C . Activated Sorting ( FACS ) experiments showed that control ALX1165L / 165L NCC ( green ) exhibited increased expression of mesenchymal markers CD90 , CD105 , CD73 with culture time ( passages 1 through 4 ) , whereas homozygous ALX1165F / 165F NCC ( blue ) showed a consistent expression of 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 same level of CD57 across passages . Data are presented as mean of positive - stained across passage numbers . Data obtained of each clone from three independent experiments were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA 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 p = 0 . 0207 at passage 4 . For CD105 , p = 0 . 0016 at passage 2 , p = 0 . 00004 at passage 3 p = 0 . 0021 at passage 4 . For CD73 , p = 0 . 0060 at passage 2 , p = 0 . 00004 at passage 3 p = 0 . 0114 at passage 4 . For CD57 , p = 0 . 0026 at passage 2 , p = 0 . 000003 at passage 3 p = 0 . 000007 at passage 4 .

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

C. 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.


A . Mutant ALX1165F / 165F NCC ( blue ) exhibited a migration defect in timed coverage of central clearing of wound when compared with control ALX1165L / 165L NCC ( black ) . Data is presented as recovery of central circular clear of wound by migrating NCC at end of a 24 - hour period . For pictures , were stained in serum free media containing 3 . 6µM CellTracker Green CMFDA ( Life Technologies ) for 30 min at 37 ° C allowed to recover for 30 min before starting experiment . were acquired every 6 hours using a Keyence BZ - X800 microscope . Surface analyses percentages of coverage were measured using ImageJ software ( NIH ) . NCC were monitored over 24 hours . data are represented as average of of closure ± . Scale bar = 200µm . Data obtained of each clone from three independent experiments were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC ( p < 0 . 0001 ) .

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

A. 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 of BMP2 BMP9 in supernatant of cultured NCC showed that ALX1165F / 165F NCC ( blue ) secrete less BMP2 more BMP9 compared to control ALX1165L / 165L NCC ( green ) . Data are represented as pooled mean ± of three clones from each genotype . Statistical significance was determined with an ANOVA . A p - value < 0 . 05 was considered significant . * : Significantly different from control NCC ( p = 0 . 0424 for BMP2 p = 0 . 0192 for BMP9 ) .

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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 culture medium could partially rescue migration defect of ALX1165F / 165F NCC . At beginning of , 10 , 50 , or 100 ng / ml of soluble BMP2 , CV2 , or a combination of two at 100 ng / ml each were added to culture medium , were monitored over next 24 hours . data are represented as average of of closure ± . Scale bar : 400µm .

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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.


A . Dissected flatmount wild type alx1 - / - zebrafish larvae craniofacial cartilages after Alcian blue , anterior points to left of page in . ventral cartilages appear normal , but alx1 - / - anterior neurocranium ( ANC ) appears narrow , with midline element that is derived from frontonasal NCC being absent . Meckel ' s cartilage ( arrow , MC ) is also diminutive . Scale bar : 200 µm .

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A. 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 are similar between wild type alx1 - / - lines . Data is represented as mean of pooled from three different clutches . RT - qPCR relative expression values were normalized to elfa 18S expression using ΔΔCT method . Data from each clutch were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . Statistical significance denoted by * ; p < 0 . 0001 between WT zebrafish alx1 - / - at measured time points ; at 10 ss , 24 hpf 36 hpf for alx3 ; at 24 hpf 36 hpf for alx4a . Refer to Table EV2 for p - values .

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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 injected with Alx1DN , after Alcian blue . developed an absence of frontonasal derived median portion of anterior neurocranium ( ANC ) a profound hypoplasia of Meckel ' s ventral cartilages . In most severely affected zebrafish , a nearly abrogated ANC was observed . Scale bar : 200 µm .

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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 Alx1DN mutant revealed aberrant migration of anterior cranial NCC when alx1 is disrupted . In control , anterior cranial NCC always migrate to contribute to median portion of ANC . In contrast , anterior cranial NCC labeled in Alx1DN fail to migrate to median ANC , where ANC structure is narrower labeled cranial NCC are found in an anterior lateral ectopic location ( asterisks ) . Scale bar : 250µm .

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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.


A Representative picture of carrying pASDOG1 : : LUC sprayed with mock solution , ABA or GA imaged 24 h later . full pictures are shown in Fig EV2 Appendix Fig S1 . Scale bar , 1 cm .

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A 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 of emitted per after treatment with ABA GA , compared with mock treatment .

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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 . full pictures are shown in Fig EV3 . Scale bar , 1 cm .

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C Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.


D of emitted per for control drought - treated .

D Quantification of emitted light intensity per plant for control and drought-treated plants.


E Strand - specific RT - qPCR asDOG1 expression of 3 - day Col - 0 ( WT ) drought - treated vs . control .

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E Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.


F asDOG1 expression level ( black ) is reduced while DOG1 sense expression ( grey ) is increased after application of ABA . Leaves of mature Col - 0 ( WT ) 40 - day - old were sprayed with ABA collected 0 , 6 10 h later for extraction RT - qPCR . Signals were normalized against level of UBC transcript . data points are averages for at least three biological replicates are normalized against mock - treated value . Error bars represent standard deviation . * * * * represent t - P - values of < 0 . 05 < 0 . 001 , respectively .

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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.


A Col - 0 ( WT ) dog1 - 3 mutant were either watered normally or subjected to water withdrawal for 5 days then watered again allowed to recover for 2 days . A picture of a representative experiment is shown . Scale bar , 2 cm .

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A 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 of recovered dead Col - 0 ( WT ) , null - 3 dog1 - 4 ; were scored as dead when unable to grow produced offspring after an extended recovery period .

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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 of DOG1 sense expression in drought - treated vs . control Col - 0 ( WT ) dog1 - 3 mutant .

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C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.


D dog1 - 3 mutant are defective in regulation of majority of tested drought marker genes . Col - 0 ( WT ) dog1 - 3 mutant were subjected to drought for 3 days expression of five marker genes was determined using RT - qPCR . Error bars represent standard deviation , * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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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.


A expressing LUC fused with full - DOG1 ( pDOG1LUC : : DOG1 ) were sprayed with mock solution or ABA 10 , 20 40 days after germination analysed 24 h after treatment . graphs show mean emitted per .

