Figure 2A FACS analysis of the percentages of CD44 + / CD24 − / ESA + breast CSC - like populations in CDK12 - overexpressing ZR - 75 - 30 and HCC - 1419 cells and CDK12 - siRNA - transfected BT474 and HCC - 1954 cells . Top , representative dot plot of CD44 - APC versus CD24 - PE expression for viable cells ( red ) and ESA - positive cells ( blue ) from the viable cells . Bottom , the percentage of CD44 + / CD24 - / ESA + cell population ( blue dots at lower right quadrant in the plots ) was quantified in the indicated cell lines . Mean ± s . d . of three independent samples . P - values were based on two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) and one - way ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . Amp , amplification ; non - amp , non - amplification ; Trz , trastuzumab . B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines . Cells either treated with ALDH inhibitor DEAB ( with DEAB ; negative control ) or untreated ( without DEAB ) were incubated with ALDEFLURO substrate ( BAAA ) . In the FACS analysis , the percentage of ALDEFLUOR - positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB . Mean ± s . d . . P - values were calculated based on a two - tailed Student ' s t - test cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive . C The self - renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere - formation assay . The number of tumorsphere cells ( > 100 μm in diameter ) was counted after 3 , 5 , and 7 days . Data represent the mean ± s . d . ( n = 3 ) . P - value was calculated based on a two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) or ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive . D Cell growth of the indicated stable cell lines treated with trastuzumab ( Trz ) or vehicle ( veh ) as measured by SRB assay . Trastuzumab - sensitive HER2 + breast cancer cell lines ( S ) and trastuzumab - insensitive HER2 + breast cancer cell lines ( partial responsive , PR ; resistant , R ) were treated with 1 µg / mL or 100 µg / mL trastuzumab , respectively . Data represent the mean ± s . d . P - values were calculated using RM ANOVA with a post - hoc LSD test . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive . E Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2 + breast cancer . Mice were orthotopically xenografted with the indicated HER2 + breast cancer cells and treated with 20 mg / kg trastuzumab . The growth curve of each group was analysed twice weekly for 5 to 6 weeks ( n = 8 / group ; mean ± s . e . m . ) . P - values were calculated using one - way ANOVA with a post - hoc LSD test for the last day of tumor measurement . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells R , trastuzumab - resistant S , trastuzumab - sensitive .

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

Figure 2A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.E Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2+ breast cancer. Mice were orthotopically xenografted with the indicated HER2+ breast cancer cells and treated with 20 mg/kg trastuzumab. The growth curve of each group was analysed twice weekly for 5 to 6 weeks (n = 8/group; mean ± s.e.m.). P-values were calculated using one-way ANOVA with a post-hoc LSD test for the last day of tumor measurement. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant S, trastuzumab-sensitive.


Figure 3A Heatmap presents the alteration of gene expression ( ≥ 1 . 5 - fold ) between control ( CON ) and CDK12 - overexpressing ( CDK12 ) ZR - 75 - 30 cells obtained from RNA - seq analysis . B GSEA displaying correlations of the gene - expression profiles between control and CDK12 - overexpressing cells based on RNA - seq results . D The CDK12 - target genes identified by RNA - seq were validated in CDK12 - overexpressing ZR - 75 - 30 cells using qRT - PCR analysis . Mean ± s . d . ( n = 3 ) . P - values by two - tailed Student ' s t - test . E ChIP - qPCR analysis displaying the fold - enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12 - target genes in the indicated cell lines . Mean ± s . d . ( n = 3 ) . P - values by two - tailed Student ' s t - test . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive . F Effect of CDK12 kinase inhibition on the expression of WNT1 , WNT3 , and IRS1 . Cells were treated with different doses of dinaciclib for 12 h , and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT - PCR Mean ± s . d . ( n = 3 ) . P - values by ANOVA with a post - hoc LSD test . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; S , trastuzumab - sensitive . G Cell viability was measured by SRB assay in trastuzumab - sensitive BT474 cells and trastuzumab - resistant HCC - 1954 cells treated with 1 µg / mL or 100 µg / mL trastuzumab ( Trz ) and / or 5 nM or 10 nM dinaciclib ( Dina ) , respectively . Mean ± s . d . ( n = 3 ) . P - values , RM ANOVA with a post - hoc LSD test . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells R , trastuzumab - resistant ; S , trastuzumab - sensitive . H Tumor growth curves for mice xenografted with the indicated HER2 + breast cancer cells treated with 20 mg / kg trastuzumab and / or 20 mg / kg dinaciclib ( n = 8 / group ; mean ± s . e . m . ) . P - values , one - way ANOVA with a post - hoc LSD ( BT474 ) or Dunnett T3 ( HCC - 1954 ) test for the last day of tumor measurement . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells R , trastuzumab - resistant ; S , trastuzumab - sensitive .

