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 .

O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O 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-GENEPROD O O B-TISSUE O O O O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

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 .

O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM B-GENEPROD O O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 - .

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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 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 .

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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 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 B-SUBCELLULAR O B-GENEPROD O O O O 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 B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O 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 - .

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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 ) 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 B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD 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 B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD 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-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM 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 B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O 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 B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O B-TISSUE B-EXP_ASSAY 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-ORGANISM B-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE 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 B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 ) .

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

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O B-TISSUE 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 after fusion with another lymphangioblast ( CCV - L - derived FLS tip ( pink ) to PHS - L ( B , blue ; n = 5 / 5 ) PHS - L - derived FLS tip ( blue ) to VA - L ( F , red ; n = 5 / 5 ) ) or by VA - L to PHS - L - derived FLS tip ( B , n = 5 / 5 ) .

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

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

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY 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 ) migration tracks where each coloured track depicts ( µm ) travelled over 3 . 5 hours by leading tip in a separate before after fusion with another lymphangioblast ( PHS - L - derived FLS tip ( blue ) to VA - L ( F , red ; n = 5 / 5 ) ) or by VA - L to PHS - L - derived FLS tip ( F , n = 5 / 5 ) .

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

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

O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE 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 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 VA - L begins expressing etv2 ( E ) at 29 hpf is independent of any neighbouring vessel . kdrl expression ( red arrowheads ) occurs 4 hours later in VA - L ( demarcated by green dotted ) ( E ' ) .

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

O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-TISSUE O O O O O O O O B-TISSUE O B-TISSUE O O B-EXP_ASSAY O O O O O

(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).


( A - G ) Lateral ( A - B ) , ventrolateral ( C - F ) ventral ( G ) of cranial vessels in an etv2 : EGFP ; kdrl : nlsmCherry compound transgenic , which has had etv2 - expressing angioblast population ( early VA - L ) on left lateral side of yolk sac ablated . were taken at 30 hpf before ( A ) after ( B ) ablation of early VA - L , with site of ablation indicated ( B ; blue dotted circle ) . Note that LDA VA adjacent to VA - L become photobleached during ablation process ( B ) , however these were not ablated as they can be seen developing normally at 54 hpf ( D ) . Follow - up were taken of both ( C , E ) ablated ( D , F ) sides of ' s head at 54 hpf ( C - D ) 78 hpf ( E - G ) . Although VA - L ( blue dotted ) is seen migrating towards FLS ( dotted lined ) on side at 54 hpf ( C ) , it is missing from ablated side ( D ) . Subsequently , LFL ( dotted ) of ablated side is short ( F ) compared to that of side ( E ) . In addition , HA ( pink dotted ) is underdeveloped on ablated side compared to side , as it no longer extends towards nor connects to left AA1 ( G ) . ( H ) Quantitation of of LFL ( µm ) on ( control ) side on ablated side at 78 hpf ( n = 16 ) . ( I ) Quantitation of total number of within LFL on ( control ) side from ablated side at 78 hpf ( n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-TISSUE O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O B-TISSUE O 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-TISSUE 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-TISSUE 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-TISSUE O O O O O O O O O O B-TISSUE O O 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-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O

(A-G) Lateral (A-B), ventrolateral (C-F) and ventral (G) images of the cranial vessels in an etv2:EGFP;kdrl:nlsmCherry compound transgenic embryo, which has had the etv2-expressing angioblast population (early VA-L) on the left lateral side of the yolk sac ablated. Images were taken at 30 hpf before (A) and after (B) ablation of the early VA-L, with the site of ablation indicated (B; blue dotted circle). Note that the LDA and VA adjacent to the VA-L become photobleached during the ablation process (B), however these were not ablated as they can be seen developing normally at 54 hpf (D). Follow-up images were taken of both the contralateral (C, E) and ablated (D, F) sides of the embryo's head at 54 hpf (C-D) and 78 hpf (E-G). Although the VA-L (light blue dotted line) is seen migrating towards the FLS (white dotted lined) on the contralateral side at 54 hpf (C), it is missing from the ablated side (D). Subsequently, the LFL (white dotted line) of the ablated side is short (F) compared to that of the contralateral side (E). In addition, the HA (pink dotted line) is underdeveloped on the ablated side compared to the contralateral side, as it no longer extends towards nor connects to the left AA1 (G). (H) Quantitation of the length of the LFL (µm) on the contralateral (control) side and on the ablated side at 78 hpf (n = 16). (I) Quantitation of the total number of cells within the LFL on the contralateral (control) side and from the ablated side at 78 hpf (n = 16).