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A 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 - ABA - treated pDOG1LUC : : DOG1 . full picture is shown in Fig EV2C D . Scale bar , 1 cm .

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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 DOG1 antisense promoter region ( pDOG1shDOG1 : : LUC ) is not induced by ABA is highly expressed throughout development . graphs show mean emitted per .

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

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 - ABA - treated pDOG1shDOG1 : : LUC . Scale bar : 1cm .

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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 of antisense after 10 h of ABA treatment in 10 - , 20 - 40 - day - old Col - 0 .

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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 pASDOG1 : : LUC reporter is silenced by application of ABA , while mutation of TATA elements in asDOG1 promoter ( pASDOG1 ∆ TATA : : LUC ) leads to attenuation of expression in presence absence of ABA .

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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 asDOG1 promoter leads to high - level DOG1 sense expression non - responsiveness to ABA . Error bars represent standard deviation * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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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.


A expressing truncated asDOG1 promoter - deficient version pDOG1shDOG1 : : LUC are more resistant to drought than expressing full - genomic version pDOG1 : : LUC - DOG1 . Three independent lines are shown for each construct , following 10 days of water deprivation . Scale bar , 2 cm .

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A 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 of selected drought marker genes in mock - ABA - treated Col - 0 ( WT ) two independent pDOG1shDOG1 : : LUC transgenic lines . Error bars represent standard deviation * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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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.


a , LAMP1 + lysosome quantity in NRK ( dotted outline ) starved for hours ( h ) . Error bars show s . e . m . ( n = 3 ) .

a, 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 expressing LAMP1 - YFP show tubules ( arrows ) . Box is expanded on right .

b, Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded on the right.


c , Immuno - ( gold marks LAMP1 ) of starved NRK . Continuity ( arrow ) illustrated between autolysosome ( red star ) tubule .

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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 as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a from d .

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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 ( seconds ) of starved LAMP1 - YFP - expressing NRK .

f, Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells.


g , Time - lapse ( minutes ) of starved LAMP1 - PAGFP or LAMP1 - RFP - expressing NRK after photo - activation . Scale bars , 5 µm .

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g, Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.


a - c , Starved LAMP1 - YFP NRK stained with Lysotracker or DQ - BSA . c , Right , of purified lysosomes . d , Magnification ( 4 × ) of shown in c . Arrows indicate reformation tubules arrowhead indicates autolysosome .

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a-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 ) immuno - EM ( f ) using LAMP1 antibody of starved LAMP1 - YFPNRKcells shown in c .

e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c.


g , Density gradient fractions of NRK starved for times shown blotted for LAMP2 or Cathepsin D . Middle panel shows 8 - h fractions 7 9 analysed by . Fraction 10 is gradient top .

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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 were photo - activated ( 4 h ) imaged at 12 h with Lysotracker ( upper ) or DQ - BSA ( lower ) . Boxes show enlargements . R ( r ) = Pearson ' s coefficient . Scale bars , 5 µm .

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.


a , ( bottom ; scale bar , 5 µm ) ( top ; scale bar , 10 µm ) of NRK - LC3 starved for times shown with enlargements ( boxes ) .

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a, 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 shown in a with indicated antibodies ( d , after transfection with non - specific ( NS ) or atg5 RNAi ) . c , Autophagy ( black lines show independent experiments ) phospho - S6K ( densitometry of data shown in b , red ) .

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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 starved for 2 h 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 ( g ) . Error bars , s . e . m . ; n = 3 .

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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.


a , LAMP1 / LC3NRK were starved for 4 h treated with GTPγS for 6 h of further starvation .

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a, 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 starved for 10 h .

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b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h.


c , Rab7 - CFP NRK starved for 2 h treated with 100 nM rapamycin for 8 h of further starvation .

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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 starved for times shown with 1 µg ml - 1 leupeptin blotted with indicated antibodies . e , in d imaged . .

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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 from with Scheie syndrome ( GM01256 ) or healthy controls starved blotted with indicated antibodies . g , in f imaged . Lysosome classified by Image - ProPlus ( IPP ) software .

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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.


A Representative show expression of YAP in normal cervical tissues , early - stage cervical cancer ( stage I II ) , advanced - stage cervical cancer ( stage III IV ) . Note significant increase in YAP immunosignals ( brown ) in advanced - stage tumors . Tissue : 1 . 5 mm . B Positivity ( of positively stained relative to total number of in tissue section ) of YAP immunosignals in normal cancerous tissues . Data were analyzed with unpaired t - in GraphPad Prism 5 with Welch ' s correction . Bars represent means ± ( n = 10 for normal tissues ; n = 69 for tissues ; * * * P 0 . 0001 ) .

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A 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 expression of YAP ( brown ) in normal cervical tissue . Scale bar = 20 μm . D - F Representative showing expression of YAP in ( D ) early - stage cervical cancer tissue ( stage Ib , T1N0M0 ) ; ( E ) medium - stage cervical cancer tissue ( stage IIb , T2N0M0 ) ; ( F ) advanced - stage cervical cancer tissue ( stage IIIb , T3N1M0 ) . Scale bar = 20 μm . G , H High - resolution showing location of YAP in ( G ) early - stage cervical cancer tissue ( H ) advanced - stage cervical cancer tissue . Scale bar = 20 μm .

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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 results showing cross - cancer YAP gene alteration . histogram showed frequencies of YAP gene mutation , deletion , amplification across cancers . Data were extracted from TCGA database analyzed using cBioPortal online analyzing tools . results indicate that highest alteration frequency of YAP gene occurs in cervical cancer .

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.


A , C , E Western blot showing of YAP phosphorylated YAP in ME180 lines [ ME180 - MXIV ( control ) , ME180 - YAP , ME180 - YAPS127A ] ( A ) ; HT3 lines [ HT3 - MXIV ( control ) , HT3 - YAP , HT3 - YAPS127A ] ( C ) ; Ect1 lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , Ect1 - YAPS127A ] ( E ) . β - Actin was used as a loading control .

