Figure 7SIRF analysis of RAD51 binding to forks . Representative RAD51 SIRF images at normal or stalled forks are shown . Scale bars : 10 µm . Two independent treatments were performed and scatter plot of RAD51 - bound replication sites from one experiment is shown . Mean values in each sample are listed at the top of the graph . N , number of cells analyzed in each sample in one experiment . RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi - resistant WT but not by ∆ 700N . Cells were treated with HU ( 2 mM , 16 h ) and stained with anti - RAD51 . Representative images are shown . Scale bars : 10 µm . Scatter plot of RAD51 foci number with and without HU treatment is shown . Mean values in each condition are listed at the top of the graph . DNA fiber analysis of nascent - strand DNA degradation in U2OS cells with STN1 and RAD51 co - depletion . Three independent treatments were performed and results from one biological replicate are shown . N , the number of DNA fibers analyzed in each sample in one experiment . Western blots showing protein knockdown in DNA fiber assays are included .

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

Figure 7SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment.RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. N, the number of DNA fibers analyzed in each sample in one experiment. Western blots showing protein knockdown in DNA fiber assays are included.


Figure 1F Whole - mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury . G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c ( Cytc ) at 5 days after injury . H Number of mitochondria in injured axons of Atf3 : BAC2 Tg and Rpt3 CKO mice at 5 days after injury ( n = 5 mice per group ) . I Total number of GFP - positive injured motor neurons in Atf3 : BAC2 Tg and Rpt3 CKO mice at 5 days after injury ( n = 4 mice per group ) . J Number of GFP - positive mitochondria in soma at 5 days after injury . Data are shown as the mean ± s . e . m . ( n = 3 mice ) . Data information : Data are shown as the mean ± s . e . m . , * * P = 0 . 0005 in ( H ) , no significant difference ( P = 0 . 8915 ) in ( I ) and * P = 0 . 0068 in ( J ) , determined by unpaired t - test . Dashed lines reveal hypoglossal nucleus in axon in ( F and G ) . Scale bars , 5 μm in ( F ) and 2 . 5 μm in ( G ) .

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

Figure 1F Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury. G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury. H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group). I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group). J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in axon in (F and G). Scale bars, 5 μm in (F) and 2.5 μm in (G).


Figure 2C Thionine - stained motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice at 7 , 14 and 17 days after hypoglossal nerve axotomy . Arrows indicate the loss of motor neurons . D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side . Data are shown as the mean ± s . e . m . ( * * P = 0 . 0002 , * * * P < 0 . 0001 , determined by unpaired t - test , n = 5 mice per group at each time point ) . Data information : Dashed lines outline hypoglossal nucleus in XII , hypoglossal nucleus ; cc , central canal . Scale bars , 150 μm in C )

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

Figure 2C Thionine-stained motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of motor neurons. D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice per group at each time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)


Figure 3C Hypoglossal motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice before and at 5 days ( 5d ) and 28 days ( 28d ) after injury , immunostained by AnkG and GFP . Arrows indicate the AIS stained by AnkG . Scale bars , 20 μm . D Quantification of the number of the AIS in control ( Cont ) and injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 and Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 043 , * * P = 0 . 0162 , * * * P = 0 . 008 , * * * * P < 0 . 0001 , determined by one - way ANOVA followed by Tukey post hoc analysis , n . d . , not detected , n = 5 mice for each group . E Quantification of the AIS length in control ( Cont ) and injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 and Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 0087 , * * P = 0 . 0024 , * * * P = 0 . 0007 , determined by one - way ANOVA followed by Tukey post hoc analysis , n . d . , not detected , n = 5 mice for each group .

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

Figure 3C Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm. D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group. E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.


Figure 4B Ubiquitination assay of AnkG protein . The lysates of COS - 7 cells expressing the indicated truncated AnkG tagged with GST and HA - Ubiquitin were pulled down with glutathione Sepharose beads , followed by western blotting with anti - HA and anti - GST antibodies . C Western blot of hypoglossal nuclei ( left ) and nerves ( right ) at 5 days after injury with anti - AnkG , RPT3 and GAPDH antibodies . AnkG products of 190 / 270 / 480 kDa are indicated by arrowheads . The positions of the degraded products by calpain are shown by asterisks at ~ 95 kDa and 72 kDa .

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

Figure 4B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.


Figure 5C The localization of AnkG and GFP - labeled mitochondria in the AIS of uninjured MN of Thy1 - Mito Tg mice and injured motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice . D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to ( C ) over a 20 μm line running from the soma to the axon . E Graph represents the percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of Thy1 - Mito mouse ( n = 5 mice per group ) . Data information : For ( E , data are shown as the mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in ( E determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . In closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in C , F Whole - mount observation of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in the proximal region of peripheral hypoglossal ( XII ) nerve as shown in ( A ) . Dashed lines outline the axon . G Graph shows mitochondrial number in hypoglossal ( XII ) axon ( n = 5 mice ) . Data information : For G , data are shown as the mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in G ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 5 μm inI Altered localization of the GFP - labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV - infected Rpt3 CKO mice at 5 days after injury . J Fluorescent intensity scans of GFP and AnkG corresponding to ( I ) . K Graph represents the percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of Atf3 : BAC2 Tg mouse treated with AnkG shRNA ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0064 in ( K ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . In I ) , closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in I ) L Thionine - stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury . Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs . Dashed lines outline hypoglossal nucleus . XII , hypoglossal nucleus ; cc , central canal . M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs ( n = 4 mice per group ) . Data information : For M ) , data are shown as the mean ± s . e . m . , * P = 0 . 0405 , * * P = 0 . 003 in ( M ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 100 μm in ( L ) .

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

Figure 5C The localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice. D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon. E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group). Data information: For (E, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in C,F Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon. G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice). Data information: For G, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm inI Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).


