Cell death analysis by Celigo of PI - positive HS - ES - 2R Cells were treated with the indicated agents for 48 hrs ( HS - ES - 2R ) DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive HS-ES-2R Cells were treated with the indicated agents for 48 hrs (HS-ES-2R) DMSO (Unt), zVAD (10 µM), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.


Cell death analysis by Celigo of PI - positive HS - SY - II cells . Cells were treated with the indicated agents for 24 hrs ( HS - SY - II ) . DMSO ( Unt ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * 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 O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive HS-SY-II cells. Cells were treated with the indicated agents for 24 hrs (HS-SY-II). DMSO (Unt), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.


Cell Death analysis by IncuCyte Zoom of CellTox Green - positive MET1 ( L ) , MET2 ( M ) and IC8 ( N ) cells . Cells were treated with the indicated agents for 40 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 6 , and statistical analysis was performed with a one - way Anova , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

Cell Death analysis by IncuCyte Zoom of CellTox Green-positive MET1 (L), MET2 (M) and IC8 (N) cells. Cells were treated with the indicated agents for 40 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=6, and statistical analysis was performed with a one-way Anova, *** P ≤ 0.001, **** P ≤ 0.0001.


Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001.


The addition of SM to the standard - of - care treatment ILP - TNF / Mel significantly prolonged survival compared to either modality alone ( n = 6 rats per cohort ) . Statistical analysis was performed with a log - rank test , * * P ≤ 0 . 01 .

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The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01.


Median TNF / Mel is 0 . 4472 and TNF / Mel / SM is 1 . 154 . TNF / Mel 25th percentile limit 0 . 02887 and 75th percentile limit 0 . 7627 . TNF / Mel / SM 25th percentile limit 1 . 136 and 75th percentile limit 1 . 67 . TNF / Mel Min error bar - 0 . 2389 and Max 0 . 7687 . TNF / SM / Mel Min error bar 1 . 12 and Max 1 . 813 . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * P ≤ 0 . 01 .

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

Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01.


DEVDase activity assay of parental BN175 cells and drug - tolerant tumour cell lines isolated from ILP - TNF / Mel / SM treated animals ( 124 , 133 , 136 ) , treated with the indicated drugs for 6 hrs ( n = 4 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) .

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DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).


Cell viability analysis using CellTiter - Glo of BN175 parental and 124 , 133 and 136 cells treated with the indicated drugs for 24 hrs ( n = 3 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Statistical analysis was performed with a one - way Anova , ns = not significant .

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

Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant.


Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 KO cells treated with the indicated agents for 24 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , SM ( 100 ng / ml ) and Riboxxol ( 1 µg / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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

Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP with TNF / Mel / SM significantly increased infiltration by CD3 + lymphocytes ( B ) and CD3 - CD161 + NK cells ( C ) .

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

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . While ILP treatment with TNF / Mel significantly reduced Granzyme B positive NK cells ( D ) , the numbers of Granzyme B positive NK cells were restored to normal levels upon co - treatment with SM ( TNF / Mel / SM ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . Importantly , ILP - mediated treatment with TNF / Mel / SM significantly increased the numbers of CD3 + CD8 + cytotoxic T cells ( E ) .

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

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP - TNF / Mel / SM significantly upregulated Granzyme B in CD3 + CD8 + CD161 - cytoxtoxic T

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cells


ILP - TNF / Mel / SM significantly reduced infiltration by CD3 + CD4 + Foxp3 - T cells whilst ( G ) increasing infiltrations by CD3 + CD4 + Foxp3 + T regulatory cells ( H ) .

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

ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).


A non - significant increase in T regulatory cell ICOS expression was also noted ( I ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O

A non-significant increase in T regulatory cell ICOS expression was also noted (I).


Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD .

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

Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.


The addition of either anti - CTLA - 4 or anti - PD - 1 antibodies prolonged survival in comparison to ILP - TNF / Mel / SM alone ( n = 6 animals per cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

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

The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.


Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - CTLA - 4 antibodies led to a significant increase in CD4 + Foxp3 - helper T cells ( C ) accompanied by a depletion of CD4 + Foxp3 + regulatory T cells ( D ) . SM represents Birinapant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.


Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - PD - 1 antibodies led to an increase in Granzyme B ( GzmB ) expression on NK cells ( E ) . SM represents Birinapant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant.


Tumour volumes at the time of surgery following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * * * , P ≤ 0 . 0001 .

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Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001.


Individual growth curves following surgery indicated improved control following ILP - TNF / Mel / SM ( n = 6 animals per cohort ) .

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

Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).


Tumour volume on re - challenge with BN175 sarcoma on contralateral limb of long - term survivors following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . Unless otherwise stated , SM represents Birinapant . Error bars represent S . D .

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Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.


ILP - TNF / Mel / SM significantly delayed growth leading to prolonged survival on re - challenge ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

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

ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.


