Figure 5A , B Quantification to the number of mitotic cells of dissected gonads from wild - type animals stained with either anti - phosphohistone H3 ( PH3 ) ( A ) , anti - CDK - 1 ( CDK - 1 ) ( B ) transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) and treated with gamma radiation 125 Gy . Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells Data information : Data are presented as mean ± SD ( A , B N = 2 ( A , B * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . One - way ANOVA Tukey " s multiple comparisons testFertilit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated with gamma radiatio at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified 2 - wa 2 - way ANOVA Tukey " s multiple compa ( C - H ) fecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit UV at the indicated doses Data information : Data are presented a mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compaFertilit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit Hydroxyure at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple comparisonsfecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated with gamma radiatio at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compafecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit UV at the indicated doses Data information : Data are presente as mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compafecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , an left untreated or treated wit Hydroxyure at the indicated doses Data information : Data are presente as mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

Figure 5A, B Quantification to the number of mitotic cells of dissected gonads from wild-type animals stained with either anti-phosphohistone H3 (PH3) (A), anti-CDK-1 (CDK-1) (B) transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) and treated with gamma radiation 125 Gy. Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells Data information: Data are presented as mean ± SD (A, B N=2 (A, B * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. One-way ANOVA Tukey"s multiple comparisons testFertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified 2-wa 2-way ANOVA Tukey"s multiple compa (C-H)fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presented a mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compaFertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple comparisonsfecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compafecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presente as mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compafecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, an left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presente as mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


Figure 6A Quantification of BRD - 1 protein and representative western blot from wild - type worms fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 Bar graphs are mean ± SEM Data information : Data are presented as mean ± SD N = 8 - 1 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specifiedB Quantification of mitotic nuclei in single gonad arm of wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) Data information : Data are presented as mean ± SD N = ) , 8 - 1 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specifiedQuantification of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) and left untreated ( C Bar graphs are mean ± SEM . Data information Data are presented a mean ± SEM N = 8 - 10 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonQuantification of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) an treated with ionizing radiation 125 Gy ( IR ) ( D ) . Bar graphs are mean ± SEM . Data information : Data are presented a mean ± SEM N = 8 - 10 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Figure 6A Quantification of BRD-1 protein and representative western blot from wild-type worms fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 Bar graphs are mean ± SEM Data information: Data are presented as mean ± SD N= 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specifiedB Quantification of mitotic nuclei in single gonad arm of wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) Data information: Data are presented as mean ± SD N= ), 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specifiedQuantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) and left untreated (C Bar graphs are mean ± SEM. Data information Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonQuantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) an treated with ionizing radiation 125 Gy (IR) (D). Bar graphs are mean ± SEM. Data information: Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparison


Figure 7Kaplan - Meier survival curves of wild - type ( WT ) and , ced - 9 ( n1653ts strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an ) , brc - 1 ( tm1145 ) ( B strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an brd - 1 ( gk297 ) ( C strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an glp - 1 ( e2141 ) ( D strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an glp4 ( bn2 ) ( E ) , strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1 ( F and left untreated or treated with 600 , 1200 or 2400 J / m2 UVBKaplan - Meier survival curves of wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA agains isp - 1 ( G ) and left untreated or treated with 600 , 1200 or 2400 J / m2

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

Figure 7Kaplan-Meier survival curves of wild-type (WT) and, ced-9(n1653ts strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an ), brc-1(tm1145) (B strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an brd-1(gk297) (C strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an glp-1(e2141) (D strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an glp4(bn2) (E), strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1 (F and left untreated or treated with 600, 1200 or 2400 J/m2 UVBKaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA agains isp-1 (G) and left untreated or treated with 600, 1200 or 2400 J/m2 UVB


Figure 8A Kaplan - Meier survival curves of wild - type ( WT ) and , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1B Quantification of apoptotic corpses in wild - type ( WT ) and , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed as ( A ) and left untreated or treated with 600 J / m2 UVB . Data information : Data are presented as mean ± SEM , N = 3 , 9 - 10 worms per replicate and condition * * P < 0 . 01 vs con ; # # P < 0 . 01 vs WT ; ooo P < 0 . 00 and oooo P < 0 . 0001 vs untreated . Not significant if nothing is specified . 2 - way ANOVA Tukey ' s multiple comparisons test . C

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

Figure 8A Kaplan-Meier survival curves of wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1B Quantification of apoptotic corpses in wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed as (A) and left untreated or treated with 600 J/m2 UVB. Data information: Data are presented as mean ± SEM, N=3, 9-10 worms per replicate and condition ** P < 0.01 vs con; ## P < 0.01 vs WT; ooo P < 0.00 and oooo P < 0.0001 vs untreated . Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test. C


Figure 6 Spot assay of designed TAD constructs . The spot assay was performed at 30ºC ( left ) and during heat shock at 37ºC ( 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

Figure 6 Spot assay of designed TAD constructs . The spot assay was performed at 30ºC (left) and during heat shock at 37ºC (right panel)


Figure 2 ( A ) Soft or stiff 4T1 or MCF - 7 cells were isolated by the microfluidic chip , and then seeded ( 500 cells ) in 90 Pa soft 3D fibrin gel for 3 days . The percentage of colonies formed was calculated and the colony size was recorded . Scale bar , 100 μm . Data information : Two - tailed Paired Student ' s t test The data represent mean ± SD . n = 3 independent experiments in ( A ) . ( F ) NSG mice were intravenously injected with 100 soft or stiff B16 or MP - 1 cells . Eight weeks later , the lung metastasis was analyzed by H & E staining . The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides . Scale bar , 0 . 5 mm . n = 3 ( for B16 ) or 5 ( for MP - 1 ) mice with metastatic tumor . Data information : Two - tailed Paired Student ' s t test The data represent mean ± SD .