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

Figure 3A Heatmap presents the alteration of gene expression (≥1.5-fold) between control (CON) and CDK12-overexpressing (CDK12) ZR-75-30 cells obtained from RNA-seq analysis.B GSEA displaying correlations of the gene-expression profiles between control and CDK12-overexpressing cells based on RNA-seq results.D The CDK12-target genes identified by RNA-seq were validated in CDK12-overexpressing ZR-75-30 cells using qRT-PCR analysis. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test.E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.F Effect of CDK12 kinase inhibition on the expression of WNT1, WNT3, and IRS1. Cells were treated with different doses of dinaciclib for 12 h, and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT-PCR Mean ± s.d. (n = 3). P-values by ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells ; R, trastuzumab-resistant; S, trastuzumab-sensitive.G Cell viability was measured by SRB assay in trastuzumab-sensitive BT474 cells and trastuzumab-resistant HCC-1954 cells treated with 1 µg/mL or 100 µg/mL trastuzumab (Trz) and/or 5 nM or 10 nM dinaciclib (Dina), respectively. Mean ± s.d. (n = 3). P-values, RM ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.H Tumor growth curves for mice xenografted with the indicated HER2+ breast cancer cells treated with 20 mg/kg trastuzumab and/or 20 mg/kg dinaciclib (n = 8/group; mean ± s.e.m.). P-values, one-way ANOVA with a post-hoc LSD (BT474) or Dunnett T3 (HCC-1954) test for the last day of tumor measurement. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.


Figure 4A Western blot and qRT - PCR results showing phosphorylation levels of GSK - 3β and WNT1 and WNT3 expression , respectively , in the indicated cell lines . Mean ± s . d . ( n = 3 ) . P - values by two - tailed Student ' s t - test . a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; S , trastuzumab - sensitiveSubcellular localization of β - catenin in the indicated stable cell lines were analysed by cellular fractionation ( B ) Subcellular localization of β - catenin in the indicated stable cell lines were analysed by immunofluorescence staining ( C ) . Nuclear translocation of β - catenin in control , CDK12 - overexpressing , and CDK12 - knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β - catenin ( N ) from the immunofluorescence image . Mean ± s . d . ( n = 3 ) . P - value was calculated using a two - tailed Student ' s t - test . M + C , cells containing membrane and / or cytoplasmic β - catenin ; N , cells dominantly expressing nuclear β - catenin . Green , β - catenin staining ; Blue , DAPI staining for visualization of nucleus ; Scale bar = 10 μ . a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; S , trastuzumab - sensitive . D Luciferase - reporter assay measuring the TCF / lymphoid enhancer - binding factor - promoter activity in the indicated cell lines . TOP , pTOP - flash ( wild - type TCF - binding sites ) , FOP , pFOP - flash ( mutant TCF - binding sites ) . Data represent the mean ± s . d . P - values were calculated using a two - tailed Student ' s t - test . E The mRNA levels of the WNT / β - catenin / TCF - target genes in CDK12 - overexpressing ZR - 75 - 30 cells and CDK12 - knockdown BT474 cells were analysed by qRT - PCR . Mean ± s . d . ( n = 3 ) . P - values were calculated using a two - tailed Student ' s t - test . F Effect of WNT / β - catenin / TCF pathway on the CDK12 regulation of breast CSCs . Cells were treated with DMSO ( vehicle ) or 10 nM FH535 , a β - catenin / TCF inhibitor , for 24 h , and changes in the CD44 + / CD24 - / ESA + cell population were measured using FACS analysis . Cells seen at lower right quadrant in the dot plots indicate CD44 + / CD24 - population ( red ) and CD44 + / CD24 - / ESA + population ( blue ) . Data represent the mean ± s . d . ofP - value was calculated using one - way ANOVA with a post - hoc LSD test .