Impaired wound closure in αSMA - TK mice . ( A ) IHC for αSMA + cells in wound tissue sections at day 6 post - wounding . Representative images are shown . Scale bar , 50 µm . Black boxes denote zoomed regions displayed in bottom panels . ( B ) Quantification of αSMA + cells at day 6 post - wounding . WT , N = 6 ; TK , N = 5 biological replicates . Two - tailed non - parametric Mann - Whitney test performed .

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

Impaired wound closure in αSMA-TK mice. (A) IHC for αSMA+ cells in wound tissue sections at day 6 post-wounding. Representative images are shown. Scale bar, 50 µm. Black boxes denote zoomed regions displayed in bottom panels. (B) Quantification of αSMA+ cells at day 6 post-wounding. WT, N=6; TK, N=5 biological replicates. Two-tailed non-parametric Mann-Whitney test performed.


( F ) Expression levels of ACTA2 in normal and diabetic human wound tissue . RNA - seq reads are derived from ( Davis et al . , 2020 , Data ref : Davis et al , 2020 ) . Normal , N = 3 ; diabetic , N = 4 biological replicates . Data are presented as a box and whisker plot of min to max values . Unpaired t - test performed .

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(F) Expression levels of ACTA2 in normal and diabetic human wound tissue. RNA-seq reads are derived from (Davis et al., 2020, Data ref: Davis et al, 2020). Normal, N=3; diabetic, N=4 biological replicates. Data are presented as a box and whisker plot of min to max values. Unpaired t-test performed.


( J - O ) Impaired granulation tissue formation and angiogenesis in αSMA - TK mice . ( J ) Representative H & E images of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines , triangles ( open : incomplete closure , filled : closed wounds ) denote the epithelial tongues . Epi , epidermis ; derm , dermis regions denoted . Scale bar : 200 μm . ( K ) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control ( set to 100 % ) . NA ( not assessed ) reflects the lack of granulation in αSMA - TK mice . WT , N = 5 ; TK , N = 4 biological replicates . ( L ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( M ) Quantification of CD31 + cells per visual field normalized to WT mice . WT , N = 3 ; TK , N = 5 biological replicates . ( N ) IHC for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) Quantification of hypoxyprobe area per visual field normalized to WT mice . WT , N = 5 ; TK , N = 6 biological replicates .

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

(J-O) Impaired granulation tissue formation and angiogenesis in αSMA-TK mice. (J) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis regions denoted. Scale bar: 200 μm. (K) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control (set to 100%). NA (not assessed) reflects the lack of granulation in αSMA-TK mice. WT, N=5; TK, N=4 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (M) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=3; TK, N=5 biological replicates. (N) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=5; TK, N=6 biological replicates.


( D ) Representative FACS plots of digested skin from bone marrow ( BM ) transplanted αSMA - RFP mice 17 days post - wounding . ( E ) FACS quantification of αSMA - RFP + cells in wounds of BM transplanted mice . αSMA - RFP with αSMA - RFP BM , N = 2 ; WT with αSMA - RFP BM , N = 3 ; αSMA - RFP with WT BM , N = 4 biological replicates . One - way ANOVA with Tukey ' s multiple comparison test performed comparing WT with αSMA - RFP BM and αSMA - RFP with WT BM to αSMA - RFP with αSMA - RFP BM . (

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

(D) Representative FACS plots of digested skin from bone marrow (BM) transplanted αSMA-RFP mice 17 days post-wounding. (E) FACS quantification of αSMA-RFP+ cells in wounds of BM transplanted mice. αSMA-RFP with αSMA-RFP BM, N=2; WT with αSMA -RFP BM, N=3; αSMA-RFP with WT BM, N=4 biological replicates. One-way ANOVA with Tukey's multiple comparison test performed comparing WT with αSMA-RFP BM and αSMA-RFP with WT BM to αSMA-RFP with αSMA-RFP BM. (


( H - J ) Multispectral imaging of changes in fibroblast subsets during wound repair . ( H ) Representative images ( 20x magnification ) of orthogonal wound tissue sections at day 6 post - wounding displaying αSMA , FAP , and FSP1 overlaid with DAPI in the wound bed . Subcutaneous ( subq ) , dermis ( derm ) , epidermis ( epi ) , and scar regions are denoted . Individual αSMA , FAP , and FSP1 channel images overlaid with DAPI . Scale bar , 50 µm . ( I ) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points . Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates . One - way ANOVA with Dunnett ' s multiple comparison test performed . ( J ) Percent overlap of αSMA with FAP and FSP1 throughout wound healing . αSMA + denotes cells positive for αSMA but not FAP or FSP1 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates .

O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-J) Multispectral imaging of changes in fibroblast subsets during wound repair. (H) Representative images (20x magnification) of orthogonal wound tissue sections at day 6 post-wounding displaying αSMA, FAP, and FSP1 overlaid with DAPI in the wound bed. Subcutaneous (subq), dermis (derm), epidermis (epi), and scar regions are denoted. Individual αSMA, FAP, and FSP1 channel images overlaid with DAPI. Scale bar, 50 µm. (I) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (J) Percent overlap of αSMA with FAP and FSP1 throughout wound healing. αSMA+ denotes cells positive for αSMA but not FAP or FSP1. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates.