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A, 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 lines [ ME180 - MXIV ( control ) , ME180 - YAP , ME180 - YAPS127A ] ( B ) ; HT3 lines [ HT3 - MXIV ( control ) , HT3 - YAP , HT3 - YAPS127A ] ( D ) ; Ect1 lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , Ect1 - YAPS127A ] ( F ) . Each point represents mean ± ( n = 4 ) . * * * P < 0 . 0001 , ME180 - MXIV vs . ME180 - YAP ME180 - MXIV vs . ME180 - YAPS127A on day 8 . * * * P < 0 . 0001 , HT3 - MXIV vs . HT3 - YAP HT3 - MXIV vs . HT3 - YAPS127A on day 8 . * * * P < 0 . 0001 , Ect1 - MXIV vs . Ect1 - Ect1 - YAPS127A on day 4 . * P < 0 . 0074 , Ect1 - MXIV vs . Ect1 - YAP on day 4 .

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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 in non - targeting control siRNA ( siCtrl ) - YAP siRNA ( siYAP ) - transfected HT3 .

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G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.


H Proliferation of HT3 treated with control ( siCtrl ) or YAP siRNA ( siYAP ) . Each point represents mean ± ( n = 5 ) . * * * P < 0 . 001 compared with siCtrl ( siCtrl vs . siYAP , P = 0 . 0002 ) .

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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).


A Representative showing colonies formed by Ect1 - MXIV , Ect1 - YAP , Ect1 - YAPS127A after growth in soft agar for 9 days . Scale bar : 500 μm . B Quantitative data showing colony formation in Ect1 - MXIV , Ect1 - YAP , Ect1 - YAPS127A . Each bar represents mean ± 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 ) .

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A 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 showing anchorage - independent growth of YAP - expressing HT3 ( C ) ME180 ( E ) cervical cancer lines . Anchorage - independent growth was determined by soft agar colony formation . Scale bar : 500 μm . D , F - quantitative showing differences of anchorage - independent growth in HT3 - MXIV , HT3 - YAP , HT3 - YAPS127A ( D ) ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A ( F ) . anchorage - independent growth was determined using a CytoSelect 96 - Well Transformation kit , data are presented as relative units ( RFU ) . Each bar represents mean ± 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 ) .

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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).


A , B Representative of tumorxenografts of ME180 - MXIV ( MX , right flank ) , ME180 - YAP ( A ) ( YAP , left flank ) , ME180 - YAPS127A ( B ) ( YAPS127A , left flank ) implanted in athymic nude mice . Red lines indicate edge of tumors . C growth curve of humantumorxenografts derived from control ( ME - MX ) YAP - overexpressing ME180cervical cancer lines ( ME - YAP , ME - YAPS127A ) implanted in athymic nude mice . Each point represents mean ± of six tumors ( control n = 10 ) .

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A, 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 showing relative of tumors from control YAP - overexpressing ME180 cervical cancer . E average of xenografts from control YAP - overexpressing ME180 lines . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc . Each bar represents mean ± ( n = 10 for control , n = 6 for YAP YAPS127A groups ) . Bars with different letters are significantly different from each other ( MXIV vs . YAP , P = 0 . 0008 ; MXIV vs . YAPS127A , P < 0 . 0001 ) .

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 of YAP in xenografts derived from ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A .

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 xenografts of ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A 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 significant increase in Ki67 - positive in ME180 - YAP ME180 - YAPS127A xenografts .

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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 showing YAP in ME180 transfected with lentiviral empty vector ( shCtrl ) or lentivirus - YAP shRNAs ( shYAP # 1 or shYAP # 2 ) .

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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 showing xenografts derived from ME180 - shCtrl ( left flank ) ME180 - shYAP # 1 ( right flank ) ( n = 6 ) .

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I Representative images showing tumor xenografts derived from ME180-shCtrl cells (left flank) and ME180-shYAP#1 cells (right flank) (n = 6).


J Representative showing relative of tumors derived from ME180 cervical cancer transfected with control shRNA ( left , shCtrl ) or YAP shRNA # 1 ( right , shYAP # 1 ) ( n = 6 ) .

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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).


RT - PCR results showing that YAP stimulates mRNA expressions of EGFR , AREG , TGF - α in ME180 cervical cancer . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc . Each bar represents mean ± ( 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 ) .

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RT-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 incubated in medium containing 1 % FBS in absence ( control ) or presence of 10 ng / ml TGF - α . Data were analyzed with unpaired t - in GraphPad Prism 5 with Welch ' s correction . Each point represents mean ± of four independent repeats . * * * P < 0 . 0001 versus control on day 5 .

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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 cycle progression . G1 , S , G2 indicate in G1 phase , synthesis phase , G2 / M phase , respectively , of cycle . Apo : apoptotic .

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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 showing of ME180 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 elongation of ME180 after TGF - α treatment for 24 h ( TGF - α , 24 h ) formation of multiple layers in ME180 after TGF - α treatment for 108 h ( TGF - α , 108 h ) .

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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 migration of ME180 . TGF - α treatment ( 100 ng / ml , 10 h ) drastically stimulated migration of ME180 .

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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.


Western blot showing effects of TGF - α on phosphorylation of EGFR , YAP , AKT , ERK1 / 2 . ME180 were starved for 6 h after reaching confluence , then were treated with TGF - α ( 100 ng / ml ) for 0 , 5 , 10 , 30 , 60 or 120 min .

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Western 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 showing effects of TGF - α on phosphorylation of components of Hippo signaling pathway . ME180 were starved for 6 h after reaching confluence , then were treated with TGF - α ( 100 ng / ml ) for 0 , 30 , 60 min .

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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 action of TGF - α YAP on mRNA expression of AREG . Each bar represents mean ± ( 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 ) .

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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 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 . siCTRL , a non - targeting siRNA , was used as a negative control . Each bar represents mean ± ( 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 ) .

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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 ( 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 mean ± ( 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 ) .

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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 wound - healing showing migration of ME180 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 mean ± ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


Western blot showing that AREG treatment induced dephosphorylation of LATS1 , MOB1 , YAP . ME180 were starved for 6 h after reaching confluence , then were treated with AREG ( 50 ng / ml ) for 0 , 30 , 60 min .

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

Western 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 in ME180 ( top middle panels ) significantly stimulated ME180 proliferation ( lower panel ) . Each bar represents mean ± ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 003 ) . Scale bar : 100 μm .

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

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 showing that AREG stimulates migration of ME180 cervical cancer within 10 h in serum - free medium . Scale bar : 200 μm .