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

Figure 2(A) Soft or stiff 4T1 or MCF-7 cells were isolated by the microfluidic chip, and then seeded (500 cells) in 90 Pa soft 3D fibrin gel for 3 days. The percentage of colonies formed was calculated and the colony size was recorded. Scale bar, 100 μm. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. n = 3 independent experiments in (A).(F) NSG mice were intravenously injected with 100 soft or stiff B16 or MP-1 cells. Eight weeks later, the lung metastasis was analyzed by H&E staining. The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides. Scale bar, 0.5 mm. n = 3 (for B16) or 5 (for MP-1) mice with metastatic tumor. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.


Figure 3The 100 stiff ALDH + or soft ALDH - 4T1 cells were injected into the mammary fat pads of NSG mice . Eight weeks later , the lung sections were H & E stained . metastasis index was calculated ( D ) . Scale bar , 0 . 5 mm . n = 5 mice with metastatic tumor . Data information : two - tailed Paired Student ' s t test data represent mean ± SD .

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

Figure 3The 100 stiff ALDH+ or soft ALDH- 4T1 cells were injected into the mammary fat pads of NSG mice. Eight weeks later, the lung sections were H&E stained. metastasis index was calculated (D). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor. Data information: two-tailed Paired Student's t test data represent mean ± SD.


Figure 4 ( E ) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF - 7 cells . n = 3 . The unit for the color scale was z - score of log2 expression data shown .

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Figure 4 (E) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF-7 cells. n = 3. The unit for the color scale was z-score of log2 expression data shown.


Figure 5 ( A ) The expression of BCL9L or β - catenin in stiff or soft 4T1 , MCF - 7 , B16 or MP - 1 cells was determined by western blot . ( B ) The expression of β - catenin and BCL9L from SGGFP , BCL9L - SGs - 4T1 , MCF - 7 , B16 or MP - 1 cells were determined by western blot . Data information n = 3 independent experiments in ( A and B ) . ( E , F ) The 100 soft SGGFP or BCL9L - SGs - 4T1 , B16 or MP - 1 cells were injected into the mammary fat pads ( 4T1 ) of BALB / c mice or tail vein ( B16 and MP - 1 ) of NSG mice . The tumor formation was recorded ( E ) . Six to eight weeks later , the lung sections were H & E stained . The metastatic micronodules in the lung were counted and the metastasis index was calculated ( F ) . Scale bar , 0 . 5 mm . n = 3 ( for B16 - F1 ) or 5 ( for MP - 1 ) mice with metastatic tumor . Data information : Bonferroni test The data represent mean ± SD .

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

Figure 5(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot. (B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot. Data information n = 3 independent experiments in (A and B).(E, F) The 100 soft SGGFP or BCL9L-SGs- 4T1, B16 or MP-1 cells were injected into the mammary fat pads (4T1) of BALB/c mice or tail vein (B16 and MP-1) of NSG mice. The tumor formation was recorded (E). Six to eight weeks later, the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (F). Scale bar, 0.5 mm. n = 3 (for B16-F1) or 5 (for MP-1) mice with metastatic tumor. Data information: Bonferroni test The data represent mean ± SD.


Figure 6 ( A ) The stiffness of primary breast ( HBC , n = 3 ) and colon ( HCC , n = 3 ) cancer cells screened from the outlet or backflow inlet was measured by AFM . n = 100 from each patient . Data information : Mann - Whitney test ( A ) The data represent mean ± SD . ( C ) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti - BCL9L antibody . The expression of BCL9L was observed under confocal microscope . Scale bar , 10 μ Data information : Paired Student ' s t test ( C ) The data represent mean ± SD . ( E ) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma . Data information : Pearson ' s correlation test E ) The data represent mean ± SD . ( F ) Overall survival compared to the BCL9L level in breast cancer ( BRCA , n = 536 ) or melanoma ( n = 376 ) patients . Data information : Log - rank survival analysis ( F ) . The data represent mean ± SD .

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

Figure 6(A) The stiffness of primary breast (HBC, n = 3) and colon (HCC, n = 3) cancer cells screened from the outlet or backflow inlet was measured by AFM. n = 100 from each patient. Data information: Mann-Whitney test (A) The data represent mean ± SD.(C) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti-BCL9L antibody. The expression of BCL9L was observed under confocal microscope. Scale bar, 10 μ Data information: Paired Student's t test (C) The data represent mean ± SD.(E) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma. Data information: Pearson's correlation test E) The data represent mean ± SD.(F) Overall survival compared to the BCL9L level in breast cancer (BRCA, n = 536) or melanoma (n = 376) patients. Data information: Log-rank survival analysis (F). The data represent mean ± SD.


