A Confocal showing basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day immunolabeled for Myosin 7A ( red ) p - ATM ( green ) . Scale bar = 15 µm . B Higher magnification showing representative OHC IHCnuclei from control CDDP - exposed . Scale bar = 5 µm . C Histograms displaying green signal of x - projections of OHC IHCnuclei from control CDDP - exposed . D of p - ATM foci numbers in both OHCs IHCs exposed to either culture medium alone ( blue red lines for OHCs IHCs , respectively ) or 10 µM CDDP ( blue red lines for OHCs IHCs , respectively , n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * P < 0 . 008 , * * * P = 0 . 00005 , CDDP - exposed OHCsvs . CDDP - exposed IHCs ) . E Higher magnification confocal 3D showing co - localization of p - ATM foci ( green ) with 53BP1 foci ( red ) in an OHCnucleus from basal region of organ of Corti culture treated with 10 µM CDDP for 1 day . Scale bar = 5 µm .

O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O

A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-ATM (green). Scale bar=15 µm.B Higher magnification images showing representative OHC and IHCnuclei from control and CDDP-exposed cells. Scale bar=5 µm.C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHCnuclei from control and CDDP-exposed cells.D Quantification analysis of p-ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (**P<0.008, ***P=0.00005, CDDP-exposed OHCsvs. CDDP-exposed IHCs).E Higher magnification confocal and 3D images showing the co-localization of p-ATM foci (green) with 53BP1 foci (red) in an OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day. Scale bar=5 µm.


F Confocal showing basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day immunolabeled for Myosin 7A ( red ) p - Chk2 ( green ) . Scale bar = 15 µm . G Histograms of green signal from OHC IHCnuclei of a CDDP - treated organ of Corti explant . H of Chk2 - positive nuclei in both IHCs ( red bars ) OHCs ( blue bars ) from control CDDP - treated organ of Corti cultures ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * P = 0 . 045 , * * * P = 0 . 0002 , CDDP - exposed IHCsvs . control IHCs ; CDDP - exposed OHCsvs . control OHCs ) .

O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-CELL_TYPE O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-CELL_TYPE O O

F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-Chk2 (green). Scale bar=15 µm.G Histograms of green fluorescent signal intensity from OHC and IHCnuclei of a CDDP-treated organ of Corti explant.H Quantification of Chk2-positive nuclei in both IHCs (red bars) and OHCs (blue bars) from control and CDDP-treated organ of Corti cultures (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (*P=0.045, ***P=0.0002, CDDP-exposed IHCsvs. control IHCs; CDDP-exposed OHCsvs. control OHCs).


I Representative western blots using antibodies against p - p53 ( serine 15 ) , p53 β - actin in control 10 µM CDDP - exposed whole cochlear extracts . J - K Histograms representing of total p53 ( J ) p53 phosphorylation ( K ) in control ( gray bars ) CDDP - exposed cochleae ( green bars , n = 6 cochleae per condition per time point ) . Actin served as a loading control . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( J : * P ≤ 0 . 023 ; K : * P = 0 . 032 , * * P = 0 . 007 , * * * P ≤ 0 . 0008 , cochleae at different times after CDDP exposure vs . control 12h ) .

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

I Representative western blots using antibodies against p-p53 (serine 15), p53 and β-actin in control and 10 µM CDDP-exposed whole cochlear extracts.J-K Histograms representing the levels of total p53 protein (J) and p53 phosphorylation (K) in control (gray bars) and CDDP-exposed cochleae (green bars, n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (J: *P ≤ 0.023; K: *P=0.032, **P=0.007, ***P ≤ 0.0008, cochleae at the different times after CDDP exposure vs. control 12h).


A 3D of OHCnuclei taken from basal regions of organ of Corti cultures treated with medium alone , 10 µM KU55933 , 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day immunolabeled for 53BP1 ( green ) Hoechst 33342 ( blue ) . Scale bar = 5 µm . B of 53BP1 foci number per nucleus in both IHCs ( red bars ) OHCs ( blue bars ) for conditions ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * * P ≤ 0 . 0004 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) .

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

A 3D images of OHCnuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.B Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0004, CDDPvs. control, or CDDPvs. CDDP+KU55933).


C Confocal showing basal region of organ of Corti cultures treated with medium alone , 10 µM KU55933 , 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days immunolabeled for Myosin 7A ( red ) . Scale bar = 24 µm . D Histograms representing numbers of surviving IHCs ( red bars ) OHCs ( blue bars ) for treatment conditions after 5 days ( n = 5 cochleae per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * P = 0 . 03 , * * P = 0 . 004 , * * * P ≤ 0 . 0008 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) .

O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDPvs. control, or CDDPvs. CDDP+KU55933).


E 3D of OHCnuclei from basal regions of organ of Corti cultures treated with culture medium alone , 100 µM PFT - α , 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT - α for 1 day immunolabeled for 53BP1 ( green ) Hoechst 33342 ( blue ) . Scale bar = 5 µm . F of 53BP1 foci number per nucleus in both IHCs ( red bars ) OHCs ( blue bars ) for conditions ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * * P ≤ 0 . 0007 , CDDPvs . control ) .