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

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 with AREG for 24 h significantly increased AREG mRNA expression . Each bar represents mean ± ( n = 9 ) . Bars with different letters are significantly different from each other ( P < 0 . 0001 ) .

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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 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 ± ( 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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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).


Representative showing of spheroids derived from ME180 - siCtrl ME180 - siLATS1 / 2 growing in a 3D hanging - drop culture system for 10 days . Scale bar : 1 . 0 mm .

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Representative images showing the morphology of spheroids derived from ME180-siCtrl and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system for 10 days. Scale bar: 1.0 mm.


Quantitative data showing changes in volume of spheroids derived from ME180 - siCTRL ME180 - siLATS1 / 2 growing in a 3D hanging - drop culture system . Each bar represents mean ± ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0004 ) .

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Quantitative data showing changes in the volume of spheroids derived from ME180-siCTRL and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0004).


Quantitative data showing changes in volume of spheroids derived from ME180 growing in a 3D hanging - drop culture system in absence or presence of AREG ( 20 ng / ml , 8 days ) . Each bar represents mean ± ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0030 ) .

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Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging-drop culture system in the absence or presence of AREG (20 ng/ml, 8 days). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0030).


Soft agar showing effect of AG1478 VTPF on colony formation in ME180 - MXIV , ME180 - YAP ME180 - YAPS127A . Scale bar : 500 μm .

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Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180-MXIV, ME180-YAP and ME180-YAPS127A cells. Scale bar: 500 μm.


Representative showing effect of AREG , EGFR inhibitor ( AG1478 ) , YAP antagonist verteporfin ( VTPF ) on growth of ME180 in a 3D hanging - drop culture system . ME180 were incubated in 3D hanging - drop culture system for 10 days in absence or presence of AREG , AG1478 or verteporfin for 8 days . Scale bar : 1 . 0 mm .

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

Representative images showing the effect of AREG, EGFR inhibitor (AG1478), and YAP antagonist verteporfin (VTPF) on the growth of ME180 cell in a 3D hanging-drop culture system. ME180 cells were incubated in the 3D hanging-drop culture system for 10 days in the absence or presence of AREG, AG1478 or verteporfin for 8 days. Scale bar: 1.0 mm.


Effect of recombinant HPV16 E6 on proliferation of HT3 . Each point represents mean ± ( n = 5 ) . * * * P = 0 . 0001 compared with control ( Ctrl ) on day 4 . HT3 were cultured in serum - reduced medium in presence or absence of recombinant HPV16 E6 ( 400 nM ) .

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Effect of recombinant HPV16 E6 protein on the proliferation of HT3 cells. Each point represents mean ± SEM (n = 5). ***P = 0.0001 compared with control (Ctrl) on day 4. HT3 cells were cultured in serum-reduced medium in the presence or absence of recombinant HPV16 E6 (400 nM).


Recombinant HPV16 E6 increased YAP level , but had no effect on β - actin level in HT3 . culture treatment procedure are same as described in ( A ) .

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

Recombinant HPV16 E6 protein increased YAP protein level, but had no effect on β-actin protein level in HT3 cells. Cell culture and treatment procedure are the same as described in (A).


AREG mRNA in HT3 incubated for 48 h with or without recombinant HPV E6 . Each bar represents mean ± ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0042 ) .

AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0042).


Western blotting showing effect of HPV16E6 ( 400 nM , 48 h ) on YAP in HT3 transfected with non - targeting control siRNA ( siCtrl ) or YAP siRNA ( siYAP ) .

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Western blotting analysis showing the effect of HPV16E6 (400 nM, 48 h) on YAP protein levels in HT3 cells transfected with non-targeting control siRNA (siCtrl) or YAP siRNA (siYAP).


Effect of YAP on HPV16 E6 stimulation of HT3 proliferation . siCtrl : non - targeting control siRNA ; siYAP : YAP siRNA ; E6 : 400 nM recombinant HPV16 E6 , 48 h . Each bar represents mean ± ( n = 4 ) . Bars with same letters are not significantly different from each other ( siCtrl vs . siCtrl + E6 , P = 0 . 0232 ; siCtrl + E6 vs . siYAP + E6 , P = 0 . 0011 ) .

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Effect of YAP on HPV16 E6 stimulation of HT3 cell proliferation. siCtrl: non-targeting control siRNA; siYAP: YAP siRNA; E6: 400 nM recombinant HPV16 E6, 48 h. Each bar represents mean ± SEM (n = 4). Bars with the same letters are not significantly different from each other (siCtrl vs. siCtrl+E6, P = 0.0232; siCtrl+E6 vs. siYAP+E6, P = 0.0011).


Knockdown of endogenous E6 in HeLa with HPV18 E6 siRNA ( siE6 ) reduced YAP in HeLa , while knockdown of YAP with YAP siRNA ( siYAP ) in these had no effect on mRNA level of HPV18 E6 . siCtrl : non - targeting control siRNA .

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

Knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein in HeLa cells, while knockdown of YAP with YAP siRNA (siYAP) in these cells had no effect on the mRNA level of HPV18 E6. siCtrl: non-targeting control siRNA.


Knockdown of endogenous HPV18 E6 in HeLa with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed mRNA expression of AREG . Each bar represents mean ± ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siHPV18E6 , P = 0 . 0181 ; siCtrl vs . siYAP , P = 0 . 0005 ) .

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

Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed mRNA expression of AREG. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siHPV18E6, P = 0.0181; siCtrl vs. siYAP, P = 0.0005).


Knockdown of endogenous HPV18 E6 in HeLa with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed growth ( n = 9 , siCtrl vs . siHPV18E6 , P = 0 . 0002 ; siCtrl vs . siYAP , P = 0 . 0004 ) .

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

Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed cell growth (n = 9, siCtrl vs. siHPV18E6, P = 0.0002; siCtrl vs. siYAP, P = 0.0004).


Concentrations of AREG in culture medium of HeLa transfected with non - targeting control siRNA ( siCTRL ) or HPV18 E6 siRNA ( siHPV18E6 ) . Each bar represents mean ± ( n = 6 ) . Bars with different letters are significantly different from each other ( P = 0 . 0461 ) .