Figure 1A Liver biopsies showing no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis stained by PSR . Size bar , 300 μm . B Immunohistochemistry ( brown staining ) for αSMA and SOX9 on consecutive sections from patients with no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis . Boxed areas are shown to the right at higher magnification . Bile duct ( bd ) . SOX9 detected in HSCs with elongated nuclei ( hatched line ) next to scar and in hepatocytes with rounded nuclei ( arrows ) . Size bars , 200 µm . C Immunofluorescence for SOX9 ( red ) co - stained with CK19 , CK7 , α1AT , and αSMA ( green ) . Arrowheads show elongated nuclei in αSMA - positive HSCs . D Immunohistochemistry for SOX9 and EpCAM ( brown staining ) on consecutive sections from severe ( IS6 ) fibrosis and normal fetal liver at 18 weeks post - conception ( wpc ) . Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . I - L Plots of SOX9 index ( total , HSC / Hep , and biliary ) and NI score marking progressors in green and non - progressors in red . The solid line indicates the mean in each graph . The broken line in NI dot plot ( L ) indicates the threshold ( score = 8 ) below which all NI is categorized as ' mild ' .

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

Figure 1A Liver biopsies showing no (IS0), moderate (IS3), and severe (IS6) fibrosis stained by PSR. Size bar, 300 μm.B Immunohistochemistry (brown staining) for αSMA and SOX9 on consecutive sections from patients with no (IS0), moderate (IS3), and severe (IS6) fibrosis. Boxed areas are shown to the right at higher magnification. Bile duct (bd). SOX9 detected in HSCs with elongated nuclei (hatched line) next to scar and in hepatocytes with rounded nuclei (arrows). Size bars, 200 µm.C Immunofluorescence for SOX9 (red) co-stained with CK19, CK7, α1AT, and αSMA (green). Arrowheads show elongated nuclei in αSMA-positive HSCs.D Immunohistochemistry for SOX9 and EpCAM (brown staining) on consecutive sections from severe (IS6) fibrosis and normal fetal liver at 18 weeks post-conception (wpc). Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis.E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).I-L Plots of SOX9 index (total, HSC/Hep, and biliary) and NI score marking progressors in green and non-progressors in red. The solid line indicates the mean in each graph. The broken line in NI dot plot (L) indicates the threshold (score = 8) below which all NI is categorized as 'mild'.


Figure 2A Immunohistochemistry for SOX9 ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . SOX9 positive bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated . B ( Right panel ) Immunofluorescence for SOX9 ( green ) and HNF4α ( red ) in WT fibrotic mice . Arrowheads ( orange / yellow staining ) indicate SOX9 + / HNF4α + hepatocytes and star ( * ; red ) indicates SOX - / HNF4α + hepatocytes . ( Left panel ) Immunofluorescence for SOX9 ( green nucleus ) and CK19 ( red cytoplasm ) in WT fibrotic mice . Arrowheads ( green ) indicate SOX9 + hepatocytes and star ( * ) indicates SOX - hepatocytes identified by DAPI staining in Appendix Figure S1 . C Co - localization by immunohistochemistry ( top panel ) and in situ hybridization ( lower panel ) for SOX9 ( red ) and αSMA ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control . E Immunohistochemistry for SOX9 ( brown ) in control ( Sox9fl / fl ; RosaCre - ) or Sox9 - null ( Sox9fl / fl ; RosaCre + ) mice following fibrosis . Bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated . SOX9 is absent , even in bile ducts in Sox9 - null liver . Size bar , 25 μm ( A , B and E ) , 10 μm ( C ) .

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Figure 2A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1.C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.E Immunohistochemistry for SOX9 (brown) in control (Sox9fl/fl;RosaCre-) or Sox9-null (Sox9fl/fl;RosaCre+) mice following fibrosis. Bile ducts (bd) and hepatocytes (arrowheads) indicated. SOX9 is absent, even in bile ducts in Sox9-null liver. Size bar, 25 μm (A, B and E), 10 μm (C).