a , Flow chart of an in vivo screen to identify active DUBs by using biotin - Ub - VME pull - down ( left panel ) , in which USP8 peptide was identified by mass - spectrometry ( right panel ) . Silver immunoblot ( middle panel ) of precipitants pulled down by biotin - Ub - VME probe of MDA - MB - 231 .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O

a, Flow chart of an in vivo screen to identify active DUBs by using biotin-Ub-VME pull-down assay (left panel), in which the USP8 peptide was identified by mass-spectrometry analysis (right panel). Silver staining and immunoblot (middle panel) of precipitants pulled down by biotin-Ub-VME probe of MDA-MB-231 cells.


b , Experimental procedure in vivo : flow chart pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice ( left panel ) . Low metastatic MDA - MB - 231 - Luciferase / GFP breast cancer were infected with viral vectors expressing DUB cDNAs subsequently intracardially injected into nude mice . mice were monitored for 4 by in vivo BLI : of 30 mice injected with expressing DUB cDNA pool ; 3 had metastases in brain bone ( 3 / 30 ) , 4 had metastases on back in bone ( 4 / 30 ) , 9 had micro - metastasis ( 9 / 30 ) ; 30 mice injected with control showed no detectable metastasis ( 30 / 30 ) . Right panel : hints of identified DUBs were shown .

O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE B-EXP_ASSAY O B-ORGANISM O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD B-DISEASE I-DISEASE O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O B-EXP_ASSAY O B-TISSUE O B-TISSUE O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

b, Experimental procedure in vivo: flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice (left panel). Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo BLI: of the 30 mice injected with cells expressing DUB cDNA pool; 3 had metastases in the brain and bone (3/30), 4 had metastases on the back and in the bone (4/30), and 9 had micro-metastasis (9/30); the 30 mice injected with control cells showed no detectable metastasis (30/30). Right panel: hints of the identified DUBs were shown.


c , expression distribution of USP8 gene in breast cancer tissues ( n = 1097 ) normal tissues ( n = 572 ) , where horizontal axis represents different groups of samples , vertical axis represents gene expression distribution ( left panel ) . box - - whisker plots represent medians ( middle ) , first quartiles ( lower bound ) , third quartiles ( upper bound ) , ± 1 . 5 × interquartile ranges ( whisker lines ) . Kaplan - Meier curves showing metastasis - free survival of individuals was negatively correlated with USP8 expression by log - rank ( right panel ) .

O O O O O B-GENEPROD O O B-DISEASE I-DISEASE O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O

c, The expression distribution of USP8 gene in breast cancer tumor tissues (n = 1097) and normal tissues (n = 572), where the horizontal axis represents different groups of samples, the vertical axis represents the gene expression distribution (left panel). The box-and-whisker plots represent the medians (middle line), first quartiles (lower bound line), third quartiles (upper bound line), and the ±1.5× interquartile ranges (whisker lines). Kaplan-Meier curves showing the metastasis-free survival of individuals was negatively correlated with USP8 expression by log-rank test (right panel).


d , MCF10A - RAS infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 # 2 ) were analyzed in a sphere . number of mammospheres with diameter > 60 μm was quantified . graph shows number ( No . ) of spheres per 5 × 103 seeded . Scale bar 50

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d, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were analyzed in a tumor sphere assay. The number of the mammospheres with diameter > 60 μm was quantified. The graph shows the number (No.) of tumor spheres per 5 × 103 cells seeded. Scale bar 50 μm


e , MCF10A - RAS infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 # 2 ) were subcutaneously injected into nude mice at indicated numbers . Mean of volumes at week 5 ( n = 10 mice for each group ) .

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e, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were subcutaneously injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5 (n = 10 mice for each group).


f , Microscopy pictures of representative of zebrafish tail fin injected with control or USP8 stably depleted - MDA - MB - 231 at 5 days post injection , n = 20 for each group . g , final invasive areas of zebrafish displaying invasion experimental metastasis at 5 dpi ( left panel ) ; of injected with control or USP8 stably depleted MDA - MB - 231 ( # 1 # 2 ) displaying metastasis at 1 , 3 5 days post injection ( right panel ) .