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

Concentrations of AREG in the culture medium of HeLa cells transfected with non-targeting control siRNA (siCTRL) or HPV18 E6 siRNA (siHPV18E6). Each bar represents mean ± SEM (n = 6). Bars with different letters are significantly different from each other (P = 0.0461).


Effect of YAP mRNA in HT3 treated with or without E6 ( 400 nM ) for 60 min in McCoy ' s 5A with 1 % serum . Data were normalized with 18S mRNA . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) . n = 4 , P = 0 . 9407 .

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

Effect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy's 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.


YAP mRNA in HT3 treated with or without E6 ( 400 nM ) for 48 h in McCoy ' s 5A with 1 % serum . mRNA were measured with real - time PCR . Data were normalized with 18S mRNA . Each bar represents mean ± ( n = 5 ) . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) ( P = 0 . 3233 ) .

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

YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy's 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).


Western blot results showing YAP EGFR after treatment with MG132 or cycloheximide ( CHX ) for 4 h or 8 h . β - actin was used as a loading control . Confluent HT3 were starved for 4 h before treatment with MG132 ( 10 μM ) or CHX ( 20 μg / ml ) for 0 , 4 or 8 h .

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

Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.


HPV16E6 prevented YAP from degradation . Confluent HT3 were starved for 4 h before treatment with or without CHX ( 20 μg / ml ) , HPV16E6 , or CHX combined with HPV16E6 for 8 h . Treatment of starved HT3 with HPV16E6 ( 400 nM ) for 8 h suppressed degradation of YAP , but not EGFR . Quantitative data showing relative YAP in ( D ) . were normalized with β - actin presented as ratios relative to that of control . Each bar represents mean ± ( n = 4 ) . Bars with different letters are significantly different from each other ( Ctrl vs . CHX , P = 0 . 0003 ; Ctrl vs . CHX + E6 , P = 0 . 2665 ; Ctrl vs . E6 , P = 0 . 0258 ) .

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

HPV16E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16E6, or CHX combined with HPV16E6 for 8 h. Treatment of starved HT3 cells with HPV16E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).


Western blot showing that expression of HPV16E6 in HT3 increased level of total YAP casein kinase Iε , but decreased level of SOCS6 . Importantly , HPV16E6 increased YAP phosphorylation at serine 127 , but suppressed its phosphorylation at serine 397 . HPV16E6 had no effect on level of β - TrcP , LATS1 / 2 MOB1 in HT3cervical cancer .

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

Western blot analysis showing that expression of HPV16E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3cervical cancer cells.


Western blot results showing that knockdown of endogenous E6 in HeLa with HPV18E6 siRNA ( siE6 ) reduced YAP , but increased SOCS6 .

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

Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.


Western blot showing that knockdown of SOCS6 in HT3 with SOCS6 siRNA ( siSOCS # 1 siSOCS6 # 2 ) increased total YAP , enhanced phosphorylation of YAP at serine 127 , but suppressed phosphorylation of YAP at serine 397 .

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

Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.


A - F Representative showing expression of YAP ( in red ) in wild - type mousecervical tissues ( n = 5 ) ( A ) , HPV16E6 - induced ( C ) HPV16E6 / E7 - induced ( E ) mousecervicaltumor tissue ( n = 4 each ) . Scale bars for ( A ) , ( C ) , ( E ) : 100 μm . High - resolution showing expression location of YAP in normal cervical tissues ( B ) , HPV16E6 - induced ( D ) HPV16E6 / E7 - induced ( F ) mousecervicaltumor tissue . Scale bars for ( B ) , ( D ) , ( F ) : 25 μm .

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A-F Representative images showing expression of YAP (in red) in wild-type mousecervical tissues (n = 5) (A), HPV16E6-induced (C) and HPV16E6/E7-induced (E) mousecervicaltumor tissue (n = 4 each). Scale bars for (A), (C), and (E): 100 μm. High-resolution images showing the expression and cellular location of YAP in normal cervical tissues (B), HPV16E6-induced (D) and HPV16E6/E7-induced (F) mousecervicaltumor tissue. Scale bars for (B), (D), and (F): 25 μm.


G , H Representative showing expression of YAP ( in red ) in normal ( G ) HPV16 - containing humanforeskinkeratinocyte raft cultures ( n = 5 each ) ( H ) . Scale bars : 100 μm . I , J High - resolution showing expression location of YAP in normal ( I ) HPV16 - containing humanforeskinkeratinocytes raft cultures ( J ) . Scale bars : 20 μm .

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G, H Representative images showing expression of YAP (in red) in normal (G) and HPV16-containing humanforeskinkeratinocyte raft cultures (n = 5 each) (H). Scale bars: 100 μm.I, J High-resolution images showing the expression and cellular location of YAP in normal (I) and HPV16-containing humanforeskinkeratinocytes raft cultures (J). Scale bars: 20 μm.


A Skeletal in lower legs were dissected was extracted on postnatal day 0 , 7 , 21 . expression of R3hdml was examined by RT - PCR . GAPDH was used as an internal control . MM , 100 bp marker . B of RT - PCR results . Data are expressed as means ± standard error of mean ( ) . experiments were repeated at least three times . * * * * p < 0 . 0001 vs . day 0 group ; one - way ANOVA followed by Bonferroni post - hoc .

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A Skeletal muscles in the lower legs were dissected and RNA was extracted on postnatal day 0, 7, and 21. The expression of R3hdml was examined by RT-PCR. GAPDH was used as an internal control. MM, 100 bp DNA marker. B Quantification of RT-PCR results. Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. **** p < 0.0001 vs. day 0 group; one-way ANOVA followed by Bonferroni post-hoc test.


Satellite were isolated from skeletal of adult mice by enzymatic digestion placed in culture medium . Then , differentiation was induced in satellite ( C ) .

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Satellite cells were isolated from skeletal muscles of adult mice by enzymatic digestion and placed in culture medium. Then, differentiation was induced in satellite cells (C).


were extracted at indicated time points . R3hdml expression was examined by western blotting using an anti - R3hdml antibody ( D ) . β - actin was used as internal control . of western blotting results ( E ) . Data are expressed as means ± standard error of mean ( ) . experiments were repeated at least three times . Representative data are shown . * p < 0 . 05 ; Student ' s t - . Scale bar = 500 µm .