Figure 3 A - J Representative images and quantification shown for olive oil treated ( n = 6 ) or chronic CCl4 ( n = 5 Sox9fl / fl ; RosaCreER - ; n = 8 Sox9fl / fl ; RosaCreER + ) induced fibrosis or following sham operation ( n = 5 ) or BDL ( n = 7 Sox9fl / fl ; RosaCreER - ; n = 5 Sox9fl / fl ; RosaCreER + ) ) induced fibrosis . A Picrosirius red ( PSR ) staining ( collagen deposition in red ) counterstained with fast green ( top row ) and immunohistochemistry for α - SMA ( brown staining bottom row ; activated HSC / myofibroblast marker ) in olive oil treated ( left ) or chronic CCl4 induced fibrosis ( right ) in control and Sox9 - null mice . Size bar = 200 μm . B , C Quantification of surface area covered by the PSR staining or α - SMA in control ( Cnt ) and Sox9 - null ( Null ) in ( A ) . D PSR staining ( red ; top row ) and immunohistochemistry for α - SMA ( brown ; middle row ) and CK19 ( brown ; bottom row ) in control and Sox9 - null mice following sham operation ( left ) or BDL induced fibrosis ( right ) . Size bar = 500 μm . E , F Quantification of surface area covered by the PSR staining or α - SMA in ( D ) . G , H Liver function is improved in Sox9 - null mice following CCl4 ( G ) or BDL ( H ) induced fibrosis compared to control mice . Reduction in serum alanine aminotransferase ( ALT ; G , H ) and bilirubin ( H ) down to levels shown in non - fibrotic mice ( olive oil treated groups ; Oil ) . G , H Liver function is improved in Sox9 - null mice following CCl4 ( G ) or BDL ( H ) induced fibrosis compared to control mice . Reduction in serum alanine aminotransferase ( ALT ; G , H ) and bilirubin ( H ) down to levels shown in non - fibrotic mice ( olive oil treated groups ; Oil ) . I Sox9 - loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4 . Olive oil treated mice had no bridging fibrosis , in line with a non - fibrotic liver histology in ( A ) . J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in ( D ) is reduced in Sox9 - null mice compared to control following BDL . All mice were treated with tamoxifen ( Tam ) which did not induce ectopic expression of SOX9 in non - fibrotic livers ( Figure 2A , E and Appendix Figure S3 ) . All experiments are n ≥ 5 as indicated . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A-J Representative images and quantification shown for olive oil treated (n=6) or chronic CCl4 (n=5 Sox9fl/fl;RosaCreER-; n=8 Sox9fl/fl;RosaCreER+) induced fibrosis or following sham operation (n=5) or BDL (n=7 Sox9fl/fl;RosaCreER-; n=5 Sox9fl/fl;RosaCreER+)) induced fibrosis. A Picrosirius red (PSR) staining (collagen deposition in red) counterstained with fast green (top row) and immunohistochemistry for α-SMA (brown staining bottom row; activated HSC / myofibroblast marker) in olive oil treated (left) or chronic CCl4 induced fibrosis (right) in control and Sox9-null mice. Size bar = 200 μm. B, C Quantification of surface area covered by the PSR staining or α-SMA in control (Cnt) and Sox9-null (Null) in (A).D PSR staining (red; top row) and immunohistochemistry for α-SMA (brown; middle row) and CK19 (brown; bottom row) in control and Sox9-null mice following sham operation (left) or BDL induced fibrosis (right). Size bar = 500 μm.E, F Quantification of surface area covered by the PSR staining or α-SMA in (D).G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).I Sox9-loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4. Olive oil treated mice had no bridging fibrosis, in line with a non-fibrotic liver histology in (A).J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in (D) is reduced in Sox9-null mice compared to control following BDL. All mice were treated with tamoxifen (Tam) which did not induce ectopic expression of SOX9 in non-fibrotic livers (Figure 2A, E and Appendix Figure S3). All experiments are n≥5 as indicated. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated.


Figure 4A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . D , E . Quantification of surface area covered by the PSR staining in ( C ) . D , E . Quantification of surface area covered by the PSR staining in ( C ) . F Expression of SOX9 protein by immunohistochemistry in quiescent ( Q ) and activated ( A ) HSCs extracted from Sox9fl / fl ; AlbCre + mice . G Individual fluorescent channels showing localization and expression of α - SMA ( left panel ) , SOX9 ( middle panel ) and composite image for α - SMA ( green ) and SOX9 ( red ) in right panel in activated Sox9fl / fl ; AlbCre + HSCs ( genotyping shown in Appendix Figure S9 ) . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . Size bars = 100 μm ( A ) , 10 μm ( B ) 200 μm ( C ) , 25 μm ( G ) .

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

Figure 4A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).D, E. Quantification of surface area covered by the PSR staining in (C).D, E. Quantification of surface area covered by the PSR staining in (C).F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice.G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).


Figure 5A , B Flow cytometry of CD45 + CD11B + cells in control ( Sox9fl / fl ; RosaCre - ; A ) and Sox9 - null ( Sox9fl / fl ; RosaCre + ; B ) livers stained for Ly6C and MHCII ( far left ) to identify four populations of cells with differences in their expression of MerTK , CD64 , F4 / 80 and MHCII . C Graphical representation of Ly6C and F4 / 80 staining in macrophages from population 3 and 4 . D , E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9 - null livers . Data are representative of four independent experiments . F F4 / 80 immunohistochemistry ( brown staining ) in control and Sox9 - null mice livers following 4 - weeks CCl4 induced fibrosis . Size bar = 100μm . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values indicated . All experiments are n = 4 .

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Figure 5A, B Flow cytometry of CD45+CD11B+ cells in control (Sox9fl/fl;RosaCre-; A) and Sox9-null (Sox9fl/fl;RosaCre+; B) livers stained for Ly6C and MHCII (far left) to identify four populations of cells with differences in their expression of MerTK, CD64, F4/80 and MHCII.C Graphical representation of Ly6C and F4/80 staining in macrophages from population 3 and 4.D, E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9-null livers. Data are representative of four independent experiments.F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.