Figure 6B Whole - mount visualization of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in sciatic nerve . Dashed lines indicate the boundary of axon . Scale bar , 5 μm . C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1 - Mito Tg and mtGFP in injured sciatic nerves of Atf3 ; BAC2 and Rpt3 CKO mice . D - F The number of mitochondria ( D ) , percentage of motile mitochondria ( E ) and stationary mitochondrial site ( F ) in axons . Data are shown as the mean ± s . e . m . * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( D ) , * * P = 0 . 0006 , * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( E ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected , n = 5 mice for each group .

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

Figure 6B Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.D-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.


Figure 7C Immunostaining of spinal cord in Atf3 : BAC Tg and Atf3 ; SOD1 mice at P70 . The boxed regions are magnified in lower panels . Arrows indicate GFP - and ATF3 - positive motor neurons ( MNs ) . Dashed lines outline the edge of the gray matter in spinal cord . D The percentage ( % ) of GFP - positive MNs ( black bar ) in ChAT - positive MNs of Atf3 : BAC and Atf3 ; SOD1 mice at P70 ( n = 5 mice per each group ) . E The percentage ( % ) of ATF3 , MMP - 9 or osteopontin ( OPN ) - positive MNs ( black bar ) in GFP - positive MNs of Atf3 ; SOD1 mice at P65 - 70 ( n = 5 - 7 mice per group ) . Data information : For ( D , E , data are shown as the mean ± s . e . m . , Scale bars , 50 μm in ( C ; upper panel ) , 20 μm in ( C ; lower ) F Immunostaining of AnkG in ChAT - positive spinal MN of Atf3 : BAC and SOD1 and in GFP - positive spinal MN of Atf3 ; SOD1 mouse at P70 . G Quantification of the AIS length ( n = 5 mice per group ) . Data information : For G , data are shown as the mean ± s . e . m . , no significant difference in ( G ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in ( FH The localization of AnkG and GFP - labeled mitochondria in uninjured spinal MN of Thy1 - Mito ( without ATF3 induction ) , traumatically sciatic nerve - injured MN of Atf3 : BAC ( with ATF3 induction ) and pathologically damaged MN of Atf3 ; SOD1 mice ( with ATF3 induction ) . Closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . I Fluorescent intensity scan of GFP and AnkG in corresponding to ( H ) . J The percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of uninjured MNs in Thy1 - Mito mice ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0008 , * * * P = 0 . 0007 in ( J ) , * * determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 5 μm in ( H ) . K Whole - mount observation of mtGFP or mtCFP in sciatic nerve . Dashed lines outline the axon . L The number of mtGFP or mtCFP in sciatic nerve ( n = 5 mice ) Data information : For L ) , data are shown as the mean ± s . e . m . , , * * P = 0 . 0036 , * * * * P < 0 . 0001 in ( L ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in K )

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

Figure 7C Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord. D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group). E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group). Data information: For (D, E, data are shown as the mean ± s.e.m., Scale bars , 50 μm in (C; upper panel), 20 μm in (C; lower)F Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70. G Quantification of the AIS length (n = 5 mice per group). Data information: For G, data are shown as the mean ± s.e.m., no significant difference in (G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in (FH The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. I Fluorescent intensity scan of GFP and AnkG in corresponding to (H). J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0008, *** P = 0.0007 in (J), ** determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in (H).K Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon. L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice) Data information: For L), data are shown as the mean ± s.e.m., , ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in K)


Figure 1B . Whole exome sequencing was carried out and analysis revealed a missense p . L165F variant ( c . 493 C > T ) in the ALX1 homeodomain , heterozygous in the parents ( ALX1165L / 165F ) , wildtype in the unaffected sibling ( ALX1165L / 165L ) , and homozygous in both affected subjects ( ALX1165F / 165F ) . E . Immunofluorescence staining for pluripotent markers SSEA4 , OCT4 , SOX2 , and TRA - 1 - 60 and alkaline phosphatase staining of iPSC clones . One representative iPSC clone is shown for each genotype . Scale bar : 400 µm . F . Expression of pluripotent ( OCT4 , NANOG ) , endoderm ( Endo . , AFP , GATA4 , FOXA2 ) , ectoderm ( Ecto . , NESTIN , GFAP , SOX1 ) , and mesoderm ( Meso . , BRACH . ( BRACHYURY ) , RUNX1 , CD34 ) gene markers for ALX1165L / 165L ( green ) , ALX1165L / 165F ( red ) , and ALX1165F / 165F ( blue ) iPSC relative to undifferentiated cells ( UND ) . Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype . Significance : p = 0 . 0167 for OCT4 , p = 0 . 0005 for NANOG , p = 0 . 000004 for AFP , p = 0 . 0082 for GATA4 , p = 0 . 0137 for FOXA2 , p = 0 . 00002 for NESTIN , p = 0 . 0167 for GFAP , p = 0 . 0014 for SOX1 , p = 0 . 0117 for BRACHYURY , p = 0 . 0008 for RUNX1 and p = 0 . 0068 for CD34 when comparing undifferentiated and differentiated ALX1165L / 165L iPSC . p = 0 . 0013 for OCT4 , p = 0 . 0011 for NANOG , p = 0 . 0000003 for AFP , p = 0 . 0003 for GATA4 , p = 0 . 0063 for FOXA2 , p = 0 . 0001 for NESTIN , p = 0 . 027 for GFAP , p = 0 . 000002 for SOX1 , p = 0 . 000009 for BRACHYURY , p = 3e - 9 for RUNX1 and p = 0 . 000006 for CD34 when comparing undifferentiated and differentiated ALX1165F / 165L iPSC . p = 0 . 0201 for OCT4 , p = 0 . 006 for NANOG , p = 1x10 - 12 for AFP , p = 5x10 - 13 for GATA4 , p = 0 . 0031 for FOXA2 , p = 0 . 0292 for NESTIN , p = 0 . 00001 for GFAP , p = 6x10 - 7 for SOX1 , p = 0 . 0204 for BRACHYURY , p = 0 . 0009 for RUNX1 and p = 0 . 000003 for CD34 when comparing undifferentiated and differentiated ALX1165F / 165F iPSC . Data from each clone were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant .

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

Figure 1B. Whole exome sequencing was carried out and analysis revealed a missense p.L165F variant (c.493 C>T) in the ALX1 homeodomain, heterozygous in the parents (ALX1165L/165F), wildtype in the unaffected sibling (ALX1165L/165L), and homozygous in both affected subjects (ALX1165F/165F).E. Immunofluorescence staining for pluripotent markers SSEA4, OCT4, SOX2, and TRA-1-60 and alkaline phosphatase staining of iPSC clones. One representative iPSC clone is shown for each genotype. Scale bar: 400 µm.F. Expression of pluripotent (OCT4, NANOG), endoderm (Endo., AFP, GATA4, FOXA2), ectoderm (Ecto., NESTIN, GFAP, SOX1), and mesoderm (Meso., BRACH. (BRACHYURY), RUNX1, CD34) gene markers for ALX1165L/165L (green), ALX1165L/165F(red), and ALX1165F/165F (blue) iPSC relative to undifferentiated cells (UND). Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. Significance: p=0.0167 for OCT4, p=0.0005 for NANOG, p= 0.000004 for AFP, p=0.0082 for GATA4, p=0.0137 for FOXA2, p=0.00002 for NESTIN, p=0.0167 for GFAP, p=0.0014 for SOX1, p=0.0117 for BRACHYURY, p=0.0008 for RUNX1 and p=0.0068 for CD34 when comparing undifferentiated and differentiated ALX1165L/165L iPSC. p=0.0013 for OCT4, p=0.0011 for NANOG, p=0.0000003 for AFP, p=0.0003 for GATA4, p=0.0063 for FOXA2, p=0.0001 for NESTIN, p=0.027 for GFAP, p =0.000002 for SOX1, p=0.000009 for BRACHYURY, p=3e-9 for RUNX1 and p=0.000006 for CD34 when comparing undifferentiated and differentiated ALX1165F/165L iPSC. p=0.0201 for OCT4, p=0.006 for NANOG, p=1x10-12 for AFP, p=5x10-13 for GATA4, p=0.0031 for FOXA2, p=0.0292 for NESTIN, p=0.00001 for GFAP, p=6x10-7 for SOX1, p=0.0204 for BRACHYURY, p=0.0009 for RUNX1 and p=0.000003 for CD34 when comparing undifferentiated and differentiated ALX1165F/165F iPSC. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


Figure 2B . Images of iPSC and iPSC - derived NCC at Days 0 , 14 , and passage 4 following differentiation . Scale bars : 400 µm ( Day 0 ) , 200µm ( Day 14 , passage 4 ) .

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Figure 2B. Images of iPSC and iPSC-derived NCC at Days 0, 14, and passage 4 following differentiation. Scale bars: 400 µm (Day 0), 200µm (Day 14, passage 4).


Figure 3Gene expression analysis across NCC differentiation of unaffected control ALX1165L / 165L ( green ) , heterozygous ALX1165L / 165F ( magenta ) and homozygous ALX1165F / 165F iPSC : ALX1 , neural plate border specifier genes ZIC1 , PAX7 , PAX3 , MSX1 , MSX2 , DLX5 ; neural crest specifier genes FOXD3 , P75 , TFAP2A , SNAI2 , TWIST1 ; and lineage specifier gene HAND2 . The RT - qPCR relative expression values were normalized to RPLP0 and GAPDH expression . Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype . Exact p - values are provided in Table EV1 . Data from each clone were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant .

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

Figure 3Gene expression analysis across NCC differentiation of unaffected control ALX1165L/165L (green), heterozygous ALX1165L/165F (magenta) and homozygous ALX1165F/165F iPSC: ALX1, neural plate border specifier genes ZIC1, PAX7, PAX3, MSX1, MSX2, DLX5; neural crest specifier genes FOXD3, P75, TFAP2A, SNAI2, TWIST1; and lineage specifier gene HAND2. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. Exact p-values are provided in Table EV1. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


Figure 4A . Homozygous ALX1165F / 165F NCC ( blue ) showed an increase in sensitivity to apoptosis when compared to control ALX1165L / 165L NCC ( black ) . The data on the left represents the mean percentage of Annexin V positive cells , indicative of apoptosis , as determined by FACS analysis , with the data on the right being an example of one such experiment . Apoptosis was induced by immersion in a 55 ° C water bath for ten minutes . Representative experiment for each condition is shown . Data is represented as pooled mean ± SEM of three independent experiments . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . . * : Significantly different from the basal apoptosis rate : p = 3x10 - 12 between control NCC basal apoptosis and induced apoptosis , and between ALX1165F / 165F NCC basal apoptosis and induced apoptosis . * * : Significantly different from control NCC ( p = 0 . 0004 ) . B . Expression levels of cyclins CCNA2 ( blue ) and CCND1 ( orange ) in NCC at passages 2 and 3 of ALX1165L / 165L and ALX1165F / 165F NCC . The RT - qPCR relative expression values were normalized to RPLP0 and GAPDH expression . Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype . * : Significantly different from control NCC at passage 2 ( p = 0 . 001 between control and ALX1165F / 165F NCC at passage 2 for CCNA2 , p = 0 . 0052 for CCND1 ) . * * : Significantly different from control NCC at passage 3 ( p = 0 . 0494 between control and ALX1165F / 165F NCC for CCNA2 , p = 0 . 0008 for CCND1 ) . C . Fluorescence Activated Cell Sorting ( FACS ) experiments showed that control ALX1165L / 165L NCC ( green ) exhibited increased expression of mesenchymal markers CD90 , CD105 , and CD73 with culture time ( passages 1 through 4 ) , whereas homozygous ALX1165F / 165F NCC ( blue ) showed a consistent expression of the markers expressed at passage 1 throughout . Further , control ALX1165L / 165L NCC showed a downregulation of CD57 expression with culture time , while ALX1165F / 165F NCC maintained the same level of CD57 across passages . Data are presented as the mean percentage of positive - stained cells across passage numbers . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC . For CD90 , p = 0 . 0013 at passage 3 and p = 0 . 0207 at passage 4 . For CD105 , p = 0 . 0016 at passage 2 , p = 0 . 00004 at passage 3 and p = 0 . 0021 at passage 4 . For CD73 , p = 0 . 0060 at passage 2 , p = 0 . 00004 at passage 3 and p = 0 . 0114 at passage 4 . For CD57 , p = 0 . 0026 at passage 2 , p = 0 . 000003 at passage 3 and p = 0 . 000007 at passage 4 .