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f, Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted-MDA-MB-231 cells at 5 days post injection, n = 20 for each group. g, The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi (left panel); Percentage of embryos injected with control or USP8 stably depleted MDA-MB-231 cells (# 1 and # 2) displaying metastasis at 1, 3 and 5 days post injection (right panel).


h , Bioluminescent of three representative mice from each group ( n = 6 ) at week 6 injected into left heart ventricle with control or MDA - MB - 231 stably depleted of endogenous USP8 with two independent shRNA ( # 1 # 2 ) . i , BLI signal ( left panel ) , number of bone metastasis of every mouse ( middle panel ) of bone metastasis free mice in each experimental group ( n = 6 mice per group ) followed in time as in h ( right panel ) .

O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O B-TISSUE B-EXP_ASSAY O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

h, Bioluminescent images of three representative mice from each group (n = 6) at week 6 injected into left heart ventricle with control or MDA-MB-231 cells stably depleted of endogenous USP8 with two independent shRNA (# 1 and # 2). i, BLI signal (left panel), number of bone metastasis of every mouse (middle panel) and the percentage of bone metastasis free mice in each experimental group (n = 6 mice per group) followed in time as in h (right panel).


a , of USP8 - correlated genes in a comparison of breast cancer with USP8 - high ( n = 38 ) versus with USP8 - low ( n = 39 ) .

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a, Heatmap of USP8-correlated genes in a comparison of breast cancer patients with USP8-high (n = 38) versus with USP8-low (n = 39).


b , Pre - ranked gene set enrichment ( GSEA ) in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) breast cancer .

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b, Pre-ranked gene set enrichment analysis (GSEA) in USP8-high (n = 38) versus USP8-low (n = 39) breast cancer patients.


c , Experimental procedure in vivo : MDA - MB - 231 were intracardially injected into nude mice ( n = 10 ) . qRT - PCR of bone metastatic showing correlations between USP8 Snai1 or Snai2 ( Slug ) in isolated from mutiple metastatic nodules ( n = 21 ) from mice .

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c, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice (n = 10). qRT-PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 (Slug) in cells isolated from mutiple metastatic nodules (n = 21) from mice.


d , 3D spheroid invasion of MCF10A - Ras showing USP8 wt increased TGF - β - induced invasion . Relative invasion was quantified shown in right panel .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O

d, 3D spheroid invasion assay of MCF10A-Ras cells showing USP8 wt increased TGF-β-induced invasion. Relative invasion was quantified and shown in the right panel.


e , MCF10A - Ras stably expressing control vector ( Co . vec ) , USP8 wt or USP8 cs were injected into blood circulation of 48 - hpf zebrafish . SB431542 ( 5 μM ) was added to zebrafish environment . Representative of zebrafish at 5 dpi ( left panel ) ; experimental metastatic areas ( middle panel ) of showing metastasis ( right panel ) are shown , n = 20 .

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e, MCF10A-Ras cells stably expressing control vector (Co.vec), USP8 wt or USP8 cs were injected into the blood circulation of 48-hpf zebrafish embryos. SB431542 (5 μM) was added to the zebrafish environment. Representative images of zebrafish at 5 dpi (left panel); the experimental metastatic areas (middle panel) and percentage of embryos showing metastasis (right panel) are shown, n = 20.


f , Diagram of DUB cDNA screening data of TGF - β - induced SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional reporter in HEK293T . X - Y - axes are relative luciferase activity in two replicates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

f, Diagram of DUB cDNA screening data of TGF-β-induced SMAD3/ SMAD4-dependent CAGA12-Luc transcriptional reporter in HEK293T cells. The X- and Y-axes are the relative luciferase activity in two replicates.


g , CAGA12 - Luc transcriptional response of HEK293T transfected with USP8 wt / cs or shUSP8 ( # 1 # 2 ) as indicated treated with TGF - β ( 1 ng / ml ) overnight .

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g, CAGA12-Luc transcriptional response of HEK293T cells transfected with USP8 wt/cs or shUSP8 (#1 and #2) as indicated and treated with TGF-β (1 ng/ml) overnight.


h , of TGF - β target genes by qRT - PCR in control or MCF10A - RAS stably depleting USP8 ( # 1 # 2 shRNA ) or expressing USP8 wt / cs treated with or without TGF - β ( 2 . 5 ng / ml ) for 8 hrs . Appendix Table S2 for details .

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h, Heatmap of TGF-β target genes by qRT-PCR analysis in control or MCF10A-RAS cells stably depleting USP8 (#1 and #2 shRNA) or expressing USP8 wt/cs and treated with or without TGF-β (2.5 ng/ml) for 8 hrs. Appendix Table S2 for details.


i , Immunoblot of total lysates , immunoprecipitates derived from control USP8 stably depleted ( # 1 # 2 shRNA ) ( left panel ) USP8 wt / cs over - expressed ( right panel ) MDA - MB - 231 treated with TGF - β ( 2 . 5 ng / ml ) for indicated time points .