( L ) Representative H & E images of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines . Scale bar , 200 μm . ( M ) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 17 , FSP1 - TK , N = 9 biological replicates . ( N ) Immunofluorescent staining for CD31 ( red ) and nuclei on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) Quantification of CD31 + cells per visual field normalized to WT mice . WT , N = 4 ; FAP - TK , N = 4 ; WT , N = 3 ; FSP1 - TK , N = 3 biological replicates . ( P ) IHC for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( Q ) Quantification of hypoxyprobe area per visual field normalized to WT mice . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 3 ; FSP1 - TK , N = 4 biological replicates . ( R )

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

(L) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines. Scale bar, 200 μm. (M) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control. WT, N=3; FAP-TK, N=3; WT, N=17, FSP1-TK, N=9 biological replicates. (N) Immunofluorescent staining for CD31 (red) and nuclei on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=4; FAP-TK, N=4; WT, N=3; FSP1-TK, N=3 biological replicates. (P) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (Q) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=3; FAP-TK, N=3; WT, N=3; FSP1-TK, N=4 biological replicates. (R) W


( C ) Enrichment plot of genes in αSMA C1 ( left panel ) . Violin plots for genes enriched in αSMA C1 ( right panels ) . ( D ) Enrichment plot of genes in αSMA C2 ( left panel ) . Violin plots for genes enriched in αSMA C2 ( right panels ) . ( E ) Enrichment plot of genes in αSMA C3 ( left panel ) . Violin plots of enriched genes in the αSMA C3 cluster ( right panels ) . Raw scRNA - seq data are derived from ( Haensel et al . , 2020 , Data ref : Haensel et al 2020 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enrichment plot of genes in αSMA C1 (left panel). Violin plots for genes enriched in αSMA C1 (right panels). (D) Enrichment plot of genes in αSMA C2 (left panel). Violin plots for genes enriched in αSMA C2 (right panels). (E) Enrichment plot of genes in αSMA C3 (left panel). Violin plots of enriched genes in the αSMA C3 cluster (right panels). Raw scRNA-seq data are derived from (Haensel et al., 2020, Data ref: Haensel et al 2020). Da


( A ) IHC for collagen I on wound tissue sections at day 17 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 50 μm . ( B ) Quantification of collagen I positive area normalized to the average area in WT controls . WT , N = 5 ; TK , N = 5 biological replicates . ( C ) Polarized light images of picrosirius red stained wound tissues at day 17 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( D ) Quantification of collagen fiber ( picrosirius red ) percent area normalized to the average area in WT controls . WT , N = 3 ; TK , N = 3 biological replicates . ( E -

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(A) IHC for collagen I on wound tissue sections at day 17 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 50 μm. (B) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=5; TK, N=5 biological replicates. (C) Polarized light images of picrosirius red stained wound tissues at day 17 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (D) Quantification of collagen fiber (picrosirius red) percent area normalized to the average area in WT controls. WT, N=3; TK, N=3 biological replicates. (E-


( E - H ) Reduced collagen deposition in Col1α1cKO wounds . ( E ) IHC for collagen I on wound tissue sections at day 14 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 25 μm . ( F ) Quantification of collagen I positive area normalized to the average area in WT controls . WT , N = 7 ; Col1α1cKO , N = 4 biological replicates . ( G ) Polarized light images of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( H ) Quantification of collagen fiber percent area and fiber width normalized to the average area and width , respectively , in WT controls . WT , N = 4 ; Col1α1cKO , N = 5 biological replicates .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(E-H) Reduced collagen deposition in Col1α1cKO wounds. (E) IHC for collagen I on wound tissue sections at day 14 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 25 μm. (F) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=7; Col1α1cKO, N=4 biological replicates. (G) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (H) Quantification of collagen fiber percent area and fiber width normalized to the average area and width, respectively, in WT controls. WT, N=4; Col1α1cKO, N=5 biological replicates.


( J - M ) Reepithelization and vascularization in WT and Col1α1cKO mice . ( J ) Representative images of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( K ) Quantification of epidermal thickness . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates . ( L ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( M ) Quantification of CD31 + area per visual field normalized to WT mice . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(J-M) Reepithelization and vascularization in WT and Col1α1cKO mice. (J) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (K) Quantification of epidermal thickness. WT, N=6; Col1α1cKO, N=5 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (M) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Col1α1cKO, N=5 biological replicates.


( C ) IF for β1 integrin ( red ) and αSMA ( green ) on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar : 20 μm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IF for β1 integrin (red) and αSMA (green) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar: 20 μm.


