Chromatin IP analysis of binding of Glis2 to Stat3 promoter ( see Appendix Fig S9D ) in the absence or presence of SHH at different concentration as indicated on triplicated samples , using specific anti - Glis2 antibodies and rabbit IgG as control .

Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control.


A ) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks ( GW ) , 10 GW , and 12 GW and in the adult human thyroid : quantitative PCR results normalized to one of three thyroid tissues at 8 GW value ( in black ) and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus 8 GW ( in black ) using ANOVA test showed no significant

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A) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences


B ) Tubb1 is expressed in the developing mouse thyroid at E13 . 5 , E15 . 5 , and E17 . 5 and in the adult mouse thyroid : quantitative PCR results normalized to one of three thyroid tissues at E13 . 5 value and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus E13 . 5 ( black ) , * * p < 0 . 01 using ANOVA

B) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA test


C ) Sorted mouse thyroid cells , Tubb1 expression in endothelial cells ( Pecam - positive cells ) , epithelial cells ( Epcam ) , fibroblasts ( Pdgfra ) , and leucocytes ( CD45 ) at E17 . 5 and adulthood : quantitative PCR results ( normalized to Epcam - positive cells at adult stage and peptidylprolyl isomerase A ) . Mean ± SEM of three independent cell - sorting experiments . Statistical comparisons versus epithelial cells ( in black ) at each stage , * p < 0 . 05 using ANOVA

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C) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA test


D ) Immunohistofluorescence of β1 - tubulin ( in red ) , thyroglobulin ( TG , in green ) , and both merged ( from left to right ) at 12 GW in human thyroid tissue . The boxes delineate the enlarged areas shown

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D) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown below


F ) Localization of transfected Myc - tagged wild - type and mutant ( P160L ) β1 - tubulin in Nthy cells . Immunostaining with anti - Myc antibody ( in green ) and microtubule cytoskeleton stained with α - tubulin antibody ( in red ) . Note that β1 - tubulin - P160L is found throughout the cytoplasm showing puncta appearance , whereas wild - type β1 - tubulin colocalizes with the

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F) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules


A ) Thyroid morphology was investigated using Nkx2 - 1 staining at E9 . 5 and E11 . 5 ( sagittal sections ) and at E13 . 5 , E15 . 5 , and E17 . 5 ( transverse sections ) in wild - type ( wt ) and Tubb1 - / - littermates . Nkx2 - 1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes . Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11 . 5 and E13 . 5 ( arrow ) , respectively , in Tubb1 - / - mice . At E11 . 5 , stained migrating thyroid cells were visible along the tract only in Tubb1 - / - littermates ( arrow ) . At E17 . 5 , a pyramidal lobe was visible in Tubb1 - / - littermates ( arrow ) . tr : trachea ; ub : ultimobranchial body . Scale bar : 50

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A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ


B ) Total thyroid surface area ( μm2 ) was quantified using Nkx2 - 1 staining at each embryonic stage . Top : thyroid surface area normalised for weight of each embryo . Bottom : thyroid surface area in μm2 . Right : proliferation ratio calculated as the proportion of Nkx2 - 1 - positive cells labelled with Ki67 at E9 . 5 in wt and Tubb1 - / - thyroid anlages . ma : median anlage . Note that the thyroid ma is larger and the proliferation ratio higher at E . 9 . 5 in Tubb1 - / - mice , whereas from E13 . 5 onwards the Tubb1 - / - thyroids were hypoplastic . Wt in black , KO , Tubb1 - / - in

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B) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in gre


C ) Thyroid marker expression by quantitative PCR in thyroid tissue at E15 . 5 and E17 . 5 in Tubb1 - / - versus wt mice normalised for peptidylprolyl isomerase A . Note the decreases in thyroid differentiation markers at E15 . 5 and E17 . 5 and also in thyroid transcription factors at E17 .

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C) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5


D ) Staining of Nkx2 - 1 and T4 at E17 . 5 and surface area quantification demonstrating T4 retention in Tubb1 - / - versus wt thyroids . The data are the percentage of T4 or calcitonin ( CT ) surface area versus total thyroid area ( estimated from the Nkx2 - 1 - stained surface area ) . Scale bar : 50 μm . Student " s test , * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . Experiments were done on at least three tissues per stage for each

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D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen


A ) Serum TSH and T4 levels in 3 - month - old Tubb1 - / - and wild - type mice . Numbers of animals tested were 10 wild - type males and 14 Tubb1 - / - males for TSH and for 14 wild - type and 22 Tubb1 - / - males for T4 . Tubb1 - / - mice had hypothyroidism with elevated TSH and decreased T4 versus wild - type mice . Student " s test , * * p <

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A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<


B ) Nkx2 - 1 ( in brown ) immunostaining of adult thyroid tissue . Note the disorganisation of the thyroid tissue in Tubb1 - / - versus wild - type