O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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-SMALL_MOLECULE O O O O

E 3D images of OHCnuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 µM PFT-α, 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT-α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.F Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0007, CDDPvs. control).


A Representative auditory brainstem response ( ABR ) waveforms evoked by 16 kHz tone - bursts in p53wt mice treated with saline ( black plot ) or CDDP ( green plot ) , p53 - / - mice treated with saline ( blue plot ) or CDDP ( blue plot ) for 5 days . B ABR thresholds recorded in p53wt mice before ( gray plot ) after 5 days of saline ( black plot ) or CDDP treatments ( green plot ) , ABR thresholds recorded in p53 - / - mice before ( gray plot ) , after 5 days of saline ( blue plot ) or CDDP treatment ( blue plot ) . C Mean ABR threshold from 4 kHz to 32 kHz derived from B . Data are expressed as mean ± ( saline treated - group : n = 7 , CDDP treated - group : n = 12 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt , before or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O

A Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone-bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53-/- mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days.B ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53-/- mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot).C Mean ABR threshold from 4 kHz to 32 kHz derived from B. Data are expressed as mean ± SEM (saline treated-group: n=7, CDDP treated-group: n=12). One-way ANOVA test followed by post hoc Tukey's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt, before or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).


D Representative scanning electron microscopy showing basal regions of cochleae from CDDP - treated p53wt p53 - / - mice after 5 days . Scale bar = 15µm .

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

D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP-treated p53wt and p53-/- mice after 5 days. Scale bar=15µm.


E Cytocochleograms representing of surviving hair in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from cochlear apex provided from saline - treated p53wt mice ( black bars ) , CDDP - treated p53wt mice ( green bars ) or p53 - / - mice ( blue bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt + saline , d5 or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) .

O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O

E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apex provided from saline-treated p53wt mice (black bars), CDDP-treated p53wt mice (green bars) or p53-/- mice (blue bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt+saline, d5 or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).


F ABR thresholds from p53wt mice recorded prior to ( gray plot ) or after 5 days systemic treatment with : DMSO ( yellow plot ) , CDDP + DMSO ( green plot ) , CDDP + PFT - α ( pink plot ) . G Mean ABR threshold from 4 kHz to 32 kHz derived from F . Data are expressed as mean ± ( DMSO treated - group : n = 7 ; CDDP + DMSO treated - group : n = 12 ; CDDP + PFT - α - treated group : n = 12 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * * P ≤ 0 . 0005 , CDDP + DMSO , d5 vs . before or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) .

O B-EXP_ASSAY O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

F ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP+DMSO (green plot), CDDP+PFT-α (pink plot).G Mean ABR threshold from 4 kHz to 32 kHz derived from F. Data are expressed as mean ± SEM (DMSO treated-group: n=7; CDDP+DMSO treated-group: n=12; CDDP+ PFT-α-treated group: n=12). One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0005, CDDP+DMSO, d5 vs. before or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).


H Cytocochleograms representing of surviving hair in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from cochlear apical end taken from p53wt mice treated with : DMSO ( yellow bars ) , CDDP + DMSO ( green bars ) , CDDP + PFT - α ( pink bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * P ≤ 0 . 008 , CDDP + DMSO , d5 vs . DMSO , d5 or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) .

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

H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP+DMSO (green bars), CDDP+PFT-α (pink bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP+DMSO, d5 vs. DMSO, d5 or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).


I ABR thresholds from p53wt mice recorded prior to ( gray plot black plot for left right ear , respectively ) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into left ear ( green plot ) , of PFT - α into right ear ( pink plot ) . J Mean ABR threshold from 4 kHz to 32 kHz derived from I . Data are expressed as mean ± ( n = 14 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * * P = 0 . 0003 , CDDP + left ear , DMSOvs . before , left ear or CDDP + left ear , DMSOvs . CDDP + right ear , PFT - α .

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

I ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot), and of PFT-α into the right ear (pink plot).J Mean ABR threshold from 4 kHz to 32 kHz derived from I. Data are expressed as mean ± SEM (n=14). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003 , CDDP+left ear, DMSOvs. before, left ear or CDDP + left ear, DMSOvs. CDDP + right ear, PFT-α.


K Cytocochleograms representing of surviving hair in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from cochlear apical end taken from DMSO - treated left ear ( green bars ) PFT - α treated right ear ( pink bars ) of same CDDP - treated p53wt mice after 5 days ( n = 5 per group ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * P ≤ 0 . 008 , CDDP + right ear , PFT - α vs . CDDP + left ear , DMSO ) .

O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-TISSUE O B-SMALL_MOLECULE O O

K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from the DMSO-treated left ear (green bars) and PFT-α treated right ear (pink bars) of the same CDDP-treated p53wt mice after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP + right ear, PFT-α vs. CDDP+left ear, DMSO).