( F - K ) Reepithelization , granulation tissue formation , and vascularization in WT and Itgb1cKO mice . ( F ) Representative images of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( G ) Quantification of epidermal thickness . WT , N = 4 ; Itgb1cKO , N = 6 biological replicates . ( H ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( I ) Quantification of CD31 + area per visual field normalized to WT mice . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( J ) Polarized light images of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in right panels . Scale bar , 50 µm . ( K ) Quantification of collagen fiber percent area normalized to the average area in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( L ) Quantification of collagen fiber width normalized to the average width in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(F-K) Reepithelization, granulation tissue formation, and vascularization in WT and Itgb1cKO mice. (F) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (G) Quantification of epidermal thickness. WT, N=4; Itgb1cKO, N=6 biological replicates. (H) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (I) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Itgb1cKO, N=6 biological replicates. (J) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in right panels. Scale bar, 50 µm. (K) Quantification of collagen fiber percent area normalized to the average area in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. (L) Quantification of collagen fiber width normalized to the average width in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. D


Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 )

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

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 )

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


Ttyh1 - expressing cells in E15 . 5 embryonic brains that were intraventricularly electroporated at E13 . 5 were immunostained using an anti - GFP primary ( reporter gene ) and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . Quantification of ( E )

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

Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Quantification of (E)


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm .

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

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm . Quantification of ( G ) ( n = 3 ) .

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

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm. Quantification of (G) (n = 3).


Immunolabeling of E14 . 5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1 - resistant Ttyh1 ( Ttyh1res ) expression vector at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . The DAPI nuclear counterstain is shown in blue . Scale bar : 100 μm . Quantification of ( I )

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

Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)


Two days after E14 . 5 primary neural progenitor cells were transduced with retroviral vectors expressing ( A ) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by

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

Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCR


Two days after E14 . 5 primary neural progenitor cells were transduced with shTtyh1 , mRNA expression levels of each indicated Notch target gene were measured by

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

Two days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR


Effects of dominant negative MAML1 ( dnMAML1 ) on Ttyh1 - enhanced Notch target gene expression were tested by qPCR using E14 . 5 primary neural progenitors at 2 days posttransduction ( n = 4 ) .

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

Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).


The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13 . 5 and cell localization was analyzed at E15 . 5 by anti - GFP immunostaining . GFP and DAPI signals are shown in green and blue , respectively . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . E Quantification of GFP + cell positions in ( D ) ( n = 3 ) .

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

The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3).


The NICD plasmid was electroporated with the Ttyh1 knockdown vector ( shTtyh1 ) into embryonic brains at E13 . 5 and cell localization was analyzed at E14 . 5 by anti - GFP immunostaining . Scale bar : 100 μm . G Quantification of GFP + cell positions in ( F ) ( n = 3 ) .

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

The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).


Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . Quantification of band

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

Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensities


Effects of Ttyh1 knockdown ( C ) on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . ( B , D ) Quantification of band

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

Effects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities


Ttyh1 - transduced E14 . 5 neural progenitors were incubated with DAPT for 2 days , then cells were harvested and lysates were used for luciferase assay ( n = 3 ) .

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

Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).


Effects of Ttyh1 - knockdown on γ - secretase activity was also assessed using E14 . 5 neural progenitor

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

Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for Western blotting using anti - NICD

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

Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for qPCR for Notch target genes ( I )

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Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)


The indicated plasmids were electroporated into E13 . 5 brains , and brain samples were harvested at E15 . 5 for anti - GFP immunolabeling . GFP and DAPI signals are shown in green and blue , respectively . Quantification of GFP + cell localization in ( J ) dnPS1 , dominant negative presenilin 1 ; VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Effects of the ion selectivity - perturbing R371Q mutation in Ttyh1 E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Effects of the ion selectivity - perturbing R371Q mutation in other Ttyh family members on Notch - dependent transcription . E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Immunolabeling of E15 . 5 brain sections electroporated in utero with indicated plasmids at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . ( D ) Quantification of GFP + cell localization in ( C ) ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


series of Ttyh1 mutants with deletions in the C - terminal cytoplasmic tail region , and ( F ) their effects on cell distribution assessed by intraventricular in utero electroporation at E13 . 5 followed by immunolabeling at E15 . 5 using anti - GFP antibody . Scale bar : 100 μm .

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series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region , and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.


Representative Western blots showing coimmunoprecipitation ( coIP ) of HA - tagged Rer1 with Myc - tagged Ttyh1 using the HEK293T cell line . WCL , whole - cell lysates .

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Representative Western blots showing coimmunoprecipitation (coIP) of HA-tagged Rer1 with Myc-tagged Ttyh1 using the HEK293T cell line. WCL, whole-cell lysates.


Venus fluorescent protein - based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction ( green ) in living COS - 7 cells by fluorescence microscopy . Ttyh1 and Rer1 were tagged with N - and C - terminal fragments of the Venus fluorescent protein ( VN and VC ) , respectively . DsRed2 - ER was cotransfected to label the endoplasmic reticulum ( ER , red ) . Scale bar : 25 μm .

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Venus fluorescent protein-based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction (green) in living COS-7 cells by fluorescence microscopy. Ttyh1 and Rer1 were tagged with N- and C-terminal fragments of the Venus fluorescent protein (VN and VC), respectively. DsRed2-ER was cotransfected to label the endoplasmic reticulum (ER, red). Scale bar: 25 μm.