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B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type mice


C ) Ultrastructural alterations shown by electron microscopy in wild - type and Tubb1 - / - thyroid tissues . In Tubb1 - / - tissues , note the disorganisation of secretion vesicles ( white asterisks ) and rods of identical density to secretion vesicles ( white arrow ) . The ER is considerably dilated in Tubb1 - / - thyrocytes . Representative views . Scale bars at the bottom left for each view . Co :

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C) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloi


D ) ER stress in Tubb1 - / - thyroid tissue . Top : Chop and XBPs expression by quantitative PCR in adults , normalized to peptidylprolyl isomerase A . Experiments with at least three tissues per stage for each genotype ; wt in black and Tubb1 - / - in grey . Bottom : Chop and Actin protein expression by western blotting in three representative wild - type and Tubb1 - / - mice . The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1 - / - versus wild - type thyroids . All lanes are from the same blot , which was cut where indicated . Student " s test , * p < 0 . 05 and * * * p <

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D) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<


E ) Co - immunostaining of endoplasmic reticulum ( ER ) marker ( KDEL , in red ) , thyroglobulin ( Tg in green ) , and both merged ( from left to right ) in adult thyroid tissue . Note the thyrocyte disorganisation in Tubb1 - / -

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E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice


A ) Representative blood smears of patients with TUBB1 mutations ( P1 and P3 homozygous for p . P160L , P4 heterozygous for p . Y106X , and P6 and P7 heterozygous for c . 35delG ) after May - Grünwald Giemsa staining . Variable platelet size in patients was observed with all three mutations with presence of macroplatelets ( arrows )

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A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)


B ) Transmission electron microscopy ( TEM ) analysis of platelet ultrastructure in the patients . The results confirm the abnormal platelet

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B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphology


C ) Representative images of proplatelet formation in P1 and P4 . Cultured control and patient megakaryocytes ( MKs ) after thrombopoietin - induced differentiation were spread over a BSA - coated coverslip on day 10 . On day 13 or 14 , MKs were fixed ( 4 % paraformaldehyde ) and proplatelet structure was observed after β - tubulin staining . Scale bars : 20 μm . Only P1 and P4 could be studied . The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions ( left ) and the diameter of coiled elements ( right ) of the future proplatelets , both of which were significantly increased in the patients , indicating that the TUBB1 mutations ( homozygous p . P160L and heterozygous p . Y106X ) affected proplatelet morphology . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * * p < 0 . 001 ; 20 - 60 MKs were analysed /

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C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat


A ) Relative expression of α - tubulin and β1 - tubulin in platelets from P1 and P3 ( homozygous p . P160L ) , P4 ( heterozygous p . Y106X ) , and P6 and P7 ( heterozygous c . 35delG ) was quantified by Western blotting using specific antibodies against α - tubulin or β1 - tubulin . With both mutations , α - tubulin expression was normal but β1 - tubulin expression was significantly decreased . The β1 - tubulin antibody recognizes C - terminal

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A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domain


B ) αIIbβ3 integrin activation was evaluated by flow cytometry ( % of positive platelets and mean fluorescence intensity ) in whole blood from P1 and P3 ( homozygous p . P160L ) and from P6 and P7 ( heterozygous c . 35delG ) ; P4 could not be studied . While no αIIbβ3 integrin activations were detected in P6 and P7 ( family 3 ) , P1 and P3 ( family 1 ) exhibited a significant increase of αIIbβ3 activation , indicating basal platelet

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B) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activation


C ) Aggregation of washed platelets induced by ADP ( 5 or 10 μM ) or collagen ( 0 . 6 , 0 . 8 or 2 μg / mL ) was evaluated in at least one member of each family . Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls , suggesting that TUBB1 affected by homozygous p . P160L and heterozygous p . Y106X mutations affected platelet function . Platelet aggregation to both agonists was normal in both patients from family 3 ( heterozygous c . 35delG ) . Family F1 , n = 3 independent experiments ; Family F2 , n = 1 experiment ; Family F3 , n = 2 independent experiments . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , nd : statistical significance not determined

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C) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because


( A ) Srcap expression in murine different tissues was examined by Northern blot . A 277 nt probe of Srcap ( 81 - 358nt ) was labeled for Northern blot analysis . 18S RNA was used as a loading control .

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(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.


( B ) Two - week - old SRCAP - C - HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining . A global look of the section was shown . Scale bar : 500 μm . Green : EpCAM , Red : HA tag , Nuclei were counterstained by DAPI .

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(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.


( C ) Murine intestine tissues were fixed and stained according to iDISCO staining protocol . SRCAP was highly expressed in vast majority of crypts in mouse intestine . Scale bar : 50 μm . Green : EpCAM , Red : SRCAP , Nuclei were counterstained by DAPI .