Figure 6 A , B Immunofluorescence of activated mouse HSCs cultured on plastic ( A ) or hydrogels ( A , B ) . Individual fluorescent channels showing localization and expression of SOX9 ( left ) , YAP ( middle ) and composite image for SOX9 ( red ) and YAP ( green ) in right panel . ( B ) Individual and composite images for αSMA ( green ) and TAZ ( red ) or TEAD ( red ) . Size bar , 25μm . C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin ( VP ) in activated mouse HSCs expressed relative to DMSO control ( n = 3 ) . D Immunohistochemistry for SOX9 ( brown ) following CCl4 or BDL induced fibrosis in mice treated with DMSO ( CCl4 n = 4 , BDL n = 7 ) or verteporfin ( CCl4 n = 3 , BDL n = 5 ) . E , F quantification of SOX9 cell numbers in the HSC / Hep population . Size bar , 50 μm . E , F quantification of SOX9 cell numbers in the HSC / Hep population . Size bar , 50 μm .

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Figure 6 A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm. C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.


Figure 1 ( C ) Survival curves after the injection of EL4 lymphoma cells ( n = 6 / group ) . ( H ) Ifng protein and Ifng mRNA levels ( n = 4 / group ) were determined by ELISA and quantitative PCR .

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Figure 1(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.


Figure 2 ( A ) m6A dot blot assay using total RNA from NK92 - siCTRL or NK92 - siFTO cells . ( D ) Western blotting for the detection of GZMB in NK92 - siCTRL and NK92 - siFTO cells after treatment with IL - 2 . ( E ) Levels of IFNγ ( n = 5 biological replicates ) and TNFα ( n = 3 biological replicates ) in the culture media 24 hours after IL - 2 treatment ( left ) or co - culture media with K562 cells for 4 hours ( right ) were determined by ELISA .

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Figure 2(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.


Figure 3 ( B ) Western blotting analysis . NK92 - siCTRL or NK92 - siFTO cells were incubated with 20 ng / ml IL - 2 for 24 hours , washed , and cultured without IL - 2 ( IL - 2 depletion ) for the indicated times ( n = 3 ) . ( C ) A western blotting analysis of NK92 + MOCK or NK92 + FTO cells incubated with 20 ng / ml IL - 2 for the indicated times was performed ( n = 3 ) .

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Figure 3(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3). (C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3). D


Figure 4 ( C ) m6A methylated RNA immunoprecipitation & quantitative PCR ( MeRIP - qPCR ) analysis using a m6A antibody in NK92 - siFTO ( upper ) or NK92 + FTO ( lower ) cells compared to NK92 - siCTRL or NK92 + MOCK cells . ( D ) SOCS3 3 ' - UTR reporter assay using the AANAT reporter system . Plasmid information for the AANAT reporter system with or without the SOCS3 3 ' - UTR sequence ( upper ) . Levels of Aanat protein ( left ) and Aanat mRNA ( right ) expression in HEK - 293T cells co - transfected with plasmid - expressing AANAT reporter containing SOCS3 3 ' - UTR and siCTRL or siFTO .

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Figure 4(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.


Figure 5 ( B ) Relative mRNA expression levels of JAK / STAT target genes , IFNγ ( n = 10 biological replicates ) , GZMB ( n = 6 biological replicates ) and PRF1 ( n = 8 biological replicates ) were determined by qPCR in CD56 + - siCTRL or CD56 + - siFTO cells . ( C ) Western blotting of CD56 + - siCTRL or CD56 + - siFTO cells incubated with 20 ng / ml IL - 15 and 20 ng / ml IL - 21 for the indicated times was performed . Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated ( p - ) and total proteins ( n = 2 ~ 3 ) . Data are representative of three times independent experiments with similar results . The protein levels were expressed as the relative band density of the corresponding protein ( lower ) .

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Figure 5(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).


Figure 6 ( E ) The images of the ventral bioluminescence ( BLI ) following the injection of NK92 - TRC or NK92 - shFTO cells . ( G ) The survival curves after the injection of K562 - Luc cells .

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Figure 6(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.(G) The survival curves after the injection of K562-Luc cells.