CoIP assays were performed with anti - Ttyh1 and anti - Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells .

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CoIP assays were performed with anti-Ttyh1 and anti-Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells.


Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing ( D ) Ttyh1 Quantification of relative band

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Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing (D) Ttyh1 Quantification of relative band intensities


D - G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1 . Quantification of relative band

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D-G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1. Quantification of relative band intensities


Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin ( 10 μM ) , were immunoblotted with an antibody to Rer1 .

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Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.


HEK293T cells were transfected with Ttyh1 - Myc and Rer1 - HA expression plasmids , and after 24 h , cells were treated with cycloheximide ( CHX ) ( 100 μg / ml ) and then harvested at the indicated time intervals . Cell lysates were analyzed by Western blotting using anti - HA antibody . ( J ) Quantification of band intensities in ( I ) ( n = 3 ) .

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HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).


γ - secretase - dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction ( n = 3 ) .

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γ-secretase-dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction (n = 3).


Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13 . 5 brains and subsequent GFP immunofluorescence of E15 . 5 brain sections . GFP and DAPI signals are shown in green and blue , respectively . ( M ) Distribution of GFP + cells in each brain layer in ( L ) was quantified ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


( b , c ) Effects of miR - 212 and miR - 132 precursors and inhibitors ( anti ) on cardiomyocyte cell size as compared with the effects of scrambled ( Scr ) controls ( n = 5 - 13 ) . Representative images used for quantification of cardiomyocyte cell size are shown in c .

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(b,c) Effects of miR-212 and miR-132 precursors and inhibitors (anti) on cardiomyocyte cell size as compared with the effects of scrambled (Scr) controls (n=5-13). Representative images used for quantification of cardiomyocyte cell size are shown in c.


( d ) Effects of various pro - hypertrophic stimuli on miR - 212 and miR - 132 expression in neonatal cardiomyocytes ( n = 6 - 10 ) .

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(d) Effects of various pro-hypertrophic stimuli on miR-212 and miR-132 expression in neonatal cardiomyocytes (n=6-10).


( e ) miR - 212 and miR - 132 expression levels during pressure - induced left ventricular hypertrophy 3 and 21 days after transaortic constriction ( TAC ) surgery of mice ( n = 4 ) .

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(e) miR-212 and miR-132 expression levels during pressure-induced left ventricular hypertrophy 3 and 21 days after transaortic constriction (TAC) surgery of mice (n=4).


( f ) Cardiomyocyte diameters after Sham operation or 3 , 14 and 35 days after TAC ( n = 4 per group ) .

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(f) Cardiomyocyte diameters after Sham operation or 3, 14 and 35 days after TAC (n=4 per group).


( g , h ) miR - 212 and miR - 132 expression ( normalized to sno - 202 levels ) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bar in c represents 50 μm . ACTN2 , α - cardiac actinin ; Ang II , angiotensin - 2 ; a . u . , arbitrary unit ; d , day ; DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; NS , no significant difference ; P / I , phenylephrine / isoprenaline .

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(g,h) miR-212 and miR-132 expression (normalized to sno-202 levels) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bar in c represents 50 μm. ACTN2, α-cardiac actinin; Ang II, angiotensin-2; a.u., arbitrary unit; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; NS, no significant difference; P/I, phenylephrine/isoprenaline.


( b ) Expression levels of miR - 212 and miR - 132 in heart samples of individual wild - type ( WT ) and miR - 212 / 132 transgenic ( TG ) mice as assayed by RT - PCR analysis against the mature forms of corresponding microRNAs . Rnu6b was used as housekeeping control .

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(b) Expression levels of miR-212 and miR-132 in heart samples of individual wild-type (WT) and miR-212/132 transgenic (TG) mice as assayed by RT-PCR analysis against the mature forms of corresponding microRNAs. Rnu6b was used as housekeeping control.


( c ) Survival rate of two different miR - 212 / 132 TG mouse families ( TG - Fam23 , TG - Fam43 ) versus WT controls was analysed by Kaplan - Meier survival assay ( n = 87 , 65 and 53 for WT , TG - Fam23 and TG - Fam43 , respectively ) .

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(c) Survival rate of two different miR-212/132 TG mouse families (TG-Fam23, TG-Fam43) versus WT controls was analysed by Kaplan-Meier survival assay (n=87, 65 and 53 for WT, TG-Fam23 and TG-Fam43, respectively).


( d ) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth . Scale bar , 1 cm .

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(d) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth. Scale bar, 1 cm.


( e , f ) Heart - to - body - weight ratios ( e ) and cardiomyocyte diameter ( f ) in 8 - week - old α - MHC - miR - 212 / 132 transgenic mice compared with their wild - type littermates ( n = 5 - 7 ) . Scale bar , 50 μm .

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(e,f) Heart-to-body-weight ratios (e) and cardiomyocyte diameter (f) in 8-week-old α-MHC-miR-212/132 transgenic mice compared with their wild-type littermates (n=5-7). Scale bar, 50 μm.