(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.


( D ) Duodenum region of intestinal tissues were obtained from Lgr5GFP - CreERT2 ( Lgr5GFP ) mice for immunofluorescence staining by indicated antibodies with OpalTM 7 - color fIHC kit . Nuclei were counterstained with DAPI . Scale bar : 50 μm .

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(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.


( E ) ISCs from Lgr5GFP mice were collected for organoid formation . Organoids were fixed for immunofluorescence staining with indicated antibodies . Scale bar : 50 μm . Data information : Green : EpCAM , Red : SRCAP , Purple : PCNA , Nuclei were counterstained by DAPI .

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(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.


( A ) Representative images of intestines from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) was measured and shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi .

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(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) Intestinal tissues from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) were collected for immunofluorescence staining with indicated antibodies . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm .

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(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( C ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( D ) Srcap + / + and Srcap - / - mice were treated with 10 Gy ' s radiation and sacrificed at different time points . Intestinal tissues were collected for H & E staining . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 100 μm .

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(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - YFP ( LRYFP ) were crossed with Srcapflox / flox mice , followed by administration of tamoxifen ( TAM ) for lineage tracing analysis . Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained . Numbers of traced crypt - villus units were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 50 μm .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.


( F ) Proliferation of Srcap + / + and Srcap - / - ISCs were examined by Ki67 staining followed by flow cytometry analysis . Percentages of proliferated cells were counted as means ± S . D ( lower panel ) . * * P < 0 . 01 by two - tailed Student ' s t - test . n = 5 per group .

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(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.


( G ) Multiple color staining was performed in Srcap + / + and Srcap - / - intestine sections . AP - Red , DAB and AP - Blue were used for immunohistochemistray coloration . Numbers of each kind of epithelial cells from 50 crypts or 50 villi ( 10 crypts and 10 villi per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bars , 50 μm .

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(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.


( A ) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti - SRCAP antibody or rabbit IgG followed by ADASDS - PAGE , sliver staining and mass spectrometry .

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(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.


( B ) SRCAP and REST were stained in murine intestinal crypts . Scale bar , 10 μm .

(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.


( D ) Flag - tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti - Flag antibody . REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting .

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(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.


( E ) C - terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay .

(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.


( F ) Intestinal crypt lysates were incubated with anti - REST antibody , followed by immunoblotting with indicated antibodies .

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(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.


( G ) Intestinal crypt lysates were incubated with anti - REST antibody and protein A / G beads in 4 ℃ overnight . Recombinant SRCAP protein was added with different concentrations , followed by immunoblotting .

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(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.


( B ) Transcriptome microarray analysis between Srcap - / - ISCs and WT ISCs . Top ten downregulated TFs in Srcap - / - ISCs were listed .

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(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.


( C ) Prdm16 expression was tested in Srcap + / + and Srcap - / - ISCs by Western blotting .

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(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.


( D ) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining . Green : GFP ; Red : SRCAP ; Blue : Prdm16 . Scale bar , 10 μm .

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(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.


( E ) Prdm16 promoter ( - 875 ~ - 854 ) region was used as probe to perform DNA - pulldown assay with murine intestinal crypt lysates followed by immunoblotting .

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(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.


( F , G ) Indicated promoter activation was analyzed with luciferase assay . DKK1 was used as a control . Results of relative fold changes are shown as means ± S . D . Data represent five independent replicates . . * * p < 0 . 01 by two - tailed Student ' s t - test . .

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(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .


( H ) 1 × 104 ISCs from indicated mice were collected for organoid formation . For gene depletion or restoration , ISCs were infected with lentivirus . Scale bar , 200 μm . Data information : All data represent five independent experiments .

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(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.


( A ) WT and Srcap - / - ISCs were conducted for transcriptome microarray , followed by Gene Set Enrichment Analysis ( GSEA ) analysis for PPARδ target genes .

(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.


( B ) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.


( C ) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining . Scale bar , 50 μm .

(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.


( D ) ISCs were collected for organoid formation followed by quantitative RT - PCR . For Prdm16 overexpression , ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.


( E ) mRNA levels of ISC signature genes were examined by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates .

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(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.


( F ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test . ( lower panel ) . n = 6 for each group . GW501516 ( 10 μM ) were added directly into organoid culture medium .

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(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.


( A ) Representative images of intestines from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) were measured in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi .

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(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( C ) Intestine tissues from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) were collected for immunohistochemistry staining . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm .

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(C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( D ) Immunofluorescence staining of intestine tissues from Srcapfl / fl ; Lgr5GFP - CreERT2 and Restfl / fl ; Lgr5GFP - CreERT2 mice . Scale bar , 50 μm .