O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

i, Immunoblot analysis of total cell lysates, immunoprecipitates derived from control and USP8 stably depleted (#1 and #2 shRNA) (left panel) and USP8 wt/cs over-expressed (right panel) MDA-MB-231 cells treated with TGF-β (2.5 ng/ml) for indicated time points.


j , Immunofluorescence 4 , 6 - diamidino - 2 - phenylindole ( DAPI ) of HaCaT infected with control or USP8 wt / cs treated with TGF - β ( 2 . 5 ng / ml ) SB431542 ( 10 μM ) for 72 hrs . Scale bar , 20 μm .

O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

j, Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HaCaT cells infected with control or USP8 wt/cs and treated with TGF-β (2.5 ng/ml) and SB431542 (10 μM) for 72 hrs. Scale bar, 20 μm.


k , Immunoblot of lysate of control USP8 stably depleted - MCF10AR - RAS ( MII ) treated with TGF - β ( 5 ng / ml ) for 48 hrs ( left ) ; Immunoblot of lysate of control USP8 wt or USP8 cs stably expressed - MCF10AR - RAS ( MII ) treated with TGF - β ( 5 ng / ml ) or SB431542 ( 10 μM ) for 48 hrs ( right ) .

O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

k, Immunoblot analysis of cell lysate of control and USP8 stably depleted-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) for 48 hrs (left); Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) or SB431542 (10 μM) for 48 hrs (right).


a , Immunofluorescence DAPI of HeLa transfected with TβRII - HA Flag - USP8 . Scale bar , 10 μm .

O O B-EXP_ASSAY B-SMALL_MOLECULE O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O

a, Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII-HA and Flag-USP8. Scale bar, 10 μm.


b , USP8 interacts with TβRII . Purified prokaryotically expressed USP8 recombinant GST - TβRII - ICD were incubated in vitro immunoprecipitated with anti - USP8 antibody ; immunoprecipitates were then immunoblotted for TβRII - ICD USP8 ( top panel ) ; Immunoblot of total lysate derived from MDA - MB - 231 treated with TGF - β as indicated immunoprecipitated with control IgG ( ns ) or anti - TβRII antibody ( bottom panel ) .

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O

b, USP8 interacts with TβRII. Purified prokaryotically expressed USP8 and recombinant GST-TβRII-ICD were incubated in vitro and immunoprecipitated with anti-USP8 antibody; immunoprecipitates were then immunoblotted for TβRII-ICD and USP8 (top panel); Immunoblot analysis of total cell lysate derived from MDA-MB-231 cells treated with TGF-β as indicated and immunoprecipitated with control IgG (ns) or anti-TβRII antibody (bottom panel).


c , Immunoblot of anti - TβRII immunoprecipitates from HEK293T stably expressing HA - Ub in vitro incubated with purified USP8 wt / cs .

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c, Immunoblot analysis of anti-TβRII immunoprecipitates from HEK293T cells stably expressing HA-Ub and in vitro incubated with purified USP8 wt/cs.


d , IB of Nickle - pull down precipitants derived from His - Ub stably expressed HEK293T incubated with purified USP8 as indicated time points .

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d, IB of Nickle-pull down precipitants derived from His-Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points.


e , IB of TCL immunoprecipitants derived from HEK293T transfected with indicated plasmids treat with or without MG132 ( 10 μM ) for 4 h .

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e, IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 (10 μM) for 4 h.


f , IB of TCL immunoprecipitants derived from HA - Ub stably expressed HEK293T transfected with Myc - USP8 wt / cs ( left panel ) or infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 # 2 ) ( right panel ) treated with TGF - β ( 5 ng / ml ) as indicated .

O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

f, IB of TCL and immunoprecipitants derived from HA-Ub stably expressed HEK293T cells transfected with Myc-USP8 wt/cs (left panel) or infected with control (Co.sh) or USP8 shRNA lentivirus (#1 and #2) (right panel) and treated with TGF-β (5 ng/ml) as indicated.


g , IB of HeLa stably expressing TβRII - HA transfected with control empty vector ( Co . vector ) Flag - USP8 wt / cs plasmids treated with CHX ( 20 μg / ml ) for indicated time points .