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

Figure 4A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. . *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004). B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1).C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.


Figure 5A . Mutant ALX1165F / 165F NCC ( blue ) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L / 165L NCC ( black ) . Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24 - hour period . For fluorescent pictures , cells were stained in serum free media containing 3 . 6µM CellTracker Green CMFDA ( Life Technologies ) for 30 min at 37 ° C and allowed to recover for 30 min before starting the experiment . Images were acquired every 6 hours using a Keyence BZ - X800 microscope . Surface area analyses and percentages of coverage were measured using ImageJ software ( NIH ) . The NCC were monitored over 24 hours . The data are represented as the average of the percentage of closure ± SEM . Scale bar = 200µm . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC ( p < 0 . 0001 ) . B . Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F / 165F NCC ( blue ) secrete less BMP2 and more BMP9 compared to control ALX1165L / 165L NCC ( green ) . Data are represented as pooled mean ± SEM of three clones from each genotype . Statistical significance was determined with an ANOVA test . A p - value < 0 . 05 was considered significant . * : Significantly different from control NCC ( p = 0 . 0424 for BMP2 and p = 0 . 0192 for BMP9 ) . C . Addition of soluble BMP2 or CV2 , a BMP9 antagonist , to the culture medium could partially rescue the migration defect of ALX1165F / 165F NCC . At the beginning of the assay , 10 , 50 , or 100 ng / ml of soluble BMP2 , CV2 , or a combination of the two at 100 ng / ml each were added to the culture medium , and the cells were monitored over the next 24 hours . The data are represented as the average of the percentage of closure ± SEM . Scale bar : 400µm .

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

Figure 5A. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001).B. Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A p-value <0.05 was considered significant. *: Significantly different from control NCC (p =0.0424 for BMP2 and p=0.0192 for BMP9).C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.


Figure 6A . Dissected flatmount wild type and alx1 - / - zebrafish larvae craniofacial cartilages after Alcian blue staining , the anterior points to the left of the page in all images . The ventral cartilages appear normal , but the alx1 - / - anterior neurocranium ( ANC ) appears narrow , with the midline element that is derived from the frontonasal NCC being absent . The Meckel ' s cartilage ( arrow , MC ) is also diminutive . Scale bar : 200 µm . B . Zebrafish alx1 mutants ( blue ) show reduced detectable expression of alx1 but increased expression of alx3 , alx4a compared to wild type controls ( green ) . alx4b expression levels are similar between wild type and alx1 - / - lines . Data is represented as the mean of all pooled embryos from three different clutches . The RT - qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method . Data from each clutch were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . Statistical significance denoted by * ; p < 0 . 0001 between WT zebrafish and alx1 - / - at all measured time points ; at 10 ss , 24 hpf and 36 hpf for alx3 ; and at 24 hpf and 36 hpf for alx4a . Refer to Table EV2 for p - values . C . Dissected flatmount of zebrafish embryos injected with Alx1DN , after Alcian blue staining . The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium ( ANC ) and a profound hypoplasia of the Meckel ' s and ventral cartilages . In the most severely affected zebrafish , a nearly abrogated ANC was observed . Scale bar : 200 µm . D . Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted . In the control animal , the anterior cranial NCC always migrate to contribute to the median portion of the ANC . In contrast , the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC , where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location ( white asterisks ) . Scale bar : 250µm .

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

Figure 6A. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm.B. Zebrafish alx1 mutants (blue) show reduced detectable expression of alx1 but increased expression of alx3, alx4a compared to wild type controls (green). alx4b expression levels are similar between wild type and alx1-/- lines. Data is represented as the mean of all pooled embryos from three different clutches. The RT-qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method. Data from each clutch were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. Statistical significance denoted by *; p<0.0001 between WT zebrafish and alx1-/- at all measured time points; at 10 ss, 24 hpf and 36 hpf for alx3; and at 24 hpf and 36 hpf for alx4a. Refer to Table EV2 for p-values.C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm.D. Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted. In the control animal, the anterior cranial NCC always migrate to contribute to the median portion of the ANC. In contrast, the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC, where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location (white asterisks). Scale bar: 250µm.


Figure 1A Representative picture of plants carrying pASDOG1 : : LUC sprayed with mock solution , ABA or GA and imaged 24 h later . The full pictures are shown in Fig EV2 and Appendix Fig S1 . Scale bar , 1 cm . B Quantification of emitted light intensity per plant after treatment with ABA and GA , compared with mock treatment . C Antisense expression is reduced in response to drought conditions . The full pictures are shown in Fig EV3 . Scale bar , 1 cm . D Quantification of emitted light intensity per plant for control and drought - treated plants . E Strand - specific RT - qPCR asDOG1 expression analysis of 3 - day Col - 0 ( WT ) drought - treated vs . control plants . F The asDOG1 expression level ( black line ) is reduced while DOG1 sense expression ( grey line ) is increased after application of ABA . Leaves of mature Col - 0 ( WT ) 40 - day - old plants were sprayed with ABA and collected 0 , 6 and 10 h later for RNA extraction and RT - qPCR . Signals were normalized against the level of the UBC transcript . The data points are the averages for at least three biological replicates and are normalized against the mock - treated value . Error bars represent standard deviation . * and * * * represent t - test P - values of < 0 . 05 and < 0 . 001 , respectively .

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Figure 1A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.C Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.D Quantification of emitted light intensity per plant for control and drought-treated plants.E Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.