A ABR thresholds recorded prior to ( gray plot ) or at day 28 in HBCx - 90 ( TP53 wt ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . B Mean ABR threshold from 4 kHz to 32 kHz derived from A . Data are expressed as mean ± ( before : n = 20 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 5 ; CDDP + PFT - α : n = 5 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * * P = 0 . 0002 , CDDP vs . before ; CDDP vs . CDDP + PFT - α . E ABR thresholds recorded prior to ( gray plot ) or at day 35 in HBCx - 14 ( TP53 mutant ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . F Mean ABR threshold from 4 kHz to 32 kHz derived from E . Data are expressed as mean ± ( before : n = 30 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 10 ; CDDP + PFT - α : n = 10 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * * P = 0 . 0003 , CDDPvs . before or CDDPvs . CDDP + PFT - α .

O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

A ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx-90 (TP53 wt) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).B Mean ABR threshold from 4 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (before: n=20; DMSO: n=5; PFT-α: n=5; CDDP: n=5; CDDP+PFT-α: n=5). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0002, CDDP vs. before; CDDP vs. CDDP+PFT-α.E ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx-14 (TP53 mutant) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).F Mean ABR threshold from 4 kHz to 32 kHz derived from E. Data are expressed as mean ± SEM (before: n=30; DMSO: n=5; PFT-α: n=5; CDDP: n=10; CDDP+PFT-α: n=10). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003, CDDPvs. before or CDDPvs. CDDP+PFT-α.


C Cytocochleograms representing of surviving hair in three cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 mm from cochlear apical end . These cochleae ( n = 5 per group ) were collected at 28 days from beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * P ≤ 0 . 007 , CDDP , d28 vs . DMSO , d28 or CDDP , d28 vs . CDDP + PFT - α , d28 ) . G Cytocochleograms representing of surviving hair in three cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 mm from cochlear apical end . These cochleae ( n = 5 per group ) were collected at 35 days from beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * P ≤ 0 . 008 , CDDP , d35 vs . DMSO , d35 or CDDP , d35 vs . CDDP + PFT - α , d35 ) .

O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.007, CDDP, d28 vs. DMSO, d28 or CDDP, d28 vs. CDDP+PFT-α, d28).G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP, d35 vs. DMSO, d35 or CDDP, d35 vs. CDDP+PFT-α, d35).


D growth curves alongside of dissected tumors collected on day 28 . Note partial inhibition of growth in CDDP CDDP + PFT - α - treated mice . H growth curves alongside of dissected tumors collected on day 35 . Note complete disappearance of tumors at day 35 significantly reduced regrowth up to day 70 in CDDP + PFT - α - treated mice .

O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O

D Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP+PFT- α-treated mice.H Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP+PFT- α-treated mice.


A Representative confocal of microvessels in transversal sections from HBCx - 14 ( TP53mutant ) tumors treated with either DMSO , CDDP or a combination of CDDP PFT - α . sections were immunolabeled for CD31 ( red ) viewed with a 20x objective . basal - like breast cancer were immunolabeled in green with an antibody against cytokeratin 5 8 . Upper right panel is a 2D projection from boxed in upper left panel . red corresponds to CD31 labeled endothelial represents lumen . shows vessel perimeter . Scale bars : left panel = 50 µm , right panels = 35 µm . samples were collected at day 21 . B Histograms representing of vascular calculated using 2D reconstruction image formula : vessel / vascular = of CD31 - positive objects + lumen per field × 100 % . both HBCx - 14 ( TP53mutant ) HBCx - 90 ( TP53wt ) tumors from different treated groups were collected at day 21 ( n = 4 sections / 3 - 4 tumors / group ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * * P ≤ 0 . 0004 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) .

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

A Representative confocal images of microvessels in transversal tumor sections from HBCx-14 (TP53mutant) tumors treated with either DMSO, CDDP or a combination of CDDP and PFT-α. The sections were immunolabeled for CD31 (red) and viewed with a 20x objective. The basal-like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars: left panel=50 µm, right panels=35 µm. The tumor samples were collected at day 21.B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31-positive objects + lumen area per field area × 100 %. The both HBCx-14 (TP53mutant) and HBCx-90 (TP53wt) tumors from the different treated groups were collected at day 21 (n=4 sections/tumor and 3-4 tumors/group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (***P ≤0.0004, CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).


C Representative confocal of caspase 3 positive ( red ) in transversal sections viewed with a 20x objective . stromal compartments were immunolabeled in green with an antibody against vimentin . HBCx - 14 ( TP53mutant ) tumors from different treated groups were collected at day 18 . Scale bar = 50 µm . c - cas - 3 : caspase 3 .

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

C Representative confocal images of cleaved caspase 3 positive cells (red) in the transversal tumor sections and viewed with a 20x objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx-14 (TP53mutant) tumors from the different treated groups were collected at day 18. Scale bar=50 µm. c-cas-3: cleaved caspase 3.


D Representative western blot using antibodies against β - actin , p53 p21 in extracts from HBCx - 14 ( TP53mutant ) HBCx - 90 ( TP53 wt ) tumors treated with either DMSO or CDDP collected at day 18 . Note higher CDDP - induced increase of p53 p21 expression in TP53wt HBCx - 90tumors .

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

D Representative western blot analysis using antibodies against β-actin, p53 and p21 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP-induced increase of p53 and p21 expression in TP53wt HBCx-90tumors.