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g, IB of HeLa cells stably expressing TβRII-HA and transfected with control empty vector (Co.vector) and Flag-USP8 wt/cs plasmids and treated with CHX (20 μg/ml) for indicated time points.


h , Immunoblot of MCF10A - RAS stably expressing USP8 wt / cs or USP8 shRNA ( # 1 # 2 ) .

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h, Immunoblot analysis of MCF10A-RAS cells stably expressing USP8 wt/cs or USP8 shRNA (#1 and #2).


i , Immunoblot of biotinylated TβRII in MDA - MB - 231 ectopically expressing USP8 wt / cs treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . of band intensities is shown in right panel . Band was normalized to t = 0 controls ( right panel ) . n = 2 technical replicates .

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i, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells ectopically expressing USP8 wt/cs and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel). n = 2 technical replicates.


j , Immunoblot of biotinylated TβRII in MDA - MB - 231 infected with lentivirus encoding control shRNA ( Co . sh ) , USP8 shRNA ( # 1 # 2 ) treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . of band intensities is shown in right panel . Band was normalized to t = 0 controls ( right panel ) .

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j, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh), USP8 shRNA (# 1 and # 2) and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel).


l , Expression of USP8 of normal tissues ( n = 11 ) breast cancer ( n = 110 ) .

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l, Expression of USP8 of normal tissues (n = 11) and breast cancer patients (n = 110).


m , Scatter - plot showing positive correlation of USP8 , TβRII p - SMAD2 expression in ( n = 110 ) . H score of every tissue samples of USP8 , TβRII p - SMAD2 was calculated .

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m, Scatter-plot showing the positive correlation of USP8, TβRII and p-SMAD2 expression in patients (n = 110). H score of every tissue samples of USP8, TβRII and p-SMAD2 was calculated.


n , of specimens displaying low or high USP8 expression compared to expression of TβRII P - SMAD2 in human breast cancer tissue ( n = 110 ) . p values from Student ' s t were indicated .

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n, Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P-SMAD2 in human breast cancer tissue (n = 110). p values from Student's t tests were indicated.


o , GEPIA curves showing metastasis - free survival of breast cancer was significantly correlated with USP8 expression by log - rank in TCGA GTEx datasets ( http : / / gepia . cancer - pku . cn / ) . Cutoff - high = 67 % , cutoff - low = 25 % .

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o, GEPIA analysis curves showing the metastasis-free survival of breast cancer patients was significantly correlated with USP8 expression by log-rank test in the TCGA and GTEx datasets (http://gepia.cancer-pku.cn/). Cutoff-high = 67%, cutoff-low = 25%.


a , Comparison of pre - treatment of USP8 mRNA in - derived tissue samples by qRT - PCR ( left panel ) , enzyme activity of USP8 in - derived tissue samples by Ub - VME ( middle panel ) TβRII + crEVs level in plasma from breast cancer by ELISA ( right panel ) between breast cancer with or without clinical response to PTX . R , responders ; n = 16 ; NR , non - responders ; n = 14 .

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a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.


b , Pearson correlation of USP8 mRNA or USP8 enzyme activity in - derived tissue samples with EV - TβRII in plasma from breast cancer without clinical response to PTX as in a ( NR , n = 14 ) .

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b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).


c , Objective response rate ( ORR ) for as in a ( n = 30 ) with high ( > 0 . 95 ng / ml ) low ( < 0 . 95 ng / ml ) pre - treatment of circulating EV - TβRII .

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c, Objective response rate (ORR) for patients as in a (n = 30) with high (>0.95 ng/ml) and low (<0.95 ng/ml) pre-treatment levels of circulating EV-TβRII.


d , Pearson correlation of of IFN - γ + of CD8 + to TβRII + crEV level in plasma from breast cancer without clinical response to PTX as in a ( NR , n = 14 ) .

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d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).


e , TβRII + crEVs ( left panel ) , USP8 enzyme activity ( midde panel ) USP8 mRNA ( right panel ) from breast cancer with or without clinical response to combined therapy using anti - PD - L1 ( Atezolizumab ) paclitaxel . R , responders ; n = 10 ( left panel ) , n = 5 ( middle right panels , only 5 - derived tissue samples obtained ) . NR , non - responders ; n = 10 ( left panel ) , n = 6 ( middle right panels , only 5 - derived tissue samples obtained ) .

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e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).