( g - i ) Echocardiography analysis of cardiac dimensions and function for α - MHC - miR - 212 / 132 TG mice and WT controls ( n = 16 - 18 ) ( g ) end - systolic area , ( h ) end - diastolic area , ( i ) fractional shortening . All values in e - i represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin ( membrane stain ) .

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(g-i) Echocardiography analysis of cardiac dimensions and function for α-MHC-miR-212/132 TG mice and WT controls (n=16-18) (g) end-systolic area, (h) end-diastolic area, (i) fractional shortening. All values in e-i represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin (membrane stain).


( a ) Heart - to - body - weight ratios for 12 - week - old miR - 212 / 132 null ( KO ) and wild - type ( WT ) mice ( n = 5 - 6 ) .

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(a) Heart-to-body-weight ratios for 12-week-old miR-212/132 null (KO) and wild-type (WT) mice (n=5-6).


( b ) Cardiomyocyte cell size of neonatal miR - 212 / 132 null and WT mice ( n = 5 - 6 isolations ) .

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(b) Cardiomyocyte cell size of neonatal miR-212/132 null and WT mice (n=5-6 isolations).


( c - f ) Heart - to - body - weight ratios ( c ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

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(c-f) Heart-to-body-weight ratios (c) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( c - f ) cardiomyocyte diameter ( d ) , cardiac fibrosis ( e ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

(c-f) cardiomyocyte diameter (d), cardiac fibrosis (e) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( c - f ) lung wet weight ( f ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice 3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

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(c-f) lung wet weight (f) in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( g - i ) Echocardiography analysis of cardiac dimensions and function in Sham - operated WT mice and miR - 212 / 132 null and WT mice 3 weeks after TAC ( n = 4 - 11 ) . ( g ) End - diastolic area , ( h ) end - systolic area and ( i ) fractional shortening . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 compared with WT TAC ; # # # P0 . 005 compared with WT TAC ; PSR , picrosirius red ( collagen stain ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin .

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(g-i) Echocardiography analysis of cardiac dimensions and function in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC (n=4-11). (g) End-diastolic area, (h) end-systolic area and (i) fractional shortening. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 compared with WT TAC; ###P0.005 compared with WT TAC; PSR, picrosirius red (collagen stain). DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin.


( a ) Luciferase activity levels upon cotransfection of a luciferase construct containing wild - type ( WT ) or mutated ( Mut . ) 3 ′ UTR of FoxO3 with scrambled ( Scr ) control ( Ctrl ) , pre - miR - 132 or pre - miR - 212 ( n = 9 ) .

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(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9).


( b , c ) Expression levels of FoxO3 on mRNA ( b ) and protein levels ( c ) in hearts of WT and α - MHC - miR - 212 / - 132 transgenic ( TG ) mice . M : Size marker ( n = 9 - 13 ) .

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(b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9-13).


( d ) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl , anti - miR - 212 and anti - miR - 132 after phenylephrine ( PE , 10 μM ) treatment ( n = 6 - 9 ; P - values against Scr + PE ) .

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(d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE).


( e - g ) Expression levels of atrogin - 1 ( e ) and Mcip1 ( f ) and calcineurin phosphatase activity levels ( g ) in hearts of WT and α - MHC - miR - 212 / 132 TG mice ( n = 5 - 9 ) .

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(e-g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5-9).


( h ) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl , pre - miR - 132 or pre - miR - 212 ( n = 5 ) .

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(h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5).


( i , j ) Mcip1 . 4 ( i ) and atrogin - 1 ( j ) mRNA levels in WT and miR - 212 / 132 null ( KO ) mice 3 weeks after transaortic constriction ( TAC ) or Sham operation ( n = 5 - 7 per group ) . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . # P0 . 05 compared with WT TAC . FC , fold change .

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(i,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5-7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.


( a ) mRNA expression levels of autophagic marker genes in hearts of wild - type ( WT ) and α - MHC - miR - 212 / 132 transgenic ( TG ) mice ( n = 9 - 10 ) .

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(a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9-10).


( b , c ) Ratio of LC3II to LC3I ( b ) and p62 protein levels ( c ) in WT , miR - 212 / 132 null ( KO ) and α - MHC miR - 212 / 132 TG mice . M : Size marker ( n = 4 - 12 ) .

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(b,c) Ratio of LC3II to LC3I (b) and p62 protein levels (c) in WT, miR-212/132 null (KO) and α-MHC miR-212/132 TG mice. M: Size marker (n=4-12).


( d , e ) Representative images ( d ) and quantification ( e ) of LC3 : mCherry puncta ( in red ) in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 30 ) . Nuclei are stained in blue with Hoechst33342 .

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(d,e) Representative images (d) and quantification (e) of LC3:mCherry puncta (in red) in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=30). Nuclei are stained in blue with Hoechst33342.


( f , g ) Representative electron microscopy images ( f ) and quantification ( g ) of autophagic vacuoles in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 20 ) .

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(f,g) Representative electron microscopy images (f) and quantification (g) of autophagic vacuoles in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=20).