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

The proteins were extracted at the indicated time points. R3hdml expression was examined by western blotting using an anti-R3hdml antibody (D). β-actin was used as internal control. Quantification of western blotting results (E). Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. Representative data are shown. * p < 0.05; Student's t-test. Scale bar = 500 µm.


A Cultured C2C12 were transfected with firefly luciferase construct containing 781 bp fragment upstream of translational start codon of R3hdml coding ( R3hdml - luc ) , together with Renilla luciferase vectors as controls ; were then differentiated in presence of 2 % horse serum . were harvested at indicated time points , after which luciferase activity of lysate was measured .

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A Cultured C2C12 cells were transfected with the firefly luciferase construct containing the 781 bp fragment upstream of the translational start codon of the R3hdml coding sequence (R3hdml-luc), together with Renilla luciferase vectors as controls; the cells were then differentiated in the presence of 2% horse serum. Cells were harvested at the indicated time points, after which luciferase activity of the cell lysate was measured.


C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 . One day 3 days after transfection , were lysed subjected to SDS - PAGE western blotting using anti - MyoD antibodies .

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

C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 cells. One day and 3 days after transfection, cells were lysed and subjected to SDS-PAGE and western blotting using anti-MyoD antibodies.


D Plasmids containing MyoD cDNA or empty vector together with R3hdml - luc Renilla luciferase vectors were transfected into C2C12 . One day 3 days after transfection , luciferase activity of lysate with or without MyoD overexpression was measured . Relative luciferase activity is expressed as fold activity above background conferred by empty vector together with R3hdml - luc .

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

D Plasmids containing MyoD cDNA or empty vector together with R3hdml-luc and Renilla luciferase vectors were transfected into C2C12 cells. One day and 3 days after transfection, the luciferase activity of the cell lysate with or without MyoD overexpression was measured. Relative luciferase activity is expressed as the fold activity above the background conferred by the empty vector together with R3hdml-luc.


A R3hdml siRNA or non - targeting siRNA was transfected into C2C12 . Then , C2C12 were differentiated into myotubes . was extracted from after induction of differentiation at indicated time points . Closed bars , control ; open bars , silenced R3hdml gene ; * * p < 0 . 01 ; * p < 0 . 05 ; Student ' s t - .

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

A R3hdml siRNA or non-targeting siRNA was transfected into C2C12 cells. Then, C2C12 cells were differentiated into myotubes. RNA was extracted from the cells after induction of differentiation at the indicated time points. Closed bars, control; open bars, silenced R3hdml gene; ** p < 0.01; * p < 0.05; Student's t-test.


B Immunofluorescence after C2C12 differentiation . MyHC is shown in green nuclei in blue ( counterstained with Hoechst ) . Representative of MyHC immunofluorescence of C2C12 myocytes transfected with siRNA or after addition of R3hdml . Scale bar = 100 µm .

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

B Immunofluorescence after C2C12 differentiation. MyHC is shown in green and the nuclei in blue (counterstained with Hoechst). Representative images of MyHC immunofluorescence of C2C12 myocytes transfected with the siRNA or after addition of R3hdml protein. Scale bar = 100 µm.


C number of nuclei per MyHC + were calculated on day 5 after differentiation . * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - hoc .

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

C The number of nuclei per MyHC+ cell were calculated on day 5 after differentiation. ** p < 0.01; one-way ANOVA followed by Bonferroni post-hoc test.


D distribution of nuclei per myotube were calculated on day 5 after differentiation . * * p < 0 . 01 ; * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - hoc .

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

D The distribution of nuclei per myotube were calculated on day 5 after differentiation. ** p < 0.01; * p < 0.05; one-way ANOVA followed by Bonferroni post-hoc test.


A Satellite were isolated by enzymatic digestion from R3hdml KO ( n = 6 ) wild type control ( n = 6 ) mice placed in culture medium . Then , proliferation was evaluated by Ki67 . nuclei were stained with DAPI . B of Ki67 positive to total ( DAPI - positive ) was evaluated . number of was counted under approximately 60 fields of view for each well . Closed columns , wild type controls ; open columns , R3hdml KO mice . * p < 0 . 05 ; Student ' s t - .

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

A Satellite cells were isolated by enzymatic digestion from R3hdml KO (n = 6) and wild type control (n = 6) mice and placed in culture medium. Then, cell proliferation was evaluated by Ki67 staining. Cell nuclei were stained with DAPI. B The ratio of Ki67 positive cells to total cells (DAPI-positive) was evaluated. The number of cells was counted under approximately 60 fields of view for each well. Closed columns, wild type controls; open columns, R3hdml KO mice. * p < 0.05; Student's t-test.


Thirty freshly isolated myofibers from 8 - 12 - week - old R3hdml KO ( n = 7 ) wild type mice ( n = 7 ) were cultured for 72 h . Then , isolated myofibers were fixed with 4 % PFA followed by immunostaining using anti Pax - 7 , anti MyoD , anti Myog antibodies . ( C , D ) Co - immunostaining parallel cultures for Pax7 MyoD showed that there were fewer with self - renewing Pax7 + / MyoD − phenotype in from R3hdml KO mice compared with control ; Pax7 + / MyoD − ( arrowheads ) , Pax7 − / MyoD + ( yellow arrows ) Pax7 + / MyoD + ( arrows ) . * p < 0 . 05 ; Student ' s t - .

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

Thirty freshly isolated myofibers from 8-12-week-old R3hdml KO (n = 7) and wild type mice (n = 7) were cultured for 72 h. Then, the isolated myofibers were fixed with 4% PFA followed by immunostaining using anti Pax-7, anti MyoD, and anti Myog antibodies. (C, D) Co-immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self-renewing Pax7+/MyoD− phenotype in muscles from R3hdml KO mice compared with control; Pax7+/MyoD− cells (arrowheads), Pax7−/MyoD+ cells (yellow arrows) and Pax7+/MyoD+ cells (white arrows). * p < 0.05; Student's t-test.


( E , F ) Co - immunostaining parallel cultures for MyoD Myog showed that there were fewer with differentiating MyoD − / Myog + phenotype in from R3hdml KO mice compared to control ; MyoD − / Myog + ( arrowheads ) , MyoD - / Myog + ( yellow arrows ) , MyoD + / Myog + ( arrows ) . * p < 0 . 05 ; Student ' s t - .

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

(E, F) Co-immunostaining parallel cultures for MyoD and Myog showed that there were fewer cells with the differentiating MyoD−/Myog+ phenotype in muscles from R3hdml KO mice compared to control; MyoD−/Myog+ cells (arrowheads), MyoD-/Myog+ cells (yellow arrows), and MyoD+/Myog+ cells (white arrows). * p < 0.05; Student's t-test.


A Seven days after CTX injection , skeletal were dissected , fixed , embedded in paraffin . Two consecutive sections were utilized , first section was subjected to in situ hybridization using an R3hdml - specific probe to detect Rh3dml , while second section was used for using anti - MyoD antibody . MyoD was visualized with DAB stain ( brown ) . R3hdml expression is highly detected in MyoD - positive . Scale bar in low magnification = 100 µm ; in high magnification = 25µm .

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

A Seven days after CTX injection, skeletal muscles were dissected, fixed, and embedded in paraffin. Two consecutive sections were utilized, and the first section was subjected to in situ hybridization using an R3hdml sequence-specific RNA probe to detect Rh3dml, while the second section was used for IHC using the anti-MyoD antibody. MyoD protein was visualized with DAB stain (brown). R3hdml expression is highly detected in MyoD-positive cells. Scale bar in low magnification images = 100 µm; in high magnification images = 25µm.


B One hundred microliters of 10 µM CTX was intramuscularly injected into forearm muscle of anesthetized R3hdml KO ( [ b ] , n = 10 ) wild type control ( [ a ] , n = 10 ) mice . Subsequently , grip was evaluated 0 , 3 , 6 , 14 days after CTX injection . R3hdml genes were overexpressed in forearm muscle of anesthetized R3hdml KO mice ( [ c ] , n = 10 ) grip was also evaluated 0 , 3 , 6 , 14 days after CTX injection . Data are expressed as means ± standard error of mean ( ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 vs . wild type control ; # p < 0 . 05 ; # # p < 0 . 01 vs . R3hdml KO mice ; two - way ANOVA followed by a Bonferroni post hoc comparison of time points .

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

B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ([b], n = 10) and wild type control ([a], n = 10) mice. Subsequently, hand grip was evaluated 0, 3, 6, and 14 days after CTX injection. R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ([c], n = 10) and hand grip was also evaluated 0, 3, 6, and 14 days after CTX injection. Data are expressed as the means ± standard error of the mean (SEM). ** p < 0.01; **** p < 0.0001 vs. wild type control; # p < 0.05; ## p < 0.01 vs. R3hdml KO mice; two-way ANOVA followed by a Bonferroni post hoc comparison of the individual time points.


C Myofibers were freshly dissected from 8 - 12 - week - old R3hdml KO ( n = 4 ) control mice ( n = 4 ) 35 days after CTX injection , number of satellite was evaluated by Pax - 7 . Arrowheads indicate Pax - 7 - positive . number of Pax - 7 - positive per myofiber was counted . At least 50 myofibers in each were evaluated . Closed columns , wild type controls ; open columns , R3hdml KO mice . Data are expressed as means ± standard error of mean ( ) . * p < 0 . 05 ; Student ' s t - .

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

C Myofibers were freshly dissected from 8-12-week-old R3hdml KO (n = 4) and control mice (n = 4) 35 days after CTX injection, and the number of satellite cells was evaluated by Pax-7 staining. Arrowheads indicate Pax-7-positive cells. The number of Pax-7-positive cells per myofiber was counted. At least 50 myofibers in each animal were evaluated. Closed columns, wild type controls; open columns, R3hdml KO mice. Data are expressed as the means ± standard error of the mean (SEM). * p < 0.05; Student's t-test.


( C - G ' ' ) Lateral of cranial vessels in lyve1b : Kaede transgenic with CCV photoconverted at 36 hpf ( C ) followed through to 48 hpf ( D ) 60 hpf ( E ) , while in a separate larva PHS has been photoconverted at 36 hpf ( F ) followed to 48 hpf ( G ) . Unconverted vessels are shown in green ( C - G ) , while photoconverted vessels are shown in red ( C ' - G ' ) . extent of lymphangioblast contribution from each source is further clarified by false colouring overlap between red green ( purple ) , with FLS demarcated ( dotted ) from adjacent veins ( C ' ' - G ' ' ) .

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

(C-G'') Lateral images of the cranial vessels in lyve1b:Kaede transgenic embryos with the CCV photoconverted at 36 hpf (C) and followed through to 48 hpf (D) and 60 hpf (E), while in a separate larva the PHS has been photoconverted at 36 hpf (F) and followed to 48 hpf (G). Unconverted vessels are shown in green (C-G), while photoconverted vessels are shown in red (C'-G'). The extent of lymphangioblast contribution from each source is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS demarcated (dotted line) from the adjacent primary veins (C''-G'').


( A - D ' ' ) Ventrolateral ( A , C , D ) lateral ( B ) of cranial vessels in lyve1b : Kaede transgenic with dorsal ( A ) ventral ( C ) portions of VA - L photoconverted at 54 hpf followed through to 72 hpf ( B ) 78 hpf ( D ) . Unconverted vessels are shown in green ( A - D ) , while photoconverted vessels are shown in red ( A ' - D ' ) . extent of lymphatic arterial contribution from VA - L to LFL ( B ' ' ) or HA ( D ' ' ) is further clarified by false colouring overlap between red green ( purple ) , with FLS ( A ' ' , C ' ' ) LFL ( B ' ' , D ' ' ) demarcated ( dotted ) from adjacent vessels . Schematic included for anatomical reference . Note , for , overlap of red green in eye ( labelled E ) is due to a combination of natural pigmentation auto - ; is not indicative of photoconverted ( C ' ' , arrowheads ) .

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

(A-D'') Ventrolateral (A, C, D) and lateral (B) images of the cranial vessels in lyve1b:Kaede transgenic embryos with the dorsal (A) and ventral (C) portions of the VA-L photoconverted at 54 hpf and followed through to 72 hpf (B) and 78 hpf (D). Unconverted vessels are shown in green (A-D), while photoconverted vessels are shown in red (A'-D'). The extent of lymphatic and arterial contribution from the VA-L to the LFL (B'') or HA (D'') is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS (A'', C'') and LFL (B'', D'') demarcated (dotted line) from the adjacent vessels. Schematic included for anatomical reference. Note, for all images, the overlap of red and green fluorescence in the eye (labelled E) is due to a combination of natural pigmentation and auto-fluorescence; and is not indicative of photoconverted cells (C'', white arrowheads).