Figure 1Kaplan - Meier analysis of overall survival in breast cancer patients with recurrent lethal disease , classified according to p - JNK expression . p - JNK was determined by immunostaining of breast cancer samples in a tissue microarray ; p - JNK low , n = 31 ; p - JNK high , n = 10 . P value and hazard ratio ( HR ) were calculated by log - rank test . Immunostaining of p - c - Jun ( arrows ) in patient - matched breast tumor and lung metastasis . Scale bar , 50 μm . Quantification of p - c - Jun positive ( p - c - Jun + ) cells per tumor area in matched primary tumors and lung metastases . Boxes depict median from 12 patients with upper and lower quartiles . Quantification of p - c - Jun positive ( p - c - Jun + ) cancer cells within mammary tumors and matched metastases in mice injected with MDA231 - LM2 cells . Immunofluorescence analysis of p - c - Jun in micrometastatic ( day 7 ) and macrometastatic ( day 21 ) nodules in a xenograft mouse model injected intravenously with MDA231 - LM2 cells . DAPI was used to stain cell nuclei , and cancer cells express GFP as a marker . Scale bar , 50 μm . Quantification of p - c - Jun + cancer cellsMammary tumor growth curves from mice described in panel G . Each value represents the mean ± SEM . For MDA231 - LM2 , n = 12 tumors in 6 mice for each experimental group . For SUM159 - LM1 , n = 16 tumors in 8 mice for each experimental group . Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 tumor - bearing mice treated with JNKi . Quantification of lung metastases . FOV , field of view ; LS , lung section . Each value represents the mean ± SEM . For metastatic foci per FOV , 8 - 10 fields were quantified per mouse . MDA231 - LM2 tumor - bearing mice : n = 6 mice per group . SUM159 - LM1 tumor - bearing mice , vehicle n = 8 mice ; JNKi n = 7 mice . Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 tumor - bearing mice treated with JNKi . Representative histology examples of metastasis in lungs , detected by expression of human vimentin . Scale bar , 100 μm . Metastatic burden determined by photon flux in lungs of MDA231 - LM2 - injected NSG mice treated with vehicle and JNKi . Boxes show the median with upper and lower quartiles . Whiskers represent minimum and maximum values . Vehicle control , n = 8 ; JNKi - treated , n = 8 . Examples of bioluminescence in

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Figure 1Kaplan-Meier analysis of overall survival in breast cancer patients with recurrent lethal disease, classified according to p-JNK expression. p-JNK was determined by immunostaining of breast cancer samples in a tissue microarray; p-JNK low, n = 31; p-JNK high, n = 10. P value and hazard ratio (HR) were calculated by log-rank test.Immunostaining of p-c-Jun (arrows) in patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.Quantification of p-c-Jun positive (p-c-Jun+) cells per tumor area in matched primary tumors and lung metastases. Boxes depict median from 12 patients with upper and lower quartiles.Quantification of p-c-Jun positive (p-c-Jun+) cancer cells within mammary tumors and matched metastases in mice injected with MDA231-LM2 cells.Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm. Quantification of p-c-Jun+ cancer cellsMammary tumor growth curves from mice described in panel G. Each value represents the mean ± SEM. For MDA231-LM2, n = 12 tumors in 6 mice for each experimental group. For SUM159-LM1, n = 16 tumors in 8 mice for each experimental group.Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Quantification of lung metastases. FOV, field of view; LS, lung section. Each value represents the mean ± SEM. For metastatic foci per FOV, 8-10 fields were quantified per mouse. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice, vehicle n = 8 mice; JNKi n = 7 mice.Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Representative histology examples of metastasis in lungs, detected by expression of human vimentin. Scale bar, 100 μm.Metastatic burden determined by photon flux in lungs of MDA231-LM2-injected NSG mice treated with vehicle and JNKi. Boxes show the median with upper and lower quartiles. Whiskers represent minimum and maximum values. Vehicle control, n = 8; JNKi-treated, n = 8. Examples of bioluminescence in mice


Figure 2Scatter plot elucidating fold change ( FC ) in gene expression between samples treated with JNKi compared to vehicle ( Y - axis ) and expression of constitutively active JNK ( MKK7 - JNK ) versus vector control ( X - axis ) from biological triplicates . Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red . GSEA graphs showing ( C ) enrichment of a mammary stem cell signature ( Pece et al , 2010 ) in MDA231 - LM2 cancer cells expressing MKK7 - JNK NES , normalized enrichment score . GSEA graphs showing under - representation of the same mammary stem cell signature in cancer cells treated with JNKi . NES , normalized enrichment score . GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231 - LM2 cells either expressing MKK7 - JNK or treated with JNKi . Mammary stem cell signatures are indicated as follows : a , up in mammary stem cells b , up in fetal mammary stem cells ; c , up in adult mammary stem cells and d , up in mammary repopulating unitsHeat map of the JNK signature composed of 68 genes that are induced by expression of MKK7 - JNK and repressed by JNKi , Proportion of JNK signature - positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK - S , JNK signature as in panel F . Subtype patient numbers : Luminal , n = 1213 ; HER2 , n = 240 ; Basal - like , n = 331 .

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Figure 2Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red.GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score.GSEA graphs showing under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score.GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating unitsHeat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi,Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.


Figure 3Gene expression profile of MDA231 - LM2 cells grown as oncospheres or as a monolayer . Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red . GSEA graph showing enrichment of JNK signature in MDA231 - LM2 cancer cells grown as oncospheres . NES , normalized enrichment score . Immunoblot of phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) and c - Jun in oncospheres from SUM159 - LM1 cells . Tubulin was included as a loading control . GSEA of a basal - like breast cancer signature induced in MDA231 - LM2 oncospheres . Normalized enrichment score ( GSEA ) of mammary stem cell signatures in oncospheres from MDA231 - LM2 cells . Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK . Values are mean from 10 wells per group ± SD . Oncosphere formation in SUM159 - LM1 cells following treatment with JNKi or vehicle control . Values are mean from 10 wells per condition ± SD . Representative images of spheresOncosphere formation in JNKi - treated cancer cells from breast cancer patients ( pleural effusions or ascites ) . ( I ) Quantification of oncospheres per well in 4 different patients . Values are mean from 10 wells ± SD . ( J ) Representative oncospheres from patient samples . Scale bar , 50 μm . Matrigel invasion assay of MDA231 - LM2 cells treated with JNKi . Left , examples of invading cancer cells stained with crystal violet ( arrows ) . Scale bar , 50 μm . Right , quantification of invading cells per field of view ( FOV ) showing mean from 8 fields ± SEM .