Experimental procedure in vivo : MDA - MB - 231 were intracardially injected into nude mice . Bioluminescent imaging ( BLI ) of representative mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . BLI are shown ( f ( left panel ) ) . BLI signal of each mice ( f ( middle panel ) ) number of bone metastasis ( f ( right panel ) ) of every

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O

Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. Bioluminescent imaging (BLI) of representative mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). BLI images are shown (f (left panel)). BLI signal of each mice (f (middle panel)) and number of bone metastasis (f (right panel)) of every mouse


Experimental procedure in vivo : MDA - MB - 231 were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . of bone metastasis free mice survival in each experimental group followed in time ( g ) .

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Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). the percentage of bone metastasis free mice survival in each experimental group followed in time (g).


Experimental procedure in vivo : MDA - MB - 231 were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . Fold change of circulating EV - TβRII by ELISA in plasma samples of mice in each experimental group at week 6 ( h ) .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O

Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). Fold change of circulating EV-TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 (h).


nude mice were tail vein - injected with 4T07 - Luc ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) Normalized BLI signals ( left panel ) representative mice ( right panel ) from each group at indicated times ( j ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j).


nude mice were tail vein - injected with 4T07 - Luc ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) Immunoblot of circulating EVs from plasma in mice ( k ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Immunoblot analysis of the circulating EVs from plasma in mice (k).


nude mice were tail vein - injected with 4T07 - Luc ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) ELISA of circulating exosomal TβRII level in plasma samples of mice at indicated times ( l ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).


a , Gene signatures indicating Golgi - associated vesicle biogenesis - trafficking - related genes are significantly enriched in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) expressing ; shown by pre - ranked gene - set enrichment ( GSEA ) .

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a, Gene signatures indicating the Golgi-associated vesicle biogenesis- and membrane trafficking-related genes are significantly enriched in USP8-high (n = 38) patients versus USP8-low (n = 39) expressing patients; shown by pre-ranked gene-set enrichment analysis (GSEA).


b , shows up - regulated genes upon USP8 over - expression down - regulated genes upon USP8 depletion when compared with control MDA - MB - 231 by - seq ( left panel ) . qRT - PCR of control or MDA - MB - 231 stably over - expressing USP8 ( USP8 OE ) or USP8 shRNA ( right panel ) .

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b, Heatmap shows the up-regulated genes upon USP8 over-expression and the down-regulated genes upon USP8 depletion when compared with control MDA-MB-231 cells by RNA-seq analysis (left panel). qRT-PCR analysis of control or MDA-MB-231 cells stably over-expressing USP8 (USP8 OE) or USP8 shRNA (right panel).


e , FACS of EVs from 4T1 MDA - MB - 231 infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA .

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e, FACS analysis of EVs from 4T1 and MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA.


f , Immunoblot of EVs from MDA - MB - 231 infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA ( # 1 & # 2 ) .

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f, Immunoblot analysis of EVs from MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA (#1 & #2).


g , Immunoblot of EVs derived from MDA - MB - 231 ectopically expressing USP8 wt / cs .

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g, Immunoblot analysis of the EVs derived from MDA-MB-231 cells ectopically expressing USP8 wt/cs.


h , Experimental procedure in vivo : BALB / c mice were nipple injected with 4T1 - Luc ( 5 × 105 per mouse ) expressing control shRNA or doxycycline - inducible shRNA targeting USP8 ( shUSP8 ) tumors were grown for 3 , followed by administration of doxycycline for 3 ( n = 6 for each group ) . i , of of TβRII + crEVs in plasma samples from mice at day 24 . j , Immunoblot of TEVs ( left panel ) of of TβRII + EVs ( right panel ) in plasma samples from mice at day 42 . k , Normalized photon ﬂux at indicated times . l ,

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h, Experimental procedure in vivo: BALB/c mice were nipple injected with 4T1-Luc cells (5 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting USP8 (shUSP8) and tumors were grown for 3 weeks, followed by the administration of doxycycline for 3 weeks (n = 6 for each group). i, Quantification of the percentage of TβRII+ crEVs in plasma samples from mice at day 24. j, Immunoblot analysis of tumor and the TEVs (left panel) and quantification of the percentage of TβRII+ EVs (right panel) in plasma samples from mice at day 42. k, Normalized photon ﬂux at the indicated times. l,


l , Representative BLI signals of in each group at day 42 after implantation of 4T1 ( 2 × 105 per mouse , n = 6 per group ) .

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l, Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells (2 × 105 cells per mouse, n = 6 per group).


m , Normalized BLI signals ( left panel ) bioluminescent ( right panel ) of lung metastasis nodules . Scale bars , 2 mm .