Figure 2A Col - 0 ( WT ) and dog1 - 3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days . A picture of a representative experiment is shown . Scale bar , 2 cm . B Quantification of recovered and dead Col - 0 ( WT ) , null - 3 and dog1 - 4 plants ; plants were scored as dead when unable to grow and produced offspring after an extended recovery period . C Analysis of DOG1 sense expression in drought - treated vs . control Col - 0 ( WT ) and dog1 - 3 mutant plants . D dog1 - 3 mutant plants are defective in the regulation of the majority of the tested drought marker genes . Col - 0 ( WT ) and dog1 - 3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT - qPCR . Error bars represent standard deviation , and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 2A Col-0 (WT) and dog1-3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days. A picture of a representative experiment is shown. Scale bar, 2 cm.B Quantification of recovered and dead Col-0 (WT), null-3 and dog1-4 plants; plants were scored as dead when unable to grow and produced offspring after an extended recovery period.C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.D dog1-3 mutant plants are defective in the regulation of the majority of the tested drought marker genes. Col-0 (WT) and dog1-3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT-qPCR. Error bars represent standard deviation, and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


Figure 3A Plants expressing LUC fused with full - length DOG1 ( pDOG1LUC : : DOG1 ) were sprayed with mock solution or ABA 10 , 20 and 40 days after germination and analysed 24 h after treatment . The graphs show mean emitted light intensity per plant . B Representative picture of mock - and ABA - treated pDOG1LUC : : DOG1 plants . The full picture is shown in Fig EV2C and D . Scale bar , 1 cm . C A truncated construct that lacks the DOG1 antisense promoter region ( pDOG1shDOG1 : : LUC ) is not induced by ABA and is highly expressed throughout development . The graphs show mean emitted light intensity per plant . D Representative picture of mock - and ABA - treated pDOG1shDOG1 : : LUC plants . Scale bar : 1cm . E RT - qPCR analysis of DOG1 sense mRNA level . F Strand - specific RT - qPCR analysis of antisense levels after 10 h of ABA treatment in 10 - , 20 - and 40 - day - old Col - 0 plants . G The pASDOG1 : : LUC reporter is silenced by the application of ABA , while mutation of TATA elements in the asDOG1 promoter ( pASDOG1 ∆ TATA : : LUC ) leads to attenuation of expression in the presence and absence of ABA . H Mutation of TATA elements in the asDOG1 promoter leads to high - level DOG1 sense expression and non - responsiveness to ABA . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 3A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.E RT-qPCR analysis of DOG1 sense mRNA level.F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


Figure 4A Plants expressing the truncated asDOG1 promoter - deficient version pDOG1shDOG1 : : LUC are more resistant to drought than plants expressing the full - length genomic version pDOG1 : : LUC - DOG1 . Three independent lines are shown for each construct , following 10 days of water deprivation . Scale bar , 2 cm . B RT - qPCR quantification of selected drought marker genes in mock - and ABA - treated Col - 0 ( WT ) and two independent pDOG1shDOG1 : : LUC transgenic lines . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 4A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


figf1a , LAMP1 + lysosome size and quantity in NRK cells ( dotted outline ) starved for hours ( h ) . Error bars show s . e . m . ( n = 3 ) . b , Starved NRK cells expressing LAMP1 - YFP show tubules ( arrows ) . Box is expanded on the right . c , Immuno - TEM ( gold marks LAMP1 ) of starved NRK cells . Continuity ( arrow ) illustrated between autolysosome ( red star ) and tubule . d , LAMP1 - YFP expressing NRK - LC3 cells as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a cell from d . f , Time - lapse images ( seconds ) of starved LAMP1 - YFP - expressing NRK cells . g , Time - lapse images ( minutes ) of starved LAMP1 - PAGFP or LAMP1 - RFP - expressing NRK cells after photo - activation . Scale bars , 5 µm .

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figf1a, LAMP1+ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bars show s.e.m. (n = 3).b, Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded on the right.c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule.d, LAMP1-YFP expressing NRK-LC3 cells as in a. Arrows show reformation tubules. e, Three-dimensional reconstruction of a cell from d.f, Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells.g, Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.


figf2a - c , Starved LAMP1 - YFP NRK cells stained with Lysotracker or DQ - BSA . c , Right , TEM of purified lysosomes . d , Magnification ( 4 × ) of images shown in c . Arrows indicate reformation tubules and arrowhead indicates autolysosome . e , f , Immunofluorescence ( e ) and immuno - EM ( f ) using LAMP1 antibody of starved LAMP1 - YFPNRKcells shown in c . g , Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D . Middle panel shows 8 - h fractions 7 and 9 analysed by TEM . Fraction 10 is the gradient top . h , Starved LAMP1 - PAGFP NRK cells were photo - activated ( 4 h ) and imaged at 12 h with Lysotracker ( upper ) or DQ - BSA ( lower ) . Boxes show enlargements . R ( r ) = Pearson ' s coefficient . Scale bars , 5 µm .

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figf2a-c, Starved LAMP1-YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome.e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c.g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top.h, Starved LAMP1-PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson's coefficient. Scale bars, 5 µm.


figf3a , TEM ( bottom ; scale bar , 5 µm ) and fluorescence ( top ; scale bar , 10 µm ) of NRK - LC3 cells starved for times shown with enlargements ( boxes ) . b , d , Immunoblots of cells shown in a with indicated antibodies ( d , after transfection with non - specific ( NS ) or atg5 RNAi ) . c , Autophagy ( black lines show independent experiments ) and phospho - S6K percentage ( densitometry of data shown in b , red line ) . e - g , LAMP1 - YFP NRK - LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h ( total , 6 h ) or 6 h ( total , 8 h ) analysed by blotting ( e ) as in b or microscopy ( f ; scale bar , 5 µm ) with quantification ( g ) . Error bars , s . e . m . ; n = 3 .

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figf3a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes).b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines  show  independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line).e-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.


figf4a , LAMP1 / LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation . b , Rab7Q67L - GFP - transfected LC3 - CFP / Lamp1 - RFP NRK cells starved for 10 h . c , Rab7 - CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation . d , LAMP1 - YFP NRK - LC3 cells starved for times shown with 1 µg ml - 1 leupeptin and blotted with indicated antibodies . e , Cells in d imaged . . f , Fibroblast cells from patients with Scheie syndrome ( GM01256 ) or healthy controls starved and blotted with indicated antibodies . g , Cells in f imaged . Lysosome size classified by Image - ProPlus ( IPP ) software .