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Figure 3Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red.GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score.Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control.GSEA of a basal-like breast cancer signature induced in MDA231-LM2 oncospheres.Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells.Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD.Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheresOncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm.Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.


Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi . SPP1 and TNC expression in MDA231 - LM2 cells transduced with pLVX - puro lentivirus without a transgene ( control ) or expressing constitutively - active JNK ( MKK7 - JNK ) or mutated inactive JNK ( MKK7 - JNK ( mut ) ) . SPP1 and TNC expression in pleural effusion ( 1 and 2 ) and ascites ( 3 and 4 ) samples from four different patients treated with JNKi ( 4 μM , 48h ) in culture . Values are mean ± SD from triplicates . The results from all four patient samples were included to calculate significance of JNKi on gene expression . SPP1 , P = 0 . 0079 ; TNC , P = 0 . 0002 . Analysis of SPP1 and TNC mRNA levels in SUM159 - LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi . For panels A - D , values represent the mean of qPCR triplicates ± SD . ChIP analysis of c - Jun binding to sequences in SPP1 or TNC promoters . Analysis of SPP1 and TNC promoter fragments pulled down by c - Jun and IgG antibodies . Values represent the mean of qPCR triplicates ± SD . Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients ( GSE14020 ) . N = 65 metastasis samples . Lung metastasis in Spp1 + / - mice implanted with MDA231 - LM2 cells expressing control shRNA ( control ) , and Spp1 - / - mice implanted with MDA231 - LM2 cells independently expressing one of two different SPP1 shRNAs ( SPP1 - deficient ) . Ex vivo lung luminescence was analyzed , panel H . Boxes show the median value with upper and lower quartiles . Whiskers represent minimum and maximum values . Control n = 16 mice , SPP1 deficient ( 1 ) n = 14 mice and SPP1 deficient ( 2 ) n = 15 mice . P values were determined by a two - tailed Mann - Whitney test . ( I ) Representative histological images of metastatic foci ( top ) and ex vivo lung bioluminescence ( bottom ) . In histological samples , immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin ( arrows ) . Scale bar , 100 µm . SUM159 - LM1 cells expressing either control shRNA ( control ) , or one of two different TNC shRNAs ( shTNC 1 and 2 ) , were injected intravenously into NSG mice . ( J ) Metastatic colonization was quantified by lung bioluminescence . Control and shTNC ( 1 ) n = 8 mice per group , shTNC ( 2 ) n = 7 mice . P value was determined by a two - tailed Mann - Whitney test . ( K ) Representative images of metastatic foci ( top ) and ex vivo lung bioluminescence ( bottom ) . Arrows denote vimentin - expressing cancer cells in metastases . Kaplan - Meier analysis of breast cancer patients ( combined GSE2603 and GSE5327 datasets ) showing a link between high SPP1 and TNC expression and poor lung metastasis - free survival . Patient samples , SPP1 and TNC low n = 70 ; SPP1 and TNC high n = 70 . HR , Hazard ratio .

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Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi.SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut)).SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002.Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD.ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD.Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples.Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm.SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases.Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.


Figure 5Gene expression profile of MDA231 - LM2 cells treated with paclitaxel ( PAX ) for 48 hours . Highlighted are examples of JNK signature genes that are induced by PAX treatment . GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231 - LM2 cells . Immunofluorescence analysis of p - c - Jun + cancer cells in lung metastases from mice injected intravenously with MDA231 - LM2 cells and treated with PAX at week 3 post injection . Lungs were harvested 72h after PAX treatment . Cancer cells express GFP as a marker . DAPI was used for nuclear staining . Scale bar , 50 µm . Quantification of cancer cells expressing p - c - Jun in panel D . Values are mean from three mice ± SEM . Nine metastatic foci were analyzed per mouse . Western blot analyzing phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) and c - Jun in MDA231 - LM2 cells treated with PAX for 48 hours . Tubulin was used as a loading control . SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD . SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of doxorubicin ( DOXO ) . qPCR triplicates ± SD . SPP1 and TNC expression in SUM159 - LM1 cancer cells treated with 4 μM 5 - Fluorouracil or 4 μM methotrexate . Cells were treated for 48 hours . qPCR triplicates ± SD . SPP1 and TNC expression in SUM159 - LM1 cells treated with paclitaxel and JNKi . qPCR triplicates ± SD . SPP1 and TNC repression by JNKi , P < 0 . 05 for both vehicle and PAX - treated cells .

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Figure 5Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse.Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.