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m, Normalized BLI signals (left panel) and bioluminescent images (right panel) of the lung metastasis nodules. Scale bars, 2 mm.


n , of CD8 + T of - draining lymph nodes ( TDLN ) at day 42 .

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n, Percentage of CD8+ T cells of tumor-draining lymph nodes (TDLN) at day 42.


b , Left : screening procedure for components critical for secretion of EV - TβRII ; Right : nanosight of GFP positive EVs derived from MDA - MB - 231 expressing TβRII - GFP pre - treated with anti - cancer compounds . n = 3 technical replicates .

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b, Left: screening procedure for cellular components critical for the secretion of EV-TβRII; Right: nanosight analysis of GFP positive EVs derived from MDA-MB-231 cells expressing TβRII-GFP and pre-treated with anti-cancer compounds. n = 3 technical replicates.


c , Immunoblot of total lysates derived from MDA - MB - 231 breast cancer treated with USP8 inhibitor TGF - β ( 2 . 5 ng / ml ) as indicated .

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c, Immunoblot analysis of total cell lysates derived from MDA-MB-231 breast cancer cell treated with USP8 inhibitor and TGF-β (2.5 ng/ml) as indicated.


d , Effect of USP8 inhibitor ( 5 μM ) on SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional response induced by TGF - β ( 2 . 5 ng / ml ) for 16 h in HEK293T transfected with control vector ( Co . vec ) or USP8 wt / cs . n = 3 biological replicates .

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d, Effect of USP8 inhibitor (5 μM) on SMAD3/SMAD4-dependent CAGA12-Luc transcriptional response induced by TGF-β (2.5 ng/ml) for 16 h in HEK293T cells transfected with control vector (Co.vec) or USP8 wt/cs. n = 3 biological replicates.


e , shows differentially expressed genes in control DMSO - treated or USP8 inhibitor ( 5 μM ) - treated MDA - MB - 231 for 24 h ( left panel ) . Volcano plot of transcriptome proﬁles between control USP8 inhibitor treated MDA - MB - 231 ( right panel ) . Red green dots represent genes significantly up - regulated down - regulated in control MDA - MB - 231 .

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e, Heatmap shows differentially expressed genes in control DMSO-treated or USP8 inhibitor (5 μM)-treated MDA-MB-231 cells for 24 h (left panel). Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA-MB-231 cells (right panel). Red and green dots represent genes significantly up-regulated and down-regulated in control MDA-MB-231 cells.


f , qRT - PCR of control ( DMSO - treated ) or USP8 inhibitor ( 5 μM for 24 h ) treated MDA - MB - 231 . Relative mRNA are shown as a .

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f, qRT-PCR analysis of control (DMSO-treated) or USP8 inhibitor (5 μM for 24 h) treated MDA-MB-231 cells. Relative mRNA levels are shown as a heatmap.


g , Gene signatures indicating up - regulated epithelial - mesenchymal - transition ( EMT ) ( upper panel ) breast cancer progenitors - related gene set ( bottom panel ) are significantly enriched in control ; shown by pre - ranked gene - set enrichment ( GSEA ) .

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g, Gene signatures indicating the up-regulated epithelial-mesenchymal-transition (EMT) (upper panel) and breast cancer progenitors-related gene set (bottom panel) are significantly enriched in control cells; shown by pre-ranked gene-set enrichment analysis (GSEA).


h , down - regulated TGF - β signaling ( upper panel ) metastasis - related gene sets ( bottom panel ) are significantly enriched in USP8 inhibitor - treated MDA - MB - 231 ; shown by pre - ranked gene - set enrichment ( GSEA ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O

h, The down-regulated TGF-β signaling (upper panel) and metastasis-related gene sets (bottom panel) are significantly enriched in USP8 inhibitor-treated MDA-MB-231 cells; shown by pre-ranked gene-set enrichment analysis (GSEA).


j , total of EVs by nanosight ( left ) of TβRII + EVs by FACS ( right ) from 4T1 MDA - MB - 231 treated with USP8 inhibitor ( 5 μM ) for 24 h . n = 3 technical replicates .