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

Figure 4A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitiveSubcellular localization of β-catenin in the indicated stable cell lines were analysed by cellular fractionation (B)Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive.D Luciferase-reporter assay measuring the TCF/ lymphoid enhancer-binding factor-promoter activity in the indicated cell lines. TOP, pTOP-flash (wild-type TCF-binding sites), FOP, pFOP-flash (mutant TCF-binding sites). Data represent the mean ± s.d. P-values were calculated using a two-tailed Student's t-test.E The mRNA levels of the WNT/β-catenin/TCF-target genes in CDK12-overexpressing ZR-75-30 cells and CDK12-knockdown BT474 cells were analysed by qRT-PCR. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test.F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test.


Figure 5A Human phospho - RTK array displaying the activation of phosphokinase - related proteins in CDK12 - knockdown BT474 cells . The bar graph shows the fold change of spot intensity . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells S , trastuzumab - sensitive . B Immunoblots showing the levels of the indicated proteins in different types of HER2 + breast cancer stably expressing CDK12 or with CDK12 knocked down . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive . Co - IP analysis showing the amounts of bindings between ErbB receptors . Cell lysates from CDK12 - overexpressing ZR - 75 - 30 cells were immunoprecipitated with anti - HER2 antibody and immunoblotted using the indicated antibodies . IgG , negative control . Cell line information : ; n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive . D Western blot and qRT - PCR analysis of the phosphorylation levels and expression of IRS1 , respectively , in the indicated cell lines . Mean ± s . d . ( n = 3 ) . P - values were calculated using a two - tailed Student ' s t - test . Cell line information : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant S , trastuzumab - sensitive . E Co - IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12 - overexpressing ZR - 75 - 30 cells . Cell line information n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive . F IRS1 effect on CDK12 - induced phosphorylation of ErbB receptors . Lysates from cells treated with either control siRNA or IRS1 siRNA ( siIRS1 ) for 24 h were subjected to immunoblotting with the indicated antibodies . Cell line information : n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive .

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

Figure 5A Human phospho-RTK array displaying the activation of phosphokinase-related proteins in CDK12-knockdown BT474 cells. The bar graph shows the fold change of spot intensity. Cell line information: a/a, CDK12-amplified/HER2-amplified cells S, trastuzumab-sensitive.B Immunoblots showing the levels of the indicated proteins in different types of HER2+ breast cancer stably expressing CDK12 or with CDK12 knocked down. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.Co-IP analysis showing the amounts of bindings between ErbB receptors. Cell lysates from CDK12-overexpressing ZR-75-30 cells were immunoprecipitated with anti-HER2 antibody and immunoblotted using the indicated antibodies. IgG, negative control. Cell line information: ; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.D Western blot and qRT-PCR analysis of the phosphorylation levels and expression of IRS1, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant S, trastuzumab-sensitive.E Co-IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12-overexpressing ZR-75-30 cells. Cell line information n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.F IRS1 effect on CDK12-induced phosphorylation of ErbB receptors. Lysates from cells treated with either control siRNA or IRS1 siRNA (siIRS1) for 24 h were subjected to immunoblotting with the indicated antibodies. Cell line information: n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


f1a , COS7 cells co - transfected with GFP - ATG14 and DsRed - ER or RFP - SEC61b ; ( an ER marker ) were immunostained for the mitochondria marker TOMM20 . Scale bars , 2 µm . b , An immunogold electron micrograph of starved HeLa cells expressing GFP - ATG14 probed with anti - GFP antibodies ( red arrows ) . Scale bar , 200 nm . c , Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP - DFCP1 . Cyto , cytosol ; micro , microsome ; mito , mitochondria ; PNS , post - nuclear supernatant .

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f1a, COS7 cells co-transfected with GFP-ATG14 and DsRed-ER or RFP-SEC61b; (an ER marker) were immunostained for the mitochondria marker TOMM20. Scale bars, 2 µm.b, An immunogold electron micrograph of starved HeLa cells expressing GFP-ATG14 probed with anti-GFP antibodies (red arrows). Scale bar, 200 nm.c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP-DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.


f2a , COS7 cells stably expressing yellow fluorescent protein ( YFP ) - ATG5 were transfected with RFP - SEC61b ; and teal fluorescent protein ( TFP ) - mito , and immunostained for TOMM20 . b , Time - lapse images of COS7 cells transfected with YFP - ATG5 , RFP - SEC61b ; and TFP - mito under starved conditions . c , d , Distribution of distances of YFP - ATG5 dots fromthe centre to the surface of the ER ( c ) and to the surface of mitochondria ( d ) . e , f , Distances of YFP - ATG5 dots over their lifetime for data from c ( e ) and d ( f ) , respectively . All scale bars , 2 µm .