E , F Representative western blot using antibodies against β - actin , p - Chk1 , Beclin 1 , LC3 - I / II Rab7 in extracts from HBCx - 14 ( TP53mutant ) HBCx - 90 ( TP53 wt ) tumors treated with either DMSO , PFT - α , CDDP or a combination of CDDP PFT - α . samples were collected at day 18 . G , H , I Histograms representing of Beclin 1 , LC3 - II Rab7 in HBCx - 14 HBCx - 90tumors treated with different regimens ( n = 3 - 4 tumors per group , experiments were performed in triplicate ) . Actin served as a loading control . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( G : * P = 0 . 03 , * * P = 0 . 006 , * * * P ≤ 0 . 0004 ; H : * P = 0 . 02 , * * * P = 0 . 0005 ; I : * P = 0 . 03 , * * * P ≤ 0 . 0006 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) .

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

E, F Representative western blot analysis using antibodies against β-actin, p-Chk1, Beclin 1, LC3-I/II and Rab7 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO, PFT-α, CDDP or a combination of CDDP and PFT-α. The tumor samples were collected at day 18.G, H, I Histograms representing the levels of Beclin 1, LC3-II and Rab7 in HBCx-14 and HBCx-90tumors treated with the different regimens (n=3-4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (G: *P = 0.03, **P =0.006 , ***P ≤ 0.0004 ; H: *P =0.02, ***P = 0.0005 ; I: *P =0.03, ***P ≤ 0.0006 , CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).


A . Representative FACS plots of freshly isolated epidermal ( Epi d0 ) day 14 ( d14 ) cultures of epidermal in different growth conditions media . KGM 2D : 2D in basal KGM ; 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . CD34 + α6 + were quantified by flow cytometry from d14 as in ( A ) ( mean ± SD ; n = 3 - 5 ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 , Kruskal - Wallis / Dunn ' s post ) . 3C 2D : 2D in KGM + Y27632 + VEGF - A + FGF - 2 ; Y : 3D - Matrigel in KGM + Y27632 ; YV : Y + VEGF - A ; YF : Y + FGF - 2 ; 3C : Y + VEGF - A + FGF - 2 . C . Absolute numbers of CD34 + α6 + from Epi d0 d14 cultures as in ( A - B ) . Fold enrichment over Epi d0 is shown ( mean ± , n = 3 - 5 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn's post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test).


D . Epidermal grown for 14 days in indicated conditions . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . 3C 2D : 2D in 3C medium . Scale bars 30µm .

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

D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm.


E . Immunofluorescence of 3C cultures showing expression of HFSC markers CD34 SOX9 in a subset of . See also Appendix Appendix figure S1 .

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

E. Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells. See also Appendix Appendix figure S1.


A . Colony forming assays show increased proliferative potential of cultured in 3C compared to freshly isolated epidermal ( Epi d0 ; control ) ( mean ± ; n = 5 ; * p ≤ 0 . 05 , Mann - Whitney U - ) . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 .

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

A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2.


B . Full thickness skin reconstitution with freshly isolated epidermal ( Epi d0 ) or 3C cultures shows that cultured in 3C retain their self - renewal capacity multipotency . A representative recipient of 4 mice / condition is shown . Right panel shows of hair follicle density . Note that 3C cultures give rise to more hair compared to controls ( mean ± ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).


C . Long - term culture of in 3C was performed as shown in left panel . 3C cultures maintain a stable population of CD34 + α6 + long term ( data are shown using a Box - - Whisker plot : box indicates 25th 75th percentiles ; error bars represent 10th 90th percentiles ; middle is median ; n = 3 - 5 independent experiments ) . p : passage .

O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage.


D . Full thickness skin reconstitution using 3C from passage 1 ( p1 ) passage 15 ( p15 ) show that cultured in 3C maintain their multipotency self - renewal capacity in long - term cultures . A representative recipient of 4 mice / condition is shown . See also Appendix figure S2 .

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D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.


B . Euclidian dendrogram of quantified transcripts from RNAseq data generated as shown in panel A . 3C cultures cluster with purified CD34 + α6 + HFSCs ( n = 3 biological replicates ) .

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B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates).


E . of selected genes differentially expressed in 3C - CD34 + α6 + 3C - CD34 ¯ α6 + . Note upregulation of multiple key HFSC lineage identity transcription factors bulge markers ( triangles ) in 3C - CD34 + α6 + , upregulation of genes implicated in lineage progression in 3C - CD34 ¯ α6 + ( circles ) .

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E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles).


G . RT - qPCR analyses of FACS - purified CD34 + α6 + CD34 - α6 + from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated 3C - CD34 + α6 + compared to freshly isolated CD34 - α6 + progenitors . 3C - CD34 - α6 + show intermediated expression ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . Both 3C - CD34 + α6 + 3C - CD34 - α6 + show upregulation of a panel of cycle genes genes shown to be enriched in HFSC progeny ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S3 Tables S1 - S3 .

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H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.


A . FACS of CD34 + α6 + over time shows that 3C cultures reach a ~ 50 : 50 population equilibrium after 10 days of culture ( mean ± ; n = 3 ) .

