Real - time PCR analysis showing that knockdown of YAP with YAP siRNA ( siYAP ) significantly suppressed TGF - α - induced ( FBS 10 % , TGF - α : 10 ng / ml for 48 h ) AREG expression in ME180 cells . siCTRL , a non - targeting siRNA , was used as a negative control . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0067 ) .

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Real-time PCR analysis showing that knockdown of YAP with YAP siRNA (siYAP) significantly suppressed TGF-α-induced (FBS 10%, TGF-α: 10 ng/ml for 48 h) AREG expression in ME180 cells. siCTRL, a non-targeting siRNA, was used as a negative control. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0067).


Proliferation of ME180 cells ( FBS 1 % ) treated with control ( siCTRL ) or YAP ( siYAP ) prior to treatment with control medium or TGF - α ( 10 ng / ml ) for 108 h . Each bar represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P = 0 . 0058 ; siYAP + CTRL vs . siYAP + TGF - α , P = 0 . 1840 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0013 ) .

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Proliferation of ME180 cells (FBS 1%) treated with control (siCTRL) or YAP (siYAP) prior to treatment with control medium or TGF-α (10 ng/ml) for 108 h. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P = 0.0058; siYAP+CTRL vs. siYAP+TGF-α, P = 0.1840; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0013).


Quantitative data of the wound - healing assay showing the migration of ME180 cells that were treated with control ( siCTRL ) or YAP siRNA ( siYAP ) prior to treatment with or without TGF - α for 12 h . Each bar in bar graphs represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0041 ) .

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Quantitative data of the wound-healing assay showing the migration of ME180 cells that were treated with control (siCTRL) or YAP siRNA (siYAP) prior to treatment with or without TGF-α for 12 h. Each bar in bar graphs represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0041).


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

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


AREG ( 50 ng / ml ) treatment induced appearance of elongated cells in ME180 cells ( top and middle panels ) and significantly stimulated ME180 cell proliferation ( lower panel ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 003 ) . Scale bar : 100 μm .

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


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

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


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

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Real-time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression. Each bar represents mean ± SEM (n = 9). Bars with different letters are significantly different from each other (P < 0.0001).


Real - time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA ( siYAP ) significantly suppressed AREG - induced AREG mRNA expression . siCTRL ( non - target siRNA ) was used as a siRNA control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + AREG , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + AREG , P < 0 . 0001 ; siCTRL + AREG vs . siYAP + AREG , P = 0 . 0037 ) .

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Real-time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA (siYAP) significantly suppressed AREG-induced AREG mRNA expression. siCTRL (non-target siRNA) was used as a siRNA control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+AREG, P < 0.0001; siYAP+CTRL vs. siYAP+AREG, P < 0.0001; siCTRL+AREG vs. siYAP+AREG, P = 0.0037).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


D Plasmids containing MyoD cDNA or empty vector together with R3hdml - luc 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 .

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

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


A R3hdml siRNA or non - targeting siRNA was transfected into C2C12 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 .

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

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


B Immunofluorescence after C2C12 differentiation . MyHC is shown in green 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 .

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

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


C 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 .

O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O 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 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 .

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

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


Thirty freshly isolated myofibers from 8 - 12 - week - old R3hdml KO ( n = 7 ) 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 .

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

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


( E , F ) Co - immunostaining parallel cultures for MyoD 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 .

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

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


A Seven days after CTX injection , skeletal 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 .

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

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


B One hundred microliters of 10 µM CTX was intramuscularly injected into 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 .

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

(F-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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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 , 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

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

f, 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 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 ) .

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

O O O O O O O O O O O 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, 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 analysis ( GSEA ) in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) breast cancer patients .

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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 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 .

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

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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 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 .

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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 cells . The X - and Y - axes are the relative luciferase activity in two replicates .

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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 cells transfected with USP8 wt / cs or shUSP8 ( # 1 and # 2 ) as indicated and 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 , 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

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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 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 ) .

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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 analysis of anti - TβRII immunoprecipitates from HEK293T cells stably expressing HA - Ub and 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 cells incubated with purified USP8 protein as indicated time points .

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 and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and 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 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 .

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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 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 .

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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 analysis of MCF10A - RAS cells stably expressing USP8 wt / cs or USP8 shRNA ( # 1 and # 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 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 .

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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 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 ) .

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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 ) and breast cancer patients ( 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 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 .

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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 , 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 .

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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 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 % .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 ) , 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 ) .

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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 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

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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 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 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 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 ) .

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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). 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 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 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 , 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 ) .

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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 analysis of EVs from 4T1 and MDA - MB - 231 cells 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.