Lateral of CCV fixed at 36 hpf ( A ) 42 hpf ( B ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) .

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

Lateral images of the CCV fixed at 36 hpf (A) and 42 hpf (B) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red).


Lateral of PHS at 36 hpf ( C ) , 42 hpf ( D ) 48 hpf ( E ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) . PHS Prox1 shown alone for time points ( C ' - E ' ) with FLS demarcated ( dotted ) from adjacent vessels , positions of PHS - LPP PHS - LPA indicated .

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

Lateral images of the PHS at 36 hpf (C), 42 hpf (D) and 48 hpf (E) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). PHS Prox1 staining shown alone for all time points (C'-E') with the FLS demarcated (dotted line) from the adjacent vessels, and the positions of the PHS-LPP and PHS-LPA indicated.


Lateral of VA - L at 36 hpf ( F ) , 42 hpf ( G ) 48 hpf ( H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) . Note Prox1 - postive ( green ) , GFP - negative structure near to VA - L is a prox1 - expressing neuromast ( NM ) .

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

Lateral images of the VA-L at 36 hpf (F), 42 hpf (G) and 48 hpf (H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). Note the Prox1-postive (green), GFP-negative structure near to the VA-L is a prox1-expressing neuromast (NM).


( A - H ) Lateral of PHS fixed at 36 hpf ( A - B , E - F ) 42 hpf ( C - D , G - H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) . These show a decrease in number of Prox1 - positive PHS ECs in ccbe1 morpholino - injected ( B , D ) sFLT4 mRNA - injected ( F , H ) compared with control morpholino - injected ( A , C ) uninjected ( E , G ) . At 42 hpf , ccbe1 morpholino - injected ( D ) sFLT4 mRNA - injected ( H ) lack PHS - LP domains present in control ( C , G ; large arrow heads ) . ( I - J ) Quantitation of of PHS ECs that are lymphatic progenitors relative to total number of PHS ECs in control morpholino - injected at 36 hpf ( n = 27 ) 42 hpf ( n = 17 ) ccbe1 morpholino - injected at 36 hpf ( n = 36 ) 42 hpf ( n = 12 ) ( I ) or in uninjected at 36 hpf ( n = 27 ) 42 hpf ( n = 21 ) sFLT4 mRNA - injected at 36 hpf ( n = 26 ) 42 hpf ( n = 17 ) ( J ) . ( K ) Quantitation of total number of PHS ECs in control morpholino - injected at 36 hpf ( n = 27 ) 42 hpf ( n = 17 ) ccbe1 morpholino - injected at 36 hpf ( n = 36 ) 42 hpf ( n = 12 ) or in uninjected at 36 hpf ( n = 27 ) 42 hpf ( n = 21 ) sFLT4 mRNA - injected at 36 hpf ( n = 26 ) 42 hpf ( n = 17 )

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

(A-H) Lateral images of the PHS fixed at 36 hpf (A-B, E-F) and 42 hpf (C-D, G-H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show a decrease in the number of Prox1-positive PHS ECs in the ccbe1 morpholino-injected (B, D) and sFLT4 mRNA-injected embryos (F, H) compared with control morpholino-injected (A, C) and uninjected embryos (E, G). At 42 hpf, ccbe1 morpholino-injected embryos (D) and sFLT4 mRNA-injected embryos (H) lack the PHS-LP domains present in the control embryos (C, G; large arrow heads). (I-J) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) (I) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (J). (K) Quantitation of the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17)


( L - O ) Lateral of FLS ( L , N ; dotted ) or dorsolateral CCV region from where FLS normally sprouts ( M , O ; dotted ) fixed at 36 hpf in lyve1b : EGFP that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) . These show no change in number of Prox1 - positive lymphatic progenitors in ccbe1 morpholino - injected ( M ) sFLT4 mRNA - injected ( O ) compared with control morpholino - injected ( L ) uninjected ( N ) . ( P ) Quantitation of of dorsolateral CCV ECs that are lymphatic progenitors relative to total number of dorsolateral CCV ECs in control morpholino - injected ( n = 26 ) , ccbe1 morpholino - injected ( n = 27 ) , uninjected ( n = 28 ) sFLT4 mRNA - injected ( n = 32 ) . ( Q ) Quantitation of total number of dorsolateral region CCV ECs in 36 hpf that were uninjected or injected with control morpholino ( n = 26 ) , ccbe1 morpholino ( n = 27 ) , or sFLT4 mRNA ( n = 32 ) .

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

(L-O) Lateral images of the FLS (L, N; dotted line) or dorsolateral CCV region from where the FLS normally sprouts (M, O; dotted line) fixed at 36 hpf in lyve1b:EGFP embryos that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show no change in the number of Prox1-positive lymphatic progenitors in the ccbe1 morpholino-injected (M) and sFLT4 mRNA-injected (O) embryos compared with control morpholino-injected (L) and uninjected (N) embryos. (P) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino-injected (n = 26), ccbe1 morpholino-injected (n = 27), uninjected (n = 28) and sFLT4 mRNA-injected embryos (n = 32). (Q) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino (n = 26), ccbe1 morpholino (n = 27), or sFLT4 mRNA (n = 32).


( R - U ) Dorsolateral of VA - L fixed at 54 hpf in lyve1b : EGFP larva that have been fluorescently immunostained with anti - PROX1 ( green ) anti - GFP ( red ) . These show that lymphatic progenitor formation still occurs within VA - L in ccbe1 morpholino - injected ( S ) sFLT4 mRNA - injected ( U ) , with Prox1 expression resembling that of controls ( R , T ) .

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

(R-U) Dorsolateral images of the VA-L fixed at 54 hpf in lyve1b:EGFP larva that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show that lymphatic progenitor formation still occurs within the VA-L in ccbe1 morpholino-injected (S) and sFLT4 mRNA-injected embryos (U), with Prox1 expression resembling that of controls (R, T).