( h , i ) Representative FACS plots ( h ) and quantification ( i ) of percent increase of autophagic flux in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 3 - 4 experiments ) . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bars , 50 μm in d and 2 μm in f . FC , fold change .

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(h,i) Representative FACS plots (h) and quantification (i) of percent increase of autophagic flux in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=3-4 experiments). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bars, 50 μm in d and 2 μm in f. FC, fold change.


( a ) Schematic representation of starvation experiment in mice . The white and grey parts on the time line represent the day and night phases , respectively . The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic representation of starvation experiment in mice. The white and grey parts on the time line represent the day and night phases, respectively. The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index.


( b ) LC3II to LC3I ratios in wild - type ( WT ) mice fed with normal diet and WT , miR - 212 / 132 − / − null and α - MHC miR - 212 / 132 transgenic ( TG ) mice under starvation for 31 h ( n = 4 ) .

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(b) LC3II to LC3I ratios in wild-type (WT) mice fed with normal diet and WT, miR-212/132−/− null and α-MHC miR-212/132 transgenic (TG) mice under starvation for 31 h (n=4).


( c ) Electron microscopy from ultrathin sections of resin - embedded heart biopsies of fed and starved WT , miR - 212 / 132 null ( KO ) and cardiomyocyte - specific miR - 212 / 132 - overexpressing ( TG ) mice . White spots around the mitochondria ( dark grey structures ) are autophagic vacuoles . The electron - dense black spots shown with white arrows are autophagosomes . Scale bars , 4 μm .

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(c) Electron microscopy from ultrathin sections of resin-embedded heart biopsies of fed and starved WT, miR-212/132 null (KO) and cardiomyocyte-specific miR-212/132-overexpressing (TG) mice. White spots around the mitochondria (dark grey structures) are autophagic vacuoles. The electron-dense black spots shown with white arrows are autophagosomes. Scale bars, 4 μm.


( d ) p - mTOR / mTOR ratios in WT and miR - 212 / 132 TG H9c2 cells 24 h after normal and starvation ( serum / glucose - deprived ) conditions ( n = 6 ) .

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(d) p-mTOR/mTOR ratios in WT and miR-212/132 TG H9c2 cells 24 h after normal and starvation (serum/glucose-deprived) conditions (n=6).


( e ) p - mTOR / mTOR ratios in hearts of WT and α - MHC miR - 212 / 132 TG mice fed with normal diet or 31 h after starvation ( n = 4 ) . M , size marker . All values represent mean ± s . e . m . * * P0 . 01 ; * * * P0 . 005 ; # P = 0 . 11 .

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(e) p-mTOR/mTOR ratios in hearts of WT and α-MHC miR-212/132 TG mice fed with normal diet or 31 h after starvation (n=4). M, size marker. All values represent mean±s.e.m. **P0.01; ***P0.005; #P=0.11.


( a - c ) Heart - to - body - weight ratios ( a ) , cardiomyocyte diameters ( b ) and cardiac fibrosis ( c ) in Sham - operated mice and mice treated with intravenous injection of either scrambled control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 weeks after TAC ( n = 4 - 11 ) . PSR , picrosirius red staining .

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

(a-c) Heart-to-body-weight ratios (a), cardiomyocyte diameters (b) and cardiac fibrosis (c) in Sham-operated mice and mice treated with intravenous injection of either scrambled control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC (n=4-11). PSR, picrosirius red staining.


( d - f ) Echocardiography analysis of cardiac dimensions and function in Sham - operated mice and mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 weeks after TAC for ( d ) fractional shortening , ( e ) end - diastolic area and ( f ) end - systolic area ( n = 4 - 9 ) .

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(d-f) Echocardiography analysis of cardiac dimensions and function in Sham-operated mice and mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC for (d) fractional shortening, (e) end-diastolic area and (f) end-systolic area (n=4-9).


( g - i ) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 weeks after TAC and treatment ( n = 4 - 8 ) . M : size marker . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin .

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(g-i) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.


( g - i ) calcineurin activity ( h ) and Mcip1 . 4 mRNA levels ( i ) in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 weeks after TAC and treatment ( n = 4 - 8 ) . M : size marker . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g-i) calcineurin activity (h) and Mcip1.4 mRNA levels (i) in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.


( A ) Examples of chromosome arms exhibiting features of telomeric semiconservative ( Semi ) replication , conservative ( Consrv ) replication of the entire telomere ( E ) and conservative replication of part of the telomere ( P ) . Idealized chromosome diagrams ( left ) and actual microscopy images ( right ) are shown . D - FISH , denaturing FISH ; ND - CO - FISH ( non - denaturing CO - FISH ) . ( B ) Percentages of chromosome arms exhibiting conservative ( Consrv ) replication of the entire telomere ( E ) , conservative replication of part of the telomere ( P ) and conservative replication of either the entire telomere of part of it ( E + P ) . NE , U2OS cells expressing normal levels of cyclin E ; OE , U2OS cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from three independent experiments . Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres : P < 0 . 025 for Consrv ( P ) and P < 0 . 05 for Consrv ( E + P ) , as calculated using the paired t test .