Figure 6Growth curves of mammary tumors in vehicle - or PAX - treated Spp1 + / - mice implanted with the indicated cells expressing control shRNA , and Spp1 - / - mice implanted with the indicated cells expressing SPP1 shRNA . MDA231 - LM2 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 14 tumors . SUM159 - LM1 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 15 tumors . Quantification of lung metastasis from the animals described in panels A and B . The number of metastatic foci per field of view ( FOV ) or per lung section ( LS ) was determined . MDA231 - LM2 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice . SUM159 - LM1 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice . Tumor weight in mice at day 40 after implantation of MDA231 - LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin ( Adriamycin ) and cyclophosphamide ( AC regimen ) . Control , AC and SPP1 def . n = 16 tumors per group , SPP1 def . + AC n = 14 tumors . Lung metastasis was quantified in mice that were implanted with MDA231 - LM2 tumors and subjected to AC chemotherapy . Each value represents the mean ± SEM ; n = 5 mice per group . Ten random fields per lung section were analyzed for percentage of metastatic area . Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E . Metastases were determined by expression of human vimentin ( arrows ) . Scale bar , 100 µm . Mammary tumor growth curves in PAX - treated NSG mice bearing control or TNC knockdown MDA231 - LM2 tumors . Values are means ± SEM , n = 16 tumors per group . * * * * P < 0 . 0001 . Lung metastases quantified in tumor bearing miceRepresentative histology of human vimentin - positive lung metastases ( arrows )

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Figure 6Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area. Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing miceRepresentative histology of human vimentin-positive lung metastases (arrows)


Figure 7Mammary tumor growth curves in NSG mice implanted with MDA231 - LM2 or SUM159 - LM1 cells and treated with paclitaxel as single agent or in combination with JNKi . Each value represents the mean ± SEM . MDA231 - LM2 : n = 12 tumors per group . SUM159 - LM1 : vehicle , JNKi and PAX n = 12 tumors per group ; PAX + JNKi n = 10 tumors . * * P < 0 . 01 , * * * P < 0 . 001 . Metastatic burden in lungs of MDA231 - LM2 and SUM159 - LM1 tumor - bearing mice treated with paclitaxel as single therapy or in combination with JNKi . Each value represents the mean ± SEM . MDA231 - LM2 tumor - bearing mice : n = 6 mice per group . SUM159 - LM1 tumor - bearing mice : vehicle , JNKi and PAX n = 6 mice per group ; PAX + JNKi n = 5 . Ten fields were quantified per lung section . FOV , field of view , LS , lung section . PAX , paclitaxel . Representative histological images of metastatic foci ( arrows ) in lungs of the MDA231 - LM2 - implanted mice described in panel C , stained for human vimentin expression in differentially treated groups . Scale bar , 100 μm . Apoptosis was determined by quantification of TUNEL - positive cells per field of view ( FOV ) in MDA231 - LM2 tumors ( from the animals described in ( C ) ) treated with JNKi and / or PAX . Values are means from 6 mice per condition ± SEM . GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial ( GSE16446 ) , stratified according to pathological response to neoadjuvant chemotherapy . NES , normalized enrichment score . pCR , pathological complete response . Kaplan - Meier analyses of chemotherapy - treated breast cancer patients associating JNK signature ( JNK - S ) with metastasis - free survival ( TOP trial data set , JNK - S low n = 54 patients ; JNK - S high n = 53 patients ( G ) ) or overall survival ( METABRIC data set , JNK - S low n = 135 patients ; JNK - S high n = 135 patients ( H ) ) . HR , hazard ratio . P values were determined by log - rank test .

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Figure 7Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response.Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.


Figure 1 ( E ) . Relative luciferase activity of the sensor ( + ) reporter or sensor ( - ) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293 - FT cells . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( F ) . Heat map of relative miRNA expression of various miRNAs in HEK - 293FT cells transfected with expression vectors of the top 5 genes from the screen , or the ctrl ( GFP ) vector . Red color represents suppressed expression compared with ctrl ( GFP ) . n = 3 .

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

Figure 1(E). Relative luciferase activity of the sensor (+) reporter or sensor (-) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293-FT cells. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.(F). Heat map of relative miRNA expression of various miRNAs in HEK-293FT cells transfected with expression vectors of the top 5 genes from the screen, or the ctrl (GFP) vector. Red color represents suppressed expression compared with ctrl (GFP). n=3.


Figure 2 ( A ) . Volcano plot of TaqMan array showing the mean average expression and p - value of miRNA expression profiles between TruB1 KD and ctrl ( scramble ) . N = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test . MiRNAs suppressed ( Relative expression TruB1 KD / ctrl < 0 . 8 and p - value < 0 . 1 ) are enclosed by the red square . ( D ) . Relative expression of let - 7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl ( siRNA ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD . ( E ) . Relative expression of primary - let - 7 family miRNAs as in ( D ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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

Figure 2(A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. Significance was assessed using 2‐tailed Student's t‐test. MiRNAs suppressed (Relative expression TruB1 KD / ctrl <0.8 and p-value < 0.1) are enclosed by the red square.(D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.(E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.