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figf4a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h.c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation.d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged..f, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software.


Figure 1A Representative images show expression of YAP in normal cervical tissues , early - stage cervical cancer ( stage I II ) , and advanced - stage cervical cancer ( stage III IV ) . Note the significant increase in YAP immunosignals ( brown staining ) in the advanced - stage tumors . Tissue core size : 1 . 5 mm . B Positivity ( percentage of positively stained cells relative to the total number of cells in the tissue section ) of YAP immunosignals in the normal and cancerous tissues . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Bars represent means ± SEM ( n = 10 for normal tissues ; n = 69 for tumor tissues ; * * * P 0 . 0001 ) . C A representative image showing the expression of YAP ( brown staining ) in normal cervical tissue . Scale bar = 20 μm . D - F Representative images showing the expression of YAP in ( D ) early - stage cervical cancer tissue ( stage Ib , T1N0M0 ) ; ( E ) medium - stage cervical cancer tissue ( stage IIb , T2N0M0 ) ; and ( F ) advanced - stage cervical cancer tissue ( stage IIIb , T3N1M0 ) . Scale bar = 20 μm . G , H High - resolution images showing the cellular location of YAP in ( G ) early - stage cervical cancer tissue and ( H ) advanced - stage cervical cancer tissue . Scale bar = 20 μm . I Multidimensional cancer genomics data analysis results showing cross - cancer YAP gene alteration . The histogram showed the frequencies of YAP gene mutation , deletion , and amplification across cancers . Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools . The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer .

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

Figure 1A Representative images show expression of YAP in normal cervical tissues, early-stage cervical cancer (stage I II), and advanced-stage cervical cancer (stage III IV). Note the significant increase in YAP immunosignals (brown staining) in the advanced-stage tumors. Tissue core size: 1.5 mm.B Positivity (percentage of positively stained cells relative to the total number of cells in the tissue section) of YAP immunosignals in the normal and cancerous tissues. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Bars represent means ± SEM (n = 10 for normal tissues; n = 69 for tumor tissues; ***P 0.0001).C A representative image showing the expression of YAP (brown staining) in normal cervical tissue. Scale bar = 20 μm.D-F Representative images showing the expression of YAP in (D) early-stage cervical cancer tissue (stage Ib, T1N0M0); (E) medium-stage cervical cancer tissue (stage IIb, T2N0M0); and (F) advanced-stage cervical cancer tissue (stage IIIb, T3N1M0). Scale bar = 20 μm.G, H High-resolution images showing the cellular location of YAP in (G) early-stage cervical cancer tissue and (H) advanced-stage cervical cancer tissue. Scale bar = 20 μm.I Multidimensional cancer genomics data analysis results showing cross-cancer YAP gene alteration. The histogram showed the frequencies of YAP gene mutation, deletion, and amplification across cancers. Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools. The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer.


Figure 2A , C , E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( A ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( C ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( E ) . β - Actin was used as a protein loading control . B , D , F Growth curves of YAP - overexpressed ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( B ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( D ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( F ) . Each point represents the mean ± SEM ( n = 4 ) . * * * P < 0 . 0001 , ME180 - MXIV vs . ME180 - YAP cells and ME180 - MXIV vs . ME180 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , HT3 - MXIV vs . HT3 - YAP cells and HT3 - MXIV vs . HT3 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , Ect1 - MXIV vs . Ect1 - Ect1 - YAPS127A cells on day 4 . * P < 0 . 0074 , Ect1 - MXIV vs . Ect1 - YAP cells on day 4 . G Western blot showing YAP levels in non - targeting control siRNA ( siCtrl ) - and YAP siRNA ( siYAP ) - transfected HT3 cells . H Proliferation of HT3 cells treated with control ( siCtrl ) or YAP siRNA ( siYAP ) . Each point represents the mean ± SEM ( n = 5 ) . * * * P < 0 . 001 compared with siCtrl ( siCtrl vs . siYAP , P = 0 . 0002 ) .

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

Figure 2A, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).


Figure 3A Representative images showing colonies formed by Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells after growth in soft agar for 9 days . Scale bar : 500 μm . B Quantitative data showing colony formation in Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells . Each bar represents mean ± SEM of five independent experiments . Bars with different letters are significantly different from each other ( Ect1 - MXIV vs . Ect1 - YAP , P = 0 . 0003 ; Ect1 - MXIV vs . Ect1 - YAPS127A , P = 0 . 0002 ) . C , E Representative images showing the anchorage - independent growth of YAP - expressing HT3 ( C ) and ME180 ( E ) cervical cancer cell lines . Anchorage - independent cell growth was determined by the soft agar colony formation assay . Scale bar : 500 μm . D , F Fluorescence - based quantitative analysis showing the differences of anchorage - independent growth in HT3 - MXIV , HT3 - YAP , and HT3 - YAPS127A ( D ) and ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells ( F ) . The anchorage - independent cell growth was determined using a CytoSelect 96 - Well Cell Transformation Assay kit , and data are presented as relative fluorescent units ( RFU ) . Each bar represents mean ± SEM of four independent experiments . Bars with different letters are significantly different from each other ( HT3 - MXIV vs . HT3 - YAP , P = 0 . 0002 ; HT3 - MXIV vs . HT3 - YAPS127A , P < 0 . 0001 ; ME - MXIV vs . ME - YAP , P = 0 . 0011 ; ME - MXIV vs . ME - YAPS127A , P = 0 . 0013 ) .