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

(A) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.


( A ) Decrease in the fraction of chromosome arms exhibiting conservative ( Consrv ) DNA replication of the entire telomere length or part of the telomere ( E + P ) in U2OS cells after depletion of POLD3 or POLD4 by siRNA . si Ctrl , control siRNA ; NE , cells expressing normal levels of cyclin E ; OE , cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from two independent experiments . For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells . PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P = 0 . 072 ; whereas for PolD4 depletion the decrease was significant at a level of P < 0 . 025 , as calculated using the paired t test . ( B ) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E ( OE ) . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 600 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) for both NE and OE cells , as determined by unpaired t tests .

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(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.


( C ) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 800 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) , as determined by unpaired t tests .

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(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.


( D ) Increased frequency of chromosome end - to - end fusions following POLD3 or POLD4 depletion . U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E for four days ( OE ) were transfected with control siRNA ( siCtrl ) os siRNAs targeting POLD3 or POLD4 . For each condition , 25 metaphases were examined ( representing about 1 , 900 high quality chromosome arms per condition ) ; the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean . For statistical analysis , the number of chromosome end - to - end fusions were compared by chi square tests among the various conditions . Cyclin E overexpression led to a higher number of fusions ( P < 0 . 0005 ) ; PolD3 and PolD4 depletion also led to a higher number of fusions ( P < 0 . 001 and P < 0 . 025 , respectively ) .

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

(D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).


A . Immunoblotting analysis of Neuro2a ( N2a ) cells expressing Sig1R - FLAG variants .

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A. Immunoblotting analysis of Neuro2a (N2a) cells expressing Sig1R-FLAG variants.


B . Cycloheximide chase analysis on N2a cells expressing Sig1R - FLAG . Quantitative data of immunoblotting for the levels of Sig1R - FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments ( upper panel ) . Representative immunoblots for the levels of Sig1R - FLAGproteins were shown ( lower panel ) . * * : p < 0 . 01 , * : p = 0 . 0216 in E102Q vs . wild - type ( WT ) ; # # : p < 0 . 01 , # : p < 0 . 05 in L95fs vs . WT . A one - way ANOVA with subsequent post hoc Tukey ' s test .

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B. Cycloheximide chase analysis on N2a cells expressing Sig1R-FLAG. Quantitative data of immunoblotting for the levels of Sig1R-FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments (upper panel). Representative immunoblots for the levels of Sig1R-FLAGproteins were shown (lower panel). **: p < 0.01, *: p = 0.0216 in E102Q vs. wild-type (WT); ##: p < 0.01, #: p < 0.05 in L95fs vs. WT. A one-way ANOVA with subsequent post hoc Tukey's test.


C . N2a cells transfected with Sig1R - FLAG variants were incubated with MG - 132 ( 10 µM ) or the combination of E64d and pepstatin A ( 5 µg / mL each ) ( E64d / PepA ) for 8 h . The relative mean levels of Sig1R - FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM ( upper panel ) . The representative immunoblots were shown ( lower panel ) . * : p < 0 . 05 vs . no inhibitor control . A one - way ANOVA with subsequent post hoc Tukey ' s test .

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C. N2a cells transfected with Sig1R-FLAG variants were incubated with MG-132 (10 µM) or the combination of E64d and pepstatin A (5 µg/mL each) (E64d/PepA) for 8 h. The relative mean levels of Sig1R-FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM (upper panel). The representative immunoblots were shown (lower panel). *: p < 0.05 vs. no inhibitor control. A one-way ANOVA with subsequent post hoc Tukey's test.


E and F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) and type 3 ( IP3R3 ) in N2a cells stably expressing human IP3R3 ( N2a - IP3R3 cells , E ) . IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated . Proper fractionation of these samples was confirmed by the fraction specific markers as indicated .

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E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in N2a cells stably expressing human IP3R3 (N2a-IP3R3 cells, E). IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated. Proper fractionation of these samples was confirmed by the fraction specific markers as indicated.


E and F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) and type 3 ( IP3R3 ) in HeLa cells ( F ) . IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated . Proper fractionation of these samples was confirmed by the fraction specific markers as indicated .

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E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in HeLa cells (F). IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated. Proper fractionation of these samples was confirmed by the fraction specific markers as indicated.


G . Interaction of IP3R3 with wild - type Sig1R or E102Q ALS - linked Sig1R mutant . Sig1R - FLAG variants were transfected in N2a - IP3R3 cells , and IP3R3 was co - immunoprecipitated using an anti - FLAG antibody and identified by immunoblotting with the specific antibodies as indicated .

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G. Interaction of IP3R3 with wild-type Sig1R or E102Q ALS-linked Sig1R mutant. Sig1R-FLAG variants were transfected in N2a-IP3R3 cells, and IP3R3 was co-immunoprecipitated using an anti-FLAG antibody and identified by immunoblotting with the specific antibodies as indicated.