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f2a, COS7 cells stably expressing yellow fluorescent protein (YFP)-ATG5 were transfected with RFP-SEC61b; and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20.b, Time-lapse images of COS7 cells transfected with YFP-ATG5, RFP-SEC61b; and TFP-mito under starved conditions.c, d, Distribution of distances of YFP-ATG5 dots fromthe centre to the surface of the ER (c) and to the surface of mitochondria (d). e, f, Distances of YFP-ATG5 dots over their lifetime for data from c (e) and d (f), respectively. All scale bars, 2 µm.


f3a , COS7 cells expressing GFP - SEC61b ; and STX17 were immunostained for TOMM20 . b , HeLa cells expressing GFP - ATG14 were immunostained for STX17 . c , Immunoblot of subcellular fractions of HEK293 cells . d , STX17 knockdown COS7 cells expressing GFP - ATG14 and RFP - SEC61b ; were immunostained for TOMM20 . Error bars denote s . d . e , Immuoblot of subcellular fractions of STX17 knockdown ( using two STX17 siRNAs , STX17 - 1 and STX17 - 2 ) HEK293 cells expressing GFP - DFCP1 . f , Co - immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag - ATG14 and Myc - STX17 . g , Pull - down of ATG14 complex from HeLa cells expressing Myc - STX17 or Myc . Scale bars , 5 µm ( a , b ) and 2 µm ( d ) .

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f3a, COS7 cells expressing GFP-SEC61b; and STX17 were immunostained for TOMM20.b, HeLa cells expressing GFP-ATG14 were immunostained for STX17.c, Immunoblot of subcellular fractions of HEK293 cells.d, STX17 knockdown COS7 cells expressing GFP-ATG14 and RFP-SEC61b; were immunostained for TOMM20. Error bars denote s.d.e, Immuoblot of subcellular fractions of STX17 knockdown (using two STX17 siRNAs, STX17-1 and STX17-2) HEK293 cells expressing GFP-DFCP1.f, Co-immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag-ATG14 and Myc-STX17.g, Pull-down of ATG14 complex from HeLa cells expressing Myc-STX17 or Myc. Scale bars, 5 µm (a, b) and 2 µm (d).


f4a , Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP - LC3 ( white arrows ) infected with GAS ( red arrows ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole . b , Electron micrographs of STX17 knockdown cells . White arrows denote autolysosomes , black arrows denote autophagosome , white arrowheads mark isolation membranes . c , Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP - LC3 . d , GFP - ATG5 dots ( arrows ) in STX17 knockdown HeLa cells . e , Quantification of staining in d . f , Immunoblot of p62 in STX17 knockdown cells . Tubulin was used as a loading control . g , Degradation of long - lived proteins in STX17 knockdown cells . St , starved ; wor , wortmannin . All error bars denote s . d . Scale bars , 1 µm ( a , b ) , 200 nm ( c ) and 5 µm ( d ) .

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f4a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP-LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole.b, Electron micrographs of STX17 knockdown cells. White arrows denote autolysosomes, black arrows denote autophagosome, white arrowheads mark isolation membranes.c, Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP-LC3.d, GFP-ATG5 dots (arrows) in STX17 knockdown HeLa cells. e, Quantification of staining in d.f, Immunoblot of p62 in STX17 knockdown cells. Tubulin was used as a loading control.g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).


Figure 2PS decarboxylase activity in wild - type ( WT , W303 ) and the indicated mutant strains expressed as ( B ) the molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) , after 20 min incubation with 13C315N - serine . ( C ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species indicated .

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Figure 2PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


Figure 3PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ( B ) the molecular species composition of * PS and * PE after 20 min incubation with 13C315N - serinePS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) the corresponding molecular signatures of PSD activity .

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Figure 3PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serinePS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


Figure 4 ( B ) Acyl chain composition of WT and psd1 ∆ cells overexpressing SCT1 ( pSCT1 ) vs . empty vector control ( pEmpty ) . Data is presented as mean ± SD ( n > 10 ) . * * * * p < 0 . 0001 , multiple t - test . ( C ) Ratio of the proportions of saturated over unsaturated acyl chains ( SFA / UFA ) of WT , psd1 ∆ , crd1 ∆ and psd2 ∆ overexpressing SCT1 vs . empty vector control . Data is presented as mean ± SD ( n ≥ 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed t - test . ( D ) OLE1 transcript levels in WT and psd1 ∆ transformed with pSCT1 vs . pEmpty as determined by RT - qPCR . Data were normalized to endogenous ACT1 mRNA , and to the average of WT pEmpty ( n = 3 ) .