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A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3).


B . were EdU - labeled for 24 h ( day 10 - 11 ) prior to by flow cytometry to quantify proliferating . of EdU + is indicated ( n = 4 ; mean ± SD ; * p ≤ 0 . 05 , Mann - Whitney U - ) .

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B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).


D . Simulated population proportions of CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs derived from mathematical model describing steady state cultures . Experimental data from ( A ) are shown as reference ( in grey ) . See also Appendix figure S4 Dataset EV4 .

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D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.


A . CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs were FACS - purified from total epidermis ( d0 ) to ≥ 98 % purity post sorting ( d0 post - sort ) , grown 14 days in 3C cultures ( d14 3C ) . A significant population of CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs is generated from both purified populations after 14 days . A representative of 3 independent experiments is shown ( mean ± SD ) .

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A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD).


B - C . Time course analyses of 3C cultures established from FACS - purified populations of CD34 + α6 + HFSCs ( B ) or CD34 ¯ α6 + non - HFSCs ( C ) from 3C cultures . Both cultures reach population equilibrium between day 6 10 ( n = 4 ; mean ± SD ) .

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B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD).


D - E . Simulated population proportions of CD34 + α6 + HFSCs ( D ) CD34 ¯ α6 + non - HFSCs ( E ) from mathematical model describing behavior of pure populations . model parameters were estimated from experimental data in B - C ( shown in grey as reference ) .

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D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference).


G . Lineage tracing of FACS - purified CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs from EGFP ¯ EGFP + 3C cultures , respectively . CD34 + α6 + CD34 ¯ α6 + were mixed at indicated ratios at d0 EGFP expression was used to trace of origin over time by flow cytometry . Presence of CD34 + α6 + EGFP + in cultures at d10 indicates non - HFSC to HFSC interconversion .

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G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion.


H . Lineage tracing as in ( F ) shows that 3C cultures reach equilibrium between day 6 10 independent of initial proportions of HFSCs non - HFSCs .

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H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs.


I . of proportion of CD34 + α6 + that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled are indicated ) .

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I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).


J . of proportion of CD34 ¯ α6 + that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled are indicated ) . Data in panels G - J show n = 4 ; mean ± SD . See also Appendix figure S5 .

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J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.


A - B . Inhibitor treatment scheme ( A ) FACS plots ( B ) of 3C cultures treated with Shh inhibitor cyclopamine or BMP inhibitor dorsomorphin ( mean ± SD ; n = 3 ) . C . of experiments as in ( B ) shows enrichment of HFSCs upon cyclopamine ( Cyclo ) treatment , whereas dorsomorphin ( Dorso ) treatment leads to depletion of HFSCs ( mean ± SD ; n = 3 ; * p ≤ 0 . 05 , Kruskal - Wallis ) .

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A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).


D . of significantly ( padj value < 0 . 05 ) differentially expressed genes from RNAseq of 3C cultures treated with Cyclo or Dorso for 48h . HFSC identity genes ( triangles ) genes implicated in HFSC lineage progression ( circles ) are indicated .

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D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.


E . RT - qPCR of 3C cultures treated as in ( D ) confirms that Id 1 , 2 3 are significantly upregulated in Cyclo treated ( mean ± ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

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E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).


F . RT - qPCR of 3C cultures treated as in ( A ) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . of experiments as in E ( mean ± SD ; n = 3 - 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S6 Dataset EV5 .

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H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.


A qPCR of relative SCTR in different tissues [ heart ( He ) , intestine ( Int ) , inguinal WAT ( Ing ) , epididymal WAT ( Ep ) , brown fat adipose ( BAT ) , liver ( Liv ) , hypothalamus ( Hypo ) , placenta ( Pla ) , skeletal muscle ( Mus ) , kidney ( Kid ) ] from C57BL / 6J mice as indicated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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A qPCR analysis of relative SCTR levels in different tissues [heart (He), intestine (Int), inguinal WAT (Ing), epididymal WAT (Ep), brown fat adipose (BAT), liver (Liv), hypothalamus (Hypo), placenta (Pla), skeletal muscle (Mus), kidney (Kid)] from C57BL/6J mice as indicated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


B Correlation of serum SCT serum triglycerides ( TG ) , total cholesterol ( TC ) nonesterified fatty ( NEFA ) in pregnant women in middle trimester to early third trimester ( n = 15 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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B Correlation of serum SCT levels and serum triglycerides (TG), total cholesterol (TC) and nonesterified fatty acids (NEFA) levels in pregnant women in the middle trimester to the early third trimester (n = 15). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


C , D Plasma SCT of dams ( C ) at gestational day 18 . 5 ( GD18 . 5 ) ( D ) at E18 . 5 ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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C, D Plasma SCT levels of dams (C) at gestational day 18.5 (GD18.5) and embryo (D) at E18.5 (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


E Representative of SCT immunohistochemical of duodenum from dams on standard ( CON ) , whole grain ( WG ) high fat ( HFD ) diet at GD18 . 5 . SCT expression of WG is highest , but lower in HFD compared with CON . Scale bar , 50 μm ( n = 10 per group ) . Right part shows of SCT positive in duodenum from dams of three groups . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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E Representative images of SCT immunohistochemical staining of duodenum from dams on standard (CON), whole grain (WG) and high fat (HFD) diet at GD18.5. SCT expression of WG is highest, but lower in HFD compared with CON. Scale bar, 50 μm (n = 10 per group). Right part shows quantification of SCT positive cells in duodenum from dams of three groups. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


B Change of of M - SCT floxed M - SCT KO mice upon HFD feeding ( n = 8 per group ) . Right part shows representative photograph of M - SCT floxed M - SCT KO mice with HFD . Data information : Statistical significance was determined using Two - Student ' s t .

