptake intermediates or vacuolar whorls . The number of GFP - Pho8 structures in WT , ∆ vps23 , ∆ snf7 and ∆ vps4 cells was 562 , 345 , 367 and 449 , respectively . The table shows the results of Chi - Square tests for independence for all pairs of strains . * * * , highly significant ( P < 0 . 00001 ) ; ns , not significant ( P > 0 . 05 ) .

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B Fraction of GFP-Pho8 structures classified as stacks, cytosolic whorls, uptake intermediates or vacuolar whorls. The number of GFP-Pho8 structures in WT, ∆vps23, ∆snf7 and ∆vps4 cells was 562, 345, 367 and 449, respectively. The table shows the results of Chi-Square tests for independence for all pairs of strains. ***, highly significant (P < 0.00001); ns, not significant (P > 0.05).


C Fraction of stacks classified as peripheral , perinuclear or vacuole - associated . The number of stacks in WT and ∆ snf7 cells was 252 and 351 , respectively . The observed distributions in the two strains were analyzed with a Chi - Square test for independence . * * * , highly significant ( P < 0 . 00001 ) .

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C Fraction of stacks classified as peripheral, perinuclear or vacuole-associated. The number of stacks in WT and ∆snf7 cells was 252 and 351, respectively. The observed distributions in the two strains were analyzed with a Chi-Square test for independence. ***, highly significant (P < 0.00001).


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . Panel ( E ) illustrates transient appearance of Snf7 at an ER whorl . Panel ( F ) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


A Electron micrographs of wild - type and ∆ snf7 cells treated with DTT for 3 h . Cy , cytosol ; V , vacuole . Scale bars : 300 nm .

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A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.


B Quantification of vacuolar whorls for which the uptake process was complete or incomplete . The observed distributions in the two strains were analyzed with a Fisher ' s exact test .

B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test.


C , D Electron micrographs of serial thin sections of a ∆ snf7 cell ( C ) or a wild - type cell ( D ) treated with DTT for 3 h . Note that the whorl appears separated from the cytosol in some sections ( e . g . section 300 nm ) but is still in contact with the cytosol ( see sections 600 nm and 700 nm ) . In the wild - type cell ( D ) , the vacuolar whorl is completely separated from the cytosol . Scale bar : 300 nm .

C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.


A . LN229 and U87 cells treated with Wnt 3a ( 0 , 10 , 20 , 50 , 100 ng / ml ) for 4 hr . The indicated proteins were analyzed by Western blotting .

A. LN229 and U87 cells treated with Wnt 3a (0, 10, 20, 50, 100 ng/ml) for 4 hr. The indicated proteins were analyzed by Western blotting.


B . LN229 cells treated with or without Wnt3a ( 50 ng / ml ) were pulsed with [ 35S ] methionine for 30 min and chased for the times indicated . Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS - PAGE and autoradiography . The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software , and the densities of the FoxM1 bands at time 0 were set as 100 % . Values are mean ± SD from two independent experiments .

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B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.


C . Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 ( 100 ng / ml ) for indicated times . Noted that the basal levels of FoxM1 and β - catenin in ( A ) and ( C ) are similar but look different due to variations on the exposure time of the Western blots .

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C. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for indicated times. Noted that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations on the exposure time of the Western blots.


D . FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl ( 10 mM ) and CHX ( 100 µg / ml ) for indicated times .

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D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.


E . GSK3 wild - type ( WT ) and knockout ( KO ) cells were treated with CHX ( 100 µg / ml ) for indicated times , and FoxM1 and GSK3α / β expression levels were determined by Western blotting .

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E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting.


F . Flag - FoxM1 and GSK3βKA or GSK3βKD plasmids were co - transfected into 293T cells for 48 hr . The cells were then treated with CHX ( 100 µg / ml ) for indicated times . Exogenous Flag - FoxM1 expression level was determined by Western blotting .

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F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.