O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O

j, The total concentration of EVs by nanosight analysis (left) and the percentage of TβRII+ EVs by FACS (right) from 4T1 and MDA-MB-231 cells treated with USP8 inhibitor (5 μM) for 24 h. n = 3 technical replicates.


k - n , Experimental procedure in vivo MDA - MB - 231 - Luc ( 1 × 105 per mouse ) were intracardially injected into nude mice ( n = 7 for each group ) . USP8 inhibitor ( 1 mg / kg ) was injected at day 1 via intraperitoneal injection every other day for three . Normalized BLI signals ( left panel ) BLI imaging of three representive nude mice from each group at week 6 ( right panel ) were shown ( k ) . of metastasis - free survival in each experimental group followed in time ( l ) . Number of bone metastasis nodules ( m ) . of TβRII + crEVs in plasma samples from mice ( left panel ) immunoblot of metastatic nodule in mice circulating EVs from plasma ( right panel ) ( n ) .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O B-GENEPROD O B-SUBCELLULAR O B-TISSUE O O B-ORGANISM O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O O O

k-n, Experimental procedure in vivo MDA-MB-231-Luc cells (1 × 105 per mouse) were intracardially injected into nude mice (n = 7 for each group). USP8 inhibitor (1 mg/kg) was injected at day 1 via intraperitoneal injection every the other day for three weeks. Normalized BLI signals (left panel) and BLI imaging of three representive nude mice from each group at week 6 (right panel) were shown (k). Percentage of metastasis-free survival in each experimental group followed in time (l). Number of bone metastasis nodules (m). Percentage of TβRII+ crEVs in plasma samples from mice (left panel) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma (right panel) (n).


( A ) of KRAS , COUP - TFII , LDHA , phosphorylated S6K ( T389 ) in KRASWT , KRASG12D , KRAS - silenced KRASG12D MEF .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O

(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRASWT, KRASG12D, and KRAS-silenced KRASG12D MEF cells.


( B ) Extracellular acidification rate ( ECAR ) in KRASWT , KRAS G12D , KRAS - or COUP - TFII - silenced KRAS G12D MEF .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_TYPE O

(B) Extracellular acidification rate (ECAR) in KRASWT, KRAS G12D, and KRAS- or COUP-TFII-silenced KRAS G12D MEF cells.


( C ) Lactate in MEF Data are expressed as mean ± of three independent experiments normalized against values measured in KRASWT MEF . * * * p < 0 . 001 .

O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O

(C) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF. ***p<0.001.


( D ) of COUP - TFII , LDHA , phosphorylated S6K ( T389 ) in COUP - TFII - silenced KRASG12D MEF .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-CELL_TYPE O

(D) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced KRASG12D MEF cells.


( A ) of KRAS , COUP - TFII , LDHA , phosphorylated S6K ( T389 ) in KRAS - silenced HCT116 DLD - 1 .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O

(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRAS-silenced HCT116 and DLD-1 cells.


( B ) of COUP - TFII , LDHA , phosphorylated S6K ( T389 ) in COUP - TFII - silenced HCT116 DLD - 1 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O

(B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells.


( C ) Effect of lactate on phosphorylated S6K ( T389 ) , 4E - BP1 ( T37 / 46 ) , ULK1 ( S757 ) in COUP - TFII - silenced HCT116 .

O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O

(C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells.


( D ) Effects of COUP - TFII inhibition of LDHA on phosphorylated S6K ( T389 ) Raptor ( S792 ) in KRAS - silenced HCT116 . Asterisk indicates exogenous HA - COUP - TFII . Arrow indicates phosphorylated Raptor ( S792 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY B-GENEPROD O O O O

(D) Effects of COUP-TFII and the inhibition of LDHA on phosphorylated S6K (T389) and Raptor (S792) in KRAS-silenced HCT116 cells. Asterisk indicates exogenous HA-COUP-TFII. Arrow indicates phosphorylated Raptor (S792).


( E ) Inhibition of COUP - TFII - silenced HCT116 , DLD - 1 , MIA PaCa - 2 proliferation its recovery by treatment with lactate . Data are expressed as mean ± of three independent experiments normalized against values measured in control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001.


( F ) Clonogenic of COUP - TFII - silenced HCT116 , DLD - 1 , MIA PaCa - 2 in presence or absence of lactate .

(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.


( A ) Effect of lactate on ERK / TSC2 AKT / TSC2 signaling pathway in serum - starved HCT116 .

O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-TISSUE O O B-CELL_LINE O

(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells.


( B ) Effect of lactate on phosphorylated S6K ( T389 ) inhibited by TSC2 overexpression in HCT116 .

O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O

(B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells.


( C ) Molecular interaction between TSC2 Rheb in presence or absence of lactate in HA - Rheb - overexpressing HCT116 after serum deprivation overnight .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-TISSUE O O O

(C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight.


( D ) Molecular interaction between TSC2 recombinant GDP - loaded Rheb in presence or absence of lactate in vitro .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O

(D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro.