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

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


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

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

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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 , TEM ( bottom ; scale bar , 5 µm ) and fluorescence ( top ; scale bar , 10 µm ) of NRK - LC3 cells 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 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 ) .

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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 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h ( total , 6 h ) or 6 h ( total , 8 h ) analysed by blotting ( e ) as in b or microscopy ( f ; scale bar , 5 µm ) with quantification ( g ) . Error bars , s . e . m . ; n = 3 .

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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 cells were starved for 4 h and 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 cells 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 cells starved for 2 h and 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 cells starved for times shown with 1 µg ml - 1 leupeptin and blotted with indicated antibodies . e , Cells 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 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 .

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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001).


F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells .

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F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells.


G Expression of Ki67 in the tumor xenografts of ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells implanted in athymic nude mice . Ki67 was visualized using an Alexa - 488 ( green ) - conjugated secondary antibody . Actin was visualized using an Alexa - 594 ( red ) - conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 20 μm . Note the significant increase in the Ki67 - positive cells in the ME180 - YAP and ME180 - YAPS127A tumor xenografts .

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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 analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector ( shCtrl ) or lentivirus - based 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 images showing tumor xenografts derived from ME180 - shCtrl cells ( left flank ) and ME180 - shYAP # 1 cells ( 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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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AREG (50 ng/ml) treatment induced appearance of elongated cells in ME180 cells (top and middle panels) and significantly stimulated ME180 cell proliferation (lower panel). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.003). Scale bar: 100 μm.


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

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


Real - time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression . Each bar represents mean ± SEM ( n = 9 ) . Bars with different letters are significantly different from each other ( P < 0 . 0001 ) .

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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 cells with YAP siRNA ( siYAP ) significantly suppressed AREG - induced AREG mRNA expression . siCTRL ( non - target siRNA ) was used as a siRNA control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + AREG , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + AREG , P < 0 . 0001 ; siCTRL + AREG vs . siYAP + AREG , P = 0 . 0037 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 assay showing the effect of AG1478 and VTPF on colony formation in ME180 - MXIV , ME180 - YAP and ME180 - YAPS127A cells . 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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 cells with HPV18E6 siRNA ( siE6 ) reduced YAP protein levels , but increased SOCS6 protein levels .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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).


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 .

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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 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 .

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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 cells . One day and 3 days after transfection , cells were lysed and subjected to SDS - PAGE and western blotting using anti - MyoD antibodies .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 ) 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 .

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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 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 ' ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O 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-MEASURED_VAR 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 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 .

O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_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

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 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 ) .

O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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 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 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 B-CONTROLLED_VAR 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 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 ) .

O O O O O 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-MEASURED_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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR 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).


( A - B ) Still images from Movie EV3 of early facial lymphatic development in a lyve1b : EGFP ( A ) , kdrl : nlsmCherry ( B ) compound transgenic from 42 - 51 . 5 hpf , with the CCV - L - derived leading tip cell ( green asterisk ) , the PHS - LP domains ( orange and yellow asterisk ) and the distal tip of the VA - L ( purple asterisk ) highlighted ( A ) . Between 42 and 46 hpf , the PHS - LPP ( orange outline ) cells divide ( dark blue parent cells , light blue daughter cells ) before sprouting as lymphangioblasts from the PHS and fusing with the tip ( pink cells ) of the CCV - derived FLS ( green outline ) at 48 hpf ( B ) . Cells ( red ) within the VA - L ( purple outline ) can also be seen migrating towards the now PHS - L - derived FLS tip by 48 hpf . The PHS - LPA is also shown ( yellow outline ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Still images from Movie EV3 of early facial lymphatic development in a lyve1b:EGFP (A), kdrl:nlsmCherry (B) compound transgenic from 42-51.5 hpf, with the CCV-L-derived leading tip cell (green asterisk), the PHS-LP domains (orange and yellow asterisk) and the distal tip of the VA-L (purple asterisk) highlighted (A). Between 42 and 46 hpf, the PHS-LPP (orange outline) cells divide (dark blue parent cells, light blue daughter cells) before sprouting as lymphangioblasts from the PHS and fusing with the tip (pink cells) of the CCV-derived FLS (green outline) at 48 hpf (B). Cells (red) within the VA-L (purple outline) can also be seen migrating towards the now PHS-L-derived FLS tip by 48 hpf. The PHS-LPA is also shown (yellow outline).


( C ) Quantitation of leading lymphangioblast velocity ( µm / min ) before and after fusion with another lymphangioblast ( CCV - L - derived FLS tip ( pink cells ) to PHS - L ( B , blue cells ; n = 5 / 5 ) and PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( B , n = 5 / 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation of leading lymphangioblast velocity (µm/min) before and after fusion with another lymphangioblast (CCV-L-derived FLS tip (pink cells) to PHS-L (B, blue cells; n = 5/5) and PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (B, n = 5/5).


( D ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV - L - derived FLS tip ( pink cells ) and the PHS - Ls ( B , blue cells ; n = 5 / 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV-L-derived FLS tip (pink cells) and the PHS-Ls (B, blue cells; n = 5/5).