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B Change of body weight of M-SCT floxed and M-SCT KO mice upon HFD feeding (n = 8 per group). Right part shows representative photograph of M-SCT floxed and M-SCT KO mice with HFD. Data information: Statistical significance was determined using Two-tailed Student's t test.


C of different tissues from M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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C The weight of different tissues from M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


D Random blood glucose in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . E Insulin in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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D Random blood glucose levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). E Insulin levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


F Intraperitoneal glucose tolerance ( GTT ) ( 1 . 0 g glucose / kg ) in M - SCT floxed M - SCT KO mice with HFD ( n = 5 per group ) AUCs were calculated . Mice were fasted for 18h before . G Intraperitoneal insulin tolerance ( ITT ) ( 0 . 75 U insulin / kg ) in M - SCT floxed M - SCT KO mice with HFD ( n = 5 per group ) AUCs were calculated . mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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F Intraperitoneal glucose tolerance test (GTT) (1.0 g glucose/ kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. Mice were fasted for 18h before assay. G Intraperitoneal insulin tolerance test (ITT) (0.75 U insulin/kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


H Serum TG , TC NEFA in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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H Serum TG, TC and NEFA levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


A , B Time - resolved oxygen consumption ( A ) heat production ( B ) in 48 - hour / cycle measured by CLAMS in M - SCT floxed M - SCT KO mice with HFD ( n = 6 per group ) , phase is marked as background . Regression - absolute oxygen consumption ( A ) or heat production ( B ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel A B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

A, B Time-resolved oxygen consumption (A) and heat production (B) in 48-hour light/dark cycle measured by CLAMS in M-SCT floxed and M-SCT KO mice with HFD (n = 6 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (A) or heat production (B) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel A and B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


C of 8 - week - old M - SCT floxed M - SCT KO offspring with chow diet during cold exposure ( 4 ℃ ; n = 8 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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C Body temperature of 8-week-old M-SCT floxed and M-SCT KO male offspring with chow diet during cold exposure (4℃; n = 8). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


D Representative HE UCP1 of Ing from M - SCT floxed M - SCT KO mice with chow diet maintained at RT or 3 days of cold exposure ( 4 ℃ ) . Scale bar , 100 μm .

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D Representative HE staining and UCP1 staining of Ing from M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or 3 days of cold exposure (4℃). Scale bar, 100 μm.


E Relative mRNA of UCP1 , CIDEA , PRDM16 , PGC1α DIO2 in iWAT from M - SCT floxed M - SCT KO mice with chow diet ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

E Relative mRNA levels of UCP1, CIDEA, PRDM16, PGC1α and DIO2 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


F Representative western blots of UCP1 , PRDM16 , PGC1α HSP90 in iWAT from M - SCT floxed M - SCT KO mice with chow diet after 3 days of cold exposure ( 4 ℃ ) . G Total UCP1 per mg iWAT of M - SCT floxed M - SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d ( 4 ℃ ) ( n = 3 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

F Representative western blots of UCP1, PRDM16, PGC1α and HSP90 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet after 3 days of cold exposure (4℃). G Total UCP1 protein per mg iWAT of M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d (4℃) (n = 3). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


H Relative mRNA of UCP1 , PGC1α , PRDM16 , CIDEA DIO2 in SVF isolated from M - SCT KO M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

H Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in SVF isolated from M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


I , J OCR of Oligomycin , FCCP Antimycin A / Rotenone - treated matured adipocytes derived from iWAT - SVF of M - SCT KO M - SCT floxed mice with HFD , AUC of OCR as indicated were calculated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

I, J OCR of Oligomycin, FCCP and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD, and the AUC of OCR as indicated were calculated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


K Glucose oxidation in adipocytes derived from iWAT - SVF of M - SCT KO M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Glucose oxidation in adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


B Stacked bars showing Proportional distribution of hypo - hypermethylated DMRs ( M - SCT KO vs M - SCT floxed ) in different regions . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Stacked bars showing the Proportional distribution of hypo- and hypermethylated DMRs (M-SCT KO vs M-SCT floxed) in different regions. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


C Heat map of DMR methylation level in mRNA between M - SCT floxed M - SCT KO . color bar indicates normalized tag in M - SCT floxed or M - SCT KO . Positive z scores , shown in table , indicate hypermethylated regions , while negative z scores imply hypomethylated regions . D As in ( C ) , for enhancer region .