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

Figure 4(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).


Figure 5 ( A ) SFA / UFA ratios of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . control ( pEmpty ) . Data is presented as mean ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n . s . not significant , unpaired two - tailed t - test of the indicated bar compared to WT pSCT1 . ( B ) Growth of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . pEmpty . Serial dilutions ( 10 - 1 - 10 - 4 ) were spotted on SGal + 0 . 05 % glucose and incubated for 3 days at 30 ° C . PS decarboxylase activity in wild - type ( WT , BY4741 ) and the indicated mutant strains transformed with pEmpty , expressed , and ( D ) molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) after 20 min incubation with 13C315N - serine . The differences in molecular species profiles of * PS and * PE between the BY4741 WT shown here and those obtained by 2H3 - serine labeling ( Figure 4A ) are attributed to the different culture media , lipid extraction procedures and mass spectrometry techniques used . ( E ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species indicated .

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Figure 5(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


Figure 6SFA / UFA - ratio of WT and indicated mutant strains overexpressing SCT1 cultured without ( data taken from Fig 5A ) and with 1 mM ethanolamine ( etn ) or 1 mM choline ( cho ) as indicated . Data is presented as average ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t - test of the indicated bar compared to the corresponding condition of WT . Underlying data for this figure can be found in Appendix Table S1 .

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Figure 6SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.


Figure 1A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions . Autophagic bodies typically contain cytosolic ribosomes . pep4 ∆ prb1 ∆ and pep4 ∆ prb1 ∆ atg2 ∆ cells were grown in SD to mid ‐ log phase ( OD600 = 1 ) and transferred to SD ( ‐ N ) for 5 h . The cells were examined by transmission electron microscopy as described in . Scale bar , 500 nm . B - D Time ‐ dependent changes in nucleoside contents under nitrogen starvation ( B ) , rapamycin treatment ( C ) , and carbon starvation ( D ) conditions . Wild ‐ type and atg2Δ cells were grown in SD and transferred to SD ( ‐ N ) , SD medium with 0 . 2 μM rapamycin , or SD ( ‐ C ) at time 0 . At the indicated time points , nucleosides were analyzed by LC / MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild ‐ type cells . All data are means of triplicate samples . The error bars represent the standard deviation .

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Figure 1A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.B-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.


Figure 2A Time ‐ dependent changes in nucleoside contents under nitrogen starvation . Nucleosides in wild ‐ type , atg2Δ , atg7Δ , atg11Δ , atg17Δ , atg19Δ , atg32Δ , nvj1Δ , and pep4prb1Δ cells under nitrogen starvation . All data are means of triplicates . The error bars represent the standard deviation . B Alkaline phosphatase ( ALP ) assay ( Pho8 ∆ 60 ) . Wild ‐ type , atg2 ∆ , atg11Δ , atg17Δ , and atg19Δ cells expressing Pho8 ∆ 60 were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , lysates were prepared and subjected to the ALP assay . The bars represent the standard deviation of three independent experiments . * P 0 . 05 ; * * P 0 . 005 ; * * * P 0 . 0005 ( paired t ‐ test , two ‐ tailed )

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Figure 2A Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.B Alkaline phosphatase (ALP) assay (Pho8∆60). Wild‐type, atg2∆, atg11Δ, atg17Δ, and atg19Δ cells expressing Pho8∆60 were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, lysates were prepared and subjected to the ALP assay. The bars represent the standard deviation of three independent experiments. *P 0.05; **P 0.005; ***P 0.0005 (paired t‐test, two‐tailed)


Figure 3A - C Wild ‐ type , atg2Δ , pho8Δ , and rny1Δ cells were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , nucleosides ( A ) were analyzed by LC / MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild ‐ type cells . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . All data are means of triplicates . The error bars represent the standard deviation . A - C Wild ‐ type , atg2Δ , pho8Δ , and rny1Δ cells were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , 5 ′ ‐ NMPs ( B ) were analyzed by LC / MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild ‐ type cells . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . All data are means of triplicates . The error bars represent the standard deviation . A - C Wild ‐ type , atg2Δ , pho8Δ , and rny1Δ cells were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , 3 ′ ‐ NMPs ( C ) were analyzed by LC / MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild ‐ type cells . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . All data are means of triplicates . The error bars represent the standard deviation .