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

Figure 3A Representative images showing colonies formed by Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells after growth in soft agar for 9 days. Scale bar: 500 μm.B Quantitative data showing colony formation in Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells. Each bar represents mean ± SEM of five independent experiments. Bars with different letters are significantly different from each other (Ect1-MXIV vs. Ect1-YAP, P = 0.0003; Ect1-MXIV vs. Ect1-YAPS127A, P = 0.0002).C, E Representative images showing the anchorage-independent growth of YAP-expressing HT3 (C) and ME180 (E) cervical cancer cell lines. Anchorage-independent cell growth was determined by the soft agar colony formation assay. Scale bar: 500 μm.D, F Fluorescence-based quantitative analysis showing the differences of anchorage-independent growth in HT3-MXIV, HT3-YAP, and HT3-YAPS127A (D) and ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells (F). The anchorage-independent cell growth was determined using a CytoSelect 96-Well Cell Transformation Assay kit, and data are presented as relative fluorescent units (RFU). Each bar represents mean ± SEM of four independent experiments. Bars with different letters are significantly different from each other (HT3-MXIV vs. HT3-YAP, P = 0.0002; HT3-MXIV vs. HT3-YAPS127A, P < 0.0001; ME-MXIV vs. ME-YAP, P = 0.0011; ME-MXIV vs. ME-YAPS127A, P = 0.0013).


Figure 4A , B Representative images of tumorxenografts of ME180 - MXIV ( MX , right flank ) , ME180 - YAP ( A ) ( YAP , left flank ) , and ME180 - YAPS127A ( B ) ( YAPS127A , left flank ) cells implanted in athymic nude mice . Red lines indicate the edge of tumors . C The growth curve of humantumorxenografts derived from control ( ME - MX ) and YAP - overexpressing ME180cervical cancer cell lines ( ME - YAP , ME - YAPS127A ) implanted in the athymic nude mice . Each point represents mean ± SEM of six tumors ( control n = 10 ) . D Representative images showing the relative size of tumors from control and YAP - overexpressing ME180 cervical cancer cells . E The average weight of tumor xenografts from the control and YAP - overexpressing ME180 cell lines . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents the mean ± SEM ( n = 10 for control , n = 6 for YAP and YAPS127A groups ) . Bars with different letters are significantly different from each other ( MXIV vs . YAP , P = 0 . 0008 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells . G Expression of Ki67 in the tumor xenografts of ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells implanted in athymic nude mice . Ki67 was visualized using an Alexa - 488 ( green ) - conjugated secondary antibody . Actin was visualized using an Alexa - 594 ( red ) - conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 20 μm . Note the significant increase in the Ki67 - positive cells in the ME180 - YAP and ME180 - YAPS127A tumor xenografts . H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector ( shCtrl ) or lentivirus - based YAP shRNAs ( shYAP # 1 or shYAP # 2 ) . I Representative images showing tumor xenografts derived from ME180 - shCtrl cells ( left flank ) and ME180 - shYAP # 1 cells ( right flank ) ( n = 6 ) . J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA ( left , shCtrl ) or YAP shRNA # 1 ( right , shYAP # 1 ) ( n = 6 ) .

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

Figure 4A, B Representative images of tumorxenografts of ME180-MXIV (MX, right flank), ME180-YAP (A) (YAP, left flank), and ME180-YAPS127A (B) (YAPS127A, left flank) cells implanted in athymic nude mice. Red lines indicate the edge of tumors.C The growth curve of humantumorxenografts derived from control (ME-MX) and YAP-overexpressing ME180cervical cancer cell lines (ME-YAP, ME-YAPS127A) implanted in the athymic nude mice. Each point represents mean ± SEM of six tumors (control n = 10).D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001).F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells.G Expression of Ki67 in the tumor xenografts of ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells implanted in athymic nude mice. Ki67 was visualized using an Alexa-488 (green)-conjugated secondary antibody. Actin was visualized using an Alexa-594 (red)-conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. Note the significant increase in the Ki67-positive cells in the ME180-YAP and ME180-YAPS127A tumor xenografts.H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector (shCtrl) or lentivirus-based YAP shRNAs (shYAP#1 or shYAP#2).I Representative images showing tumor xenografts derived from ME180-shCtrl cells (left flank) and ME180-shYAP#1 cells (right flank) (n = 6).J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA (left, shCtrl) or YAP shRNA#1 (right, shYAP#1) (n = 6).


Figure 5RT - PCR results showing that YAP stimulates the mRNA expressions of EGFR , AREG , and TGF - α in ME180 cervical cancer cell . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( YAP mRNA : MXIV vs . YAP , P = 0 . 0003 ; MXIV vs . YAPS127A , P = 0 . 0001 ; EGFR mRNA : MXIV vs . YAP , P = 0 . 0066 ; MXIV vs . YAPS127A , P = 0 . 0003 ; TGF - α mRNA : MXIV vs . YAP , P = 0 . 0235 ; MXIV vs . YAPS127A , P = 0 . 0040 ; AREG mRNA : MXIV vs . YAP , P = 0 . 0002 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . Proliferation of ME180 cells incubated in medium containing 1 % FBS in the absence ( control ) or presence of 10 ng / ml TGF - α . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Each point represents the mean ± SEM of four independent repeats . * * * P < 0 . 0001 versus control on day 5 . TGF - α treatment ( 10 ng / ml , 24 h ) promotes ME180 cell cycle progression . G1 , S , and G2 indicate cells in G1 phase , DNA synthesis phase , and the G2 / M phase , respectively , of cell cycle . Apo : apoptotic cells . Representative images showing the morphology of ME180 cells with or without TGF - α ( 10 ng / ml ) treatment for 24 h ( scale bar : 50 μm ) or 108 h ( scale bar : 25 μm ) . Please note the elongation of ME180 cells after TGF - α treatment for 24 h ( TGF - α , 24 h ) and the formation of multiple layers in ME180 cells after TGF - α treatment for 108 h ( TGF - α , 108 h ) . Effect of TGF - α on the migration of ME180 cells . TGF - α treatment ( 100 ng / ml , 10 h ) drastically stimulated the migration of ME180 cells .