B . Lysates from 293T cells expressing Flag - FoxM1 or its mutants were subjected to IP using mouse anti - Flag antibody , followed by IB with GSK3α / β antibody ( upper panel ) and rabbit anti - Flag antibody ( middle panel ) .

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B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel).


D . HA - ubiquitin and Flag - tagged FoxM1 ( WT ) or mutants FoxM1S228A , FoxM1T309A , or Flag - FoxM1S474A plasmid were co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody , followed by IB with Flag and HA antibody .

D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.


E . HA - ubiquitin was co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 phospho - S474 antibody , followed by IB with FoxM1 and HA antibody .

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E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.


F . HA - tagged K48 - only ubiquitin or K63 - only ubiquitin construct was transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 antibody , followed by IB with FoxM1 and HA antibody .

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F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.


G . GSK3βCA or vector and Flag - tagged FoxM1 ( WT ) , mutants S474A or S478A were co - transfected into 293T cells . Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho - S474 antibody .

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G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.


A . Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG , followed by IB with indicated antibodies .

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A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies.


B . Axin siRNA or control siRNA were transfected into LN229 cells , after 36 hours , lysate of the cells was performed with co - IP using FoxM1 antibody followed by IB with indicated antibodies .

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B. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies.


C . Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours . Lysates of the cells were subjected to IB with FoxM1 phospho - S474 antibody .

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C. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody.


D . Cell lysates of LN229 cells with or without Wnt - 3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG , followed by IB with FoxM1 and Axin antibodies . .

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D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies..


E . Axin siRNA or control siRNA with HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

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E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


F . Wild type Flag - FoxM1 or S474A mutant was transfected into 293T cells after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies .

F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.


G . HA - Ubiquitin and / or FBXW7 , GSK3β CA , Flag - FoxM1 were co - transfected into 293T cells , after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies .

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G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.


H . FBXW7 siRNA or control siRNA and HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


A . 293T cells were transfected with USPs cDNA or control plasmid . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a for 4 hr . FoxM1 protein levels in the cells were then determined by Western blotting .

A. 293T cells were transfected with USPs cDNA or control plasmid. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 hr. FoxM1 protein levels in the cells were then determined by Western blotting.


B . 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6 - well plates . After 36 hr of transfection , cells were treated with 100 µg / ml CHX for 0 , 2 , 4 , or 6 hr . FoxM1 protein levels in the cells were then determined by Western blotting .

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B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.


C . GSK3 - β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 4 hr . Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody .

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C. GSK3-β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 hr. Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody.


D . GSK3βCA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 6 hr . Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody .

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D. GSK3βCA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 hr. Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody.


E . USP5 and Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A or Flag - FoxM1S474E plasmids were co - transfected into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody .

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E. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.


F . GSK3β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody .

F. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.


G . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without GSK3β CA into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody .

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G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.


H . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without siUSP5 into U87 cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a and 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody .

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H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.


A . Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues . Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain . Scale bar : 50 µmB . ICAT stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . C . FoxM1 stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations .

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A. Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues. Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain. Scale bar: 50 µmB. ICAT stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.C. FoxM1 stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.


D . Increased amounts of FoxM1 plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

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D. Increased amounts of FoxM1 plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


E . Increased amounts of ICAT plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


F . FoxM1 siRNA or control siRNA was transfected into U87 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

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F. FoxM1 siRNA or control siRNA was transfected into U87 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


G . ICAT siRNA or control siRNA was transfected into LN229 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

G. ICAT siRNA or control siRNA was transfected into LN229 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


H . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , FoxM1 - or / and ICAT plasmid - transfected U87 cells . Fold was calculated relative to that in cells transfected with the vector , which was set as 1 .

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H. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, FoxM1- or/and ICAT plasmid-transfected U87 cells. Fold was calculated relative to that in cells transfected with the vector, which was set as 1.


I . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in FoxM1 siRNA - , ICAT siRNA - , control siRNA - or combination of FoxM1 siRNA and ICAT siRNA - transfected U87 cells as described in ( H ) .