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

C Heat map of DMR methylation level in mRNA between M-SCT floxed and M-SCT KO. The color bar indicates the normalized tag counts in M-SCT floxed or M-SCT KO. Positive z scores, shown in the table, indicate hypermethylated regions, while negative z scores imply hypomethylated regions. D As in (C), for enhancer region.


E Scatter plot representation of most significant difference of methylation in mRNA ( left ) enhancer ( right ) regions between M - SCT floxed M - SCT KO ( p < 0 . 005 ) . Data information : Statistical significance was determined using Two - Student ' s t .

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

E Scatter plot representation of the most significant difference of methylation in mRNA (left) and enhancer (right) regions between M-SCT floxed and M-SCT KO (p < 0.005). Data information: Statistical significance was determined using Two-tailed Student's t test.


F Volcano plot methylation represents DMRs in mRNA ( left ) enhancer ( right ) regions . Cutoffs ( vertical horizontal dashed lines ) used to identify DMRs between M - SCT floxed M - SCT KO mice . vertical lines correspond to twofold up down , respectively , horizontal represents a P value of 0 . 01 . Data information : Statistical significance was determined using Two - Student ' s t .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Volcano plot DNA methylation represents all DMRs in mRNA (left) and enhancer (right) regions. Cutoffs (vertical and horizontal dashed lines) used to identify DMRs between M-SCT floxed and M-SCT KO mice. The vertical lines correspond to twofold up and down, respectively, and the horizontal line represents a P value of 0.01. Data information: Statistical significance was determined using Two-tailed Student's t test.


I , J Schematic diagram showing CpG islands of Mus musculus PRDM16 ( I ) CIDEA ( J ) genes . conditions to be met by CpG islands identified were : 200 - 300 bp , CG content of > 50 % observed CpG content vs expected CpG content > 0 . 6 . Methylation of each CpG islands in PRDM16 ( I ) CIDEA ( J ) are presented as fold change relative to average value of M - SCT floxed group β - actin promoter was used as a negative control ( n = 3 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

I, J Schematic diagram showing the CpG islands of Mus musculus PRDM16 (I) and CIDEA (J) genes. The conditions to be met by CpG islands identified were: 200-300 bp length, CG content of > 50% and observed CpG content vs expected CpG content > 0.6. Methylation levels of each CpG islands in PRDM16 (I) and CIDEA (J) are presented as the fold change relative to the average value of the M-SCT floxed group and β-actin promoter was used as a negative control (n = 3 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


A Western blots of DNMT1 , DNMT3a , DNMT3b HSP90 in Ing from M - SCT KO M - SCT floxed mice with HFD .

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

A Western blots analysis of DNMT1, DNMT3a, DNMT3b and HSP90 in Ing from M-SCT KO and M-SCT floxed mice with HFD.


B Relative mRNA of DNMT1 , DNMT3a DNMT3b in Ing from M - SCT KO M - SCT floxed mice with HFD ( n = 8 ) . Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . ns , not statistically significant .

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

B Relative mRNA levels of DNMT1, DNMT3a and DNMT3b in Ing from M-SCT KO and M-SCT floxed mice with HFD (n = 8). Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. ns, not statistically significant.


C Western blots of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in of M - SCT KO M - SCT floxed mice at E18 . 5 .

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C Western blots analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in embryo of M-SCT KO and M-SCT floxed mice at E18.5.


D Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in mouse fibroblast ( MEF ) treated with 10 nM or 100 nM SCT .

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D Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in mouse embryo fibroblast (MEF) cells treated with 10 nM or 100 nM SCT.


E Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in MEF treated with 10 nM SCT or 20 μM compound C ( C . C . ) for 24 h .

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

E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C (C.C.) for 24 h.


F Western blot of p - AMPKα , AMPKα , DNMT1 , SCTR HSP90 . MEF were transfected with indicated siRNA then treated with 1 mM AMPK agonist ( AICAR ) for 24 h .

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

F Western blot analysis of p-AMPKα, AMPKα, DNMT1, SCTR and HSP90. MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist (AICAR) for 24 h.


G Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in AMPK + / + or AMPKα1 / α2 double knockout ( AMPK - / - ) MEF treated with 10 nM SCT for 24 h .

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

G Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in AMPK+/+ or AMPKα1/α2 double knockout (AMPK-/-) MEF cells treated with 10 nM SCT for 24 h.


H , I Western blot of Flag - DNMT1 in Flag - DNMT1 - transfected HEK293T treated with AICAR with indicated dose or time periods .

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

H, I Western blot analysis of Flag-DNMT1 in Flag-DNMT1-transfected HEK293T cells treated with AICAR with indicated dose or time periods.


J HEK293T were transfected with indicated plasmids then treated with 10 nM SCT for 24 h , followed by IP IB .

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J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h, followed by IP and IB analysis.


K HEK293T were transfected with indicated plasmids or siRNA then lysed , followed by IP IB .

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

K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed, followed by IP and IB analysis.


L AMPK + / + or AMPK - / - MEF were transfected with indicated plasmids then lysed , followed by IP IB .