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

Figure 5RT-PCR results showing that YAP stimulates the mRNA expressions of EGFR, AREG, and TGF-α in ME180 cervical cancer cell. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (YAP mRNA: MXIV vs. YAP, P = 0.0003; MXIV vs. YAPS127A, P = 0.0001; EGFR mRNA: MXIV vs. YAP, P = 0.0066; MXIV vs. YAPS127A, P = 0.0003; TGF-α mRNA: MXIV vs. YAP, P = 0.0235; MXIV vs. YAPS127A, P = 0.0040; AREG mRNA: MXIV vs. YAP, P = 0.0002; MXIV vs. YAPS127A, P < 0.0001).Proliferation of ME180 cells incubated in medium containing 1% FBS in the absence (control) or presence of 10 ng/ml TGF-α. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Each point represents the mean ± SEM of four independent repeats. ***P < 0.0001 versus control on day 5.TGF-α treatment (10 ng/ml, 24 h) promotes ME180 cell cycle progression. G1, S, and G2 indicate cells in G1 phase, DNA synthesis phase, and the G2/M phase, respectively, of cell cycle. Apo: apoptotic cells.Representative images showing the morphology of ME180 cells with or without TGF-α (10 ng/ml) treatment for 24 h (scale bar: 50 μm) or 108 h (scale bar: 25 μm). Please note the elongation of ME180 cells after TGF-α treatment for 24 h (TGF-α, 24 h) and the formation of multiple layers in ME180 cells after TGF-α treatment for 108 h (TGF-α, 108 h).Effect of TGF-α on the migration of ME180 cells. TGF-α treatment (100 ng/ml, 10 h) drastically stimulated the migration of ME180 cells.


Figure 6Western blot analysis showing effects of TGF - α on phosphorylation of EGFR , YAP , AKT , and ERK1 / 2 . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 5 , 10 , 30 , 60 or 120 min . Western blot analysis showing effects of TGF - α on the phosphorylation of the components of the Hippo signaling pathway . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 30 , 60 min . Real - time PCR determines the action of TGF - α and YAP levels on the mRNA expression of AREG . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( Ctrl vs . TGF - α , P < 0 . 0001 ; MXIV vs . YAP , P < 0 . 0001 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . Real - time PCR analysis showing that knockdown of YAP with YAP siRNA ( siYAP ) significantly suppressed TGF - α - induced ( FBS 10 % , TGF - α : 10 ng / ml for 48 h ) AREG expression in ME180 cells . siCTRL , a non - targeting siRNA , was used as a negative control . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0067 ) . Proliferation of ME180 cells ( FBS 1 % ) treated with control ( siCTRL ) or YAP ( siYAP ) prior to treatment with control medium or TGF - α ( 10 ng / ml ) for 108 h . Each bar represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P = 0 . 0058 ; siYAP + CTRL vs . siYAP + TGF - α , P = 0 . 1840 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0013 ) . Quantitative data of the wound - healing assay showing the migration of ME180 cells that were treated with control ( siCTRL ) or YAP siRNA ( siYAP ) prior to treatment with or without TGF - α for 12 h . Each bar in bar graphs represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0041 ) .

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

Figure 6Western blot analysis showing effects of TGF-α on phosphorylation of EGFR, YAP, AKT, and ERK1/2. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 5, 10, 30, 60 or 120 min.Western blot analysis showing effects of TGF-α on the phosphorylation of the components of the Hippo signaling pathway. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 30, 60 min.Real-time PCR determines the action of TGF-α and YAP levels on the mRNA expression of AREG. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (Ctrl vs. TGF-α, P < 0.0001; MXIV vs. YAP, P < 0.0001; MXIV vs. YAPS127A, P < 0.0001).Real-time PCR analysis showing that knockdown of YAP with YAP siRNA (siYAP) significantly suppressed TGF-α-induced (FBS 10%, TGF-α: 10 ng/ml for 48 h) AREG expression in ME180 cells. siCTRL, a non-targeting siRNA, was used as a negative control. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0067).Proliferation of ME180 cells (FBS 1%) treated with control (siCTRL) or YAP (siYAP) prior to treatment with control medium or TGF-α (10 ng/ml) for 108 h. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P = 0.0058; siYAP+CTRL vs. siYAP+TGF-α, P = 0.1840; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0013).Quantitative data of the wound-healing assay showing the migration of ME180 cells that were treated with control (siCTRL) or YAP siRNA (siYAP) prior to treatment with or without TGF-α for 12 h. Each bar in bar graphs represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0041).


Figure 7Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1 , MOB1 , and YAP . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with AREG ( 50 ng / ml ) for 0 , 30 , 60 min . AREG ( 50 ng / ml ) treatment induced appearance of elongated cells in ME180 cells ( top and middle panels ) and significantly stimulated ME180 cell proliferation ( lower panel ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 003 ) . Scale bar : 100 μm . Wound - healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum - free medium . Scale bar : 200 μm . Real - time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression . Each bar represents mean ± SEM ( n = 9 ) . Bars with different letters are significantly different from each other ( P < 0 . 0001 ) . Real - time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA ( siYAP ) significantly suppressed AREG - induced AREG mRNA expression . siCTRL ( non - target siRNA ) was used as a siRNA control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + AREG , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + AREG , P < 0 . 0001 ; siCTRL + AREG vs . siYAP + AREG , P = 0 . 0037 ) .

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Figure 7Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1, MOB1, and YAP. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with AREG (50 ng/ml) for 0, 30, 60 min.AREG (50 ng/ml) treatment induced appearance of elongated cells in ME180 cells (top and middle panels) and significantly stimulated ME180 cell proliferation (lower panel). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.003). Scale bar: 100 μm.Wound-healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum-free medium. Scale bar: 200 μm.Real-time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression. Each bar represents mean ± SEM (n = 9). Bars with different letters are significantly different from each other (P < 0.0001).Real-time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA (siYAP) significantly suppressed AREG-induced AREG mRNA expression. siCTRL (non-target siRNA) was used as a siRNA control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+AREG, P < 0.0001; siYAP+CTRL vs. siYAP+AREG, P < 0.0001; siCTRL+AREG vs. siYAP+AREG, P = 0.0037).