( E - F ) Still images from Movie EV4 of facial lymphatic development in a lyve1b : EGFP kdrl : nlsmCherry compound transgenic from 48 - 62 hpf showing that after the CCV - derived FLS fuses to the PHS - L , these take over as the leading tip cells ( orange asterisks ) , with one PHS - L migrating anteriorly to fuse with PHS - LPA ( yellow asterisk ) , and others migrating ventrally to fuse with the VA - L ( purple asterisk ) After the PHS - derived portion ( orange outline ) of the FLS ( green outline ) fuses to the VA - L ( purple outline ) , migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation ( dark blue and red parent cells , light blue and pink daughter cells ) before resuming migration anteriorly along the ventral base of the

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Still images from Movie EV4 of facial lymphatic development in a lyve1b:EGFP kdrl:nlsmCherry compound transgenic from 48-62 hpf showing that after the CCV-derived FLS fuses to the PHS-L, these take over as the leading tip cells (orange asterisks), with one PHS-L migrating anteriorly to fuse with PHS-LPA (yellow asterisk), and others migrating ventrally to fuse with the VA-L (purple asterisk) After the PHS-derived portion (orange outline) of the FLS (green outline) fuses to the VA-L (purple outline), migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation (dark blue and red parent cells, light blue and pink daughter cells) before resuming migration anteriorly along the ventral base of the eye


( G - H ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 . 5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast ( PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( F , n = 5 / 5 ) .

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

(G-H) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3.5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast (PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (F, n = 5/5).


( A - B ' ) Lateral ( A ) and ventrolateral ( B , B ' ) images of the cranial vessels in kdrl : nlsKaede ; kdrl : EGFP embryos , with unconverted tissue shown in green ( A - B ) while photoconverted tissue is shown in red ( B ' ) . The early kdrl - positive VA - L was photoconverted in 34 hpf embryos ( A ) , and followed through to 54 hpf ( B , B ' ) . B ' is a higher magnification image of the box in B and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA . The VA - L photoconverted cells are indicated by 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B') Lateral (A) and ventrolateral (B, B') images of the cranial vessels in kdrl:nlsKaede;kdrl:EGFP embryos, with unconverted tissue shown in green (A-B) while photoconverted tissue is shown in red (B'). The early kdrl-positive VA-L was photoconverted in 34 hpf embryos (A), and followed through to 54 hpf (B, B'). B' is a higher magnification image of the box in B and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA. The VA-L photoconverted cells are indicated by arrowheads.


( C ) Still images from Movie EV5 of VA - L development in a kdrl : EGFP ; kdrl : nlsmCherry compound transgenic from 31 - 35 . 5 hpf . This movie shows that the VA - L begins expressing kdrl just after 32 hpf , in a manner that is isolated and independent of any neighbouring kdrl - positive tissue or vessel . Putative kdrl - positive primitive myeloid cells ( light blue asterisks ) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA - L . ( D ) Still images from Movie EV6 depicting a 3D reconstruction of the 35 . 5 hpf timepoint from Movie EV5 . A dorsal view that scrolls through the Y - stack of the VA - L is shown , which illustrates that the VA - L is closely associated with , but not connected to the VA .

O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Still images from Movie EV5 of VA-L development in a kdrl:EGFP;kdrl:nlsmCherry compound transgenic from 31-35.5 hpf. This movie shows that the VA-L begins expressing kdrl just after 32 hpf, in a manner that is isolated and independent of any neighbouring kdrl-positive tissue or vessel. Putative kdrl-positive primitive myeloid cells (light blue asterisks) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA-L. (D) Still images from Movie EV6 depicting a 3D reconstruction of the 35.5 hpf timepoint from Movie EV5. A dorsal view that scrolls through the Y-stack of the VA-L is shown, which illustrates that the VA-L is closely associated with, but not connected to the VA.


( E - E ' ) Still images from Movie EV7 of VA - L development in an etv2 : EGFP ( E ) , kdrl : nlsmCherry ( E ' ) compound transgenic from 28 - 35 . 2 hpf . This movie shows that the VA - L begins expressing etv2 ( E ) at 29 hpf and is independent of any neighbouring vessel . kdrl expression ( red arrowheads ) occurs 4 hours later in the VA - L ( demarcated by the green dotted line ) ( E ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_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

(E-E') Still images from Movie EV7 of VA-L development in an etv2:EGFP (E), kdrl:nlsmCherry (E') compound transgenic from 28-35.2 hpf. This movie shows that the VA-L begins expressing etv2 (E) at 29 hpf and is independent of any neighbouring vessel. kdrl expression (red arrowheads) occurs 4 hours later in the VA-L (demarcated by the green dotted line) (E').


( F - G ' ) Lateral images of the cranial vessels in etv2 : Kaede embryos , with unconverted tissue shown in green ( F - G ) while photoconverted tissue is shown in red ( G ' ) . The earliest visible form of the VA - L was photoconverted at 29 hpf ( F ) , then followed through to 54 ( G ) . G ' is a higher magnification image of the box in G and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA , and VA - L photoconverted cells indicated ( arrowheads ) .

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(F-G') Lateral images of the cranial vessels in etv2:Kaede embryos, with unconverted tissue shown in green (F-G) while photoconverted tissue is shown in red (G'). The earliest visible form of the VA-L was photoconverted at 29 hpf (F), then followed through to 54 (G). G' is a higher magnification image of the box in G and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA, and VA-L photoconverted cells indicated (arrowheads).