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L AMPK+/+ or AMPK-/- MEF cells were transfected with indicated plasmids and then lysed, followed by IP and IB analysis.


A Serum SCT in M - HFD M - HFD + SCT mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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A Serum SCT levels in M-HFD and M-HFD+SCT mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


B Change of of M - HFD ( offspring of dams with high - fat pregnant diets ) M - HFD + SCT ( offspring of dams with high - fat pregnant diets supplied with SCT ) mice upon HFD feeding ( n = 8 per group ) . Representative photograph of M - HFD M - HFD + SCT mice after HFD feeding . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

B Change of body weight of M-HFD (offspring of dams with high-fat pregnant diets) and M-HFD+SCT (offspring of dams with high-fat pregnant diets supplied with SCT) mice upon HFD feeding (n = 8 per group). Representative photograph of M-HFD and M-HFD+SCT mice after HFD feeding. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


C , Time - resolved oxygen consumption ( C ) in 48 - hour / cycle measured by CLAMS in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) , phase is marked as background . Regression - absolute oxygen consumption ( C ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

C, Time-resolved oxygen consumption (C) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (C) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


D Time - resolved heat production ( D ) in 48 - hour / cycle measured by CLAMS in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) , phase is marked as background . Regression - absolute heat production ( D ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

D Time-resolved heat production (D) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute heat production (D) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


E GTT ( 1 g glucose / kg ) in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) AUCs were calculated in right part . Mice were fasted for 18h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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E GTT (1 g glucose/kg body weight) in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group) and AUCs were calculated in right part. Mice were fasted for 18h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


F ITT ( 0 . 75 U insulin / kg ) in M - HFD M - HFD + SCT mice with HFD , AUCs were calculated in bottom ( n = 5 per group ) . Mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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F ITT (0.75 U insulin/kg body weight) in M-HFD and M-HFD+SCT mice with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


G Serum TG , TC NEFA in M - HFD M - HFD + SCT mice with HFD . ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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G Serum TG, TC and NEFA levels in M-HFD and M-HFD+SCT mice with HFD. (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


H Representative HE UCP1 of iWAT from M - HFD M - HFD + SCT mice with HFD . Scale bar , 100 μm ( n = 5 per group ) .

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H Representative HE staining and UCP1 staining of iWAT from M-HFD and M-HFD+SCT mice with HFD. Scale bar, 100 μm (n = 5 per group).


I Relative mRNA of UCP1 , PGC1α , PRDM16 , CIDEA DIO2 in iWAT from M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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I Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in iWAT from M-HFD and M-HFD+SCT mice with HFD (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


( F ) 5 - day - old Col - 0Cas9 WT CRISPR glv seedlings were treated with 100 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 30 - 32 biological replicates from 2 pooled experiments ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 001 ) .

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(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).


( G ) Ethylene production was measured in Col - 0Cas9 WT CRISPR glv 3 . 5 h after elicitation with 500 nM flg22 or co - treatment with 1 µM GLV2 . Ethylene production was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 11 - 12 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 01 ) .

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(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).


( H ) Mean cfu of Pto DC3000 COR - 4 days after spray inoculation of Col - 0Cas9 WT CRISPR glv ; n = 12 biological replicates from 3 pooled experiments - / + SD ( Students T - , * p < 0 . 05 ) .

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(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).


( B ) ROS burst in WT after elicitation with 10 nM flg22 , 1 μM GLV2 , co - treatment or pre - treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22 . Shown is mean of total ROS production ( Total RLU ) over 40 minutes normalized to flg22 response set as 100 % - / + SD , n = 96 - 120 biological replicates from 15 pooled experiments ( one - way ANOVA , Tukey post - hoc , a - b / c / d , p < 0 . 001 ; b - c , p < 0 . 05 ; b - d , p < 0 .

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(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001


( D ) PR1 expression was measured in 10 - day - old WT seedlings treated for 24 h with 100 nM flg22 , 1 μM GLV2 or co - treatment . Expression was normalized to UBQ house - keeping gene . Shown is mean of n = 4 . Symbol colors represent different biological

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(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates


( B ) Ethylene production was measured in indicated genotypes 3 . 5 h after elicitation with mock or 500 nM flg22 . Ethylene accumulation was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 18 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 001 ; a - c , p < 0 . 001 ; b - c , p < 0 . 001 ) .

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(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).


( E ) Ethylene production was measured in WT rgi5x rgi5x pRGI3 : : RGI3 # 2 # 3 3 . 5 h after elicitation with 500 nM flg22 . Ethylene production was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 8 biological replicates from 2 pooled experiments - / + SD ( one - way ANOVA , Dunnett post - hoc , a - b p < 0 . 05 ) .

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(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).


( F ) 5 - day - old seedlings of indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 36 biological replicates from 3 pooled experiments ( Students T - , * * * p < 0 . 001 ) .

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(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).


( C ) 5 - day - old seedlings of WT , rgi5x rgi5x pRGI3 : : RGI3 # 2 # 3 were treated with 10 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 32 biological replicates pooled from 4 experiments ( Students T - , * * p < 0 . 01 ) .

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(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).