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I. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in FoxM1 siRNA-, ICAT siRNA-, control siRNA- or combination of FoxM1 siRNA and ICAT siRNA-transfected U87 cells as described in (H).


J . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmid - transfected U87 cells as described in ( H ) .

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J. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmid-transfected U87 cells as described in (H).


A . FoxM1 or / and ICAT plasmids were co - transfected with Axin - 2 promoter luciferase and TK - Renilla plasmid into U87 cells . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the vector group . Values are mean ± SD for triplicate samples .

A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples.


B . FoxM1 siRNA # 1 , # 2 or control siRNA were co - transfected to U87 cells with Axin - 2 promoter reporter . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the control siRNA group . Values are mean ± SD for triplicate samples .

B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples.


C . ICAT siRNA or control siRNA were co - transfected into LN229 cells with Axin - 2 promoter reporter . Luciferase activity was measured as in ( C ) .

C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C).


D . FoxM1 or / and ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


E . ICAT , FoxM1 or FoxM1 - ∆ FB ( FoxM1 mutant lacking the forkhead box domain ) plasmid or their combinations were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

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E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


F . FoxM1 siRNA and / or ICAT siRNA were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells , and the cells were treated with Wnt3a ( 50 ng / ml ) or control PBS . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

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F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


G . Vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in ( A ) . Values are mean ± SD triplicate samples .

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G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples.


H . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA # 1 or # 2 were determined by Western blotting .

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H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting.


I . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting .

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I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting.


J . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or / and ICAT plasmids were determined by Western blotting .

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J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.


A . A total of 1 × 104 LN229 cells were transfected with control vector , FoxM1 , and / or ICAT plasmids and treated with Wnt3a ( 50 ng / ml ) and were then plated and counted 48 hr after seeding . The proliferation percentage was calculated relative to that in cells transfected with the control vector , which was set as 100 % . Data represent the mean ± SD of three independent experiments .

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A. A total of 1 × 104 LN229 cells were transfected with control vector, FoxM1, and/or ICAT plasmids and treated with Wnt3a (50 ng/ml) and were then plated and counted 48 hr after seeding. The proliferation percentage was calculated relative to that in cells transfected with the control vector, which was set as 100%. Data represent the mean ± SD of three independent experiments.


B . LN229 cells were transfected with vector control , FoxM1 , FoxM1 plus sh - β - catenin or FoxM1 plus ICAT plasmid . The proliferation percentage was determined as described in ( A ) .

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B. LN229 cells were transfected with vector control, FoxM1, FoxM1 plus sh-β-catenin or FoxM1 plus ICAT plasmid. The proliferation percentage was determined as described in (A).


C . LN229 - shFoxM1 cells were transfected with FoxM1 - shR ( siRNA - resistant FoxM1 ) , or FoxM1 - shR plus ICAT plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) .

C. LN229-shFoxM1 cells were transfected with FoxM1-shR (siRNA-resistant FoxM1), or FoxM1-shR plus ICAT plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A).


D . LN229 cells were transfected with USP5 and / or FoxM1 siRNA . The proliferation percentage was determined as described in ( A ) .

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D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).


E . LN229 cells were transfected with USP5 siRNA and / or FoxM1 plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . Data shown in ( A ) - ( E ) represent the mean ± SD of three independent experiments .

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E. LN229 cells were transfected with USP5 siRNA and/or FoxM1 plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A). Data shown in (A)-(E) represent the mean ± SD of three independent experiments.


F - H . Expression of FoxM1 , Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens . Staining of FoxM1 , Cyclin D1 , or USP5 was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test .

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F-H. Expression of FoxM1, Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens. Staining of FoxM1, Cyclin D1, or USP5 was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


I - J . Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology . The staining was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test .

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I-J. Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology. The staining was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


B Equilibrium titration of Hsf1 binding to Alexa Fluor ® 488 - labeled HSE - DNA . Fluorescence polarization plotted versus the Hsf1 trimer concentration . KD = 4 . 7 ± 1 . 9 nM ( n = 4 ) .

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B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).