(D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - lacZ ( LRlacZ ) were crossed with Rest - / - mice , followed by TAM administration for lineage tracing for intestinal whole - mount staining for β - gal ( upper panel ) . Mice were sacrificed after one week and typical sections were shown ( middle panel ) . Scale bar , 200 μm . Numbers of traced crypt - villus units were calculated as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . 50 fields ( 10 fields per mouse ) were calculated for each group ( n = 5 ) ( lower panel ) .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).


( F ) Rest + / + and Rest - / - mice were treated with 10 Gy ' s radiation and sacrificed at the indicated time points . Intestinal tissues were collected for H & E staining . Scale bars , 100 μm . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) .

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(F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5).


( G ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . n = 6 for each group . Organoid number per well was counted as means ± S . D . ( lower panel ) . Lentivirus - carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM - treated LRCas9 mice to obtain Prdm16 - / - and Ppard - / - ISCs . 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 5 for each group .

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(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.


A , B Quantification of mitotic ( A ) and meiotic ( B ) cells in the distal germline in wild - type animals fed bacteria transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) Data information : Data are presented as mean ± SE ( A ) N = 3 at least 5 worms per replicate and condition , ( B ) N = 3 at least 10 worms for conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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A, B Quantification of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) Data information: Data are presented as mean ± SE (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained either anti - phosphohistone H3 ( anti - PH3 ) ( C relative to the total number of mitotic cells of wild - type animal Data informatio Data are presented a as mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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Quantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C relative to the total number of mitotic cells of wild-type animal Data informatio Data are presented a as mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained eithe anti - CDK - 1 ( anti - CDK - 1 ( D relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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Quantification of dissected distal germline from wild-type animal stained eithe anti-CDK-1 (anti-CDK-1 (D relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained eithe 5 - ethynyl - 2 ′ - deoxyuridine ( EdU - green ) ( E ) relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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Quantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild - type worms fed as in ( A ) Data information : Data are presented as mean ± SE ( F ) N = 5 at least 10 worms per replicate and condition . * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A) Data information: Data are presented as mean ± SE (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiation 125 Gy ( A Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation 125 Gy (A Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Quantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit ) , UVC 400J / m2 ( B Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit ), UVC 400J/m2 (B Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Quantification of apoptotic corpse in the meiotic compartment o wild - type strain relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit different doses of UVB ( C Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification of apoptotic corpse in the meiotic compartment o wild-type strain relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit different doses of UVB (C Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Quantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit 100µM cisplatin Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit 100µM cisplatin Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 ( A and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) o isp - 1 ( B ) , and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOV Tukey " s multiple

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

Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) o isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOV Tukey"s multiple comparisons


Real Time PCR gene expression analysis of egl - genes , performed in wild - type animals 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 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 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals 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 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 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression analysis of egl - genes , performed in wild - type animals 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 Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals 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 Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals 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 Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals 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 Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treate with UV Data information : Data are presented as mean ± SE N = F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s mu comparisons

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

Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treate with UV Data information: Data are presented as mean ± SE N= F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s mu comparisons test


Representative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue ) and either anti - PARP - 1 ( red ) ( A Scale bar : 20µ ( A Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

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

Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


Representative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue and eithe anti - RPA - 1 ( green ) and anti - RAD - 51 ( red ) antibodies and treated with gamma radiation ( 125 Gy ) ( B ) ( untreated animals displayed no staining of RPA - 1 and RAD - 51 , not shown ) 15µm ( B Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


Quantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treated with gamma radiation 125 G Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

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

Quantification of mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 G Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


Quantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treate with 1200 J / m2 UV Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

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

Quantification of mitotic cells in wild-type animals fed as (A) and left untreated or treate with 1200 J/m2 UV Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


A , 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 test


Fertilit 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 )

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

Fertilit 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

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

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 compa


Fertilit 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

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

Fertilit 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 comparisons


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 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

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

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 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 compa


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 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

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 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


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 , 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

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, 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


A 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 specified


B 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

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B 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 specified


Quantification 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

Quantification 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 comparison


Quantification 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

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Quantification 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


Kaplan - 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 -

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Kaplan-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-1


Kaplan - 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 -

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Kaplan-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-1


Kaplan - 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 -

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Kaplan-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-1


Kaplan - 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 -

Kaplan-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-1


Kaplan - 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 -

Kaplan-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-1


Kaplan - 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

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Kaplan-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 UVB


Kaplan - 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

Kaplan-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


A 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 -

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A 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-1


B 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

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B 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


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

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


( 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 ) .

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(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 .

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(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.


The 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 .

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The 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.


( 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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(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.


( 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 ) .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

(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.


A Liver biopsies showing no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis stained by PSR . Size bar , 300 μm .

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A 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 .

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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 .

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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 .

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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 ) .

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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 ' .

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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'.


A Immunohistochemistry for SOX9 ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . SOX9 positive bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated .

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A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.