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Figure 3A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, nucleosides (A) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 5′‐NMPs (B) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 3′‐NMPs (C) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


Figure 4A , B Expression and localization of Rny1 and Pho8 . Wild ‐ type cells and atg2Δ cells expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged proteins were observed by fluorescence microscopy ( A ) . Scale bar , 5 μm . A , B Expression and localization of Rny1 and Pho8 . Wild ‐ type cells and atg2 ∆ cells expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged proteins were analyzed by Western blot with anti ‐ GFP antibody ( B ) . C Detection of free RNA . Wild ‐ type , atg2 ∆ , rny1 ∆ , and rny1 ∆ atg2 ∆ cells grown in SD ( left ) or SD ( ‐ N ) for 2 h ( right ) were stained with FM4 ‐ 64 and GR Green and observed by fluorescence microscopy . Scale bar , 5 μm .

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

Figure 4A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2Δ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were observed by fluorescence microscopy (A). Scale bar, 5 μm.A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2∆ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were analyzed by Western blot with anti‐GFP antibody (B).C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


Figure 5A Time ‐ dependent change in nucleoside levels after nitrogen starvation . Wild ‐ type , atg2 ∆ , ubp3 ∆ , and bre5 ∆ cells were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . Metabolites were analyzed by LC / MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild ‐ type cells . All data are means of triplicates . The error bars represent the standard deviation . B Detection of free RNA within cells . rny1 ∆ , rny1 ∆ atg2 ∆ , rny1 ∆ ubp3 ∆ , and rny1 ∆ bre5 ∆ cells grown in SD ( ‐ N ) for 2 h were stained with FM4 ‐ 64 and GR Green and observed by fluorescence microscopy . Scale bar , 5 μm .

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Figure 5A Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.B Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


Figure 6B , C Nucleosides ( B ) and nucleobases ( C ) within the cells . Wild ‐ type and atg2 ∆ were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , the cells were collected ; their metabolites were extracted , and the nucleosides and nucleobases were analyzed by LC / MS . The level of each nucleoside and base is presented as its absolute concentration , as described in and Supplementary Methods . All data are means of duplicates except the wild ‐ type strain at 1 h . D , E Nucleosides ( D ) and nucleobases ( E ) in the medium . The culture medium of the above cultures was filtered , and nucleosides and nucleobases were directly analyzed by LC / MS as described in and Supplementary Methods . All data are means of duplicates . F , G Destinations of nucleosides and bases . Based on the data from ( B - E ) , the level of each nucleoside and base in cells ( open circle ) and in cells and medium ( closed circle ) of wild ‐ type strain were estimated . See Supplementary Methods in detail ( flow chart of the calculation procedure ) .

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Figure 6B, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h.D, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates.F, G Destinations of nucleosides and bases. Based on the data from (B-E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).


Figure 7Wild ‐ type and atg1Δ of S . pombe were grown in SD to mid ‐ log phase and transferred to SD ( ‐ N ) at time 0 . At the indicated time points , the cells were collected ; the metabolites in these cells were extracted and analyzed by LC / MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild ‐ type cells . All data are means of triplicates . The error bars represent the standard deviation .

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

Figure 7Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.


Figure 1Immunoprecipitation was performed with GFP - binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings , expressing either YFP - MKKK7 or Lti6B . Seedlings were treated with 1 μM flg22 for the indicated time . YFP - MKKK7 and Lti6B - GFP were detected with an anti - GFP antibody while FLS2 was detected with an FLS - specific antibody . Upper panel shows ( co ) - immunoprecipated proteins , lower panel shows input levels of protein . Arrowheads indicate the position of proteins of interest .

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Figure 1Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.


Figure 2Application of selected reaction monitoring ( SRM ) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22 . Bars represent the mean ratio of endogenous phosphopeptide versus spiked - in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM ( n = 3 . ) . Asterisks indicate significant difference level compared to t = 0 ( student t - test , * > 0 . 05 , * * > 0 . 01 and * * * > 0 . 001 ) . The color of each bar corresponds to the different time points ( 0 minutes = dark blue , 5 minutes = red , 10 minutes = green , 20 minutes = purple , 30 minutes = light blue ) . Above each graph is the protein name and the phosphorylated residue ( in brackets ) is indicated and below the corresponding phosphopeptide is shown with the Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation site indicated by " [ + 80 ] " .

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Figure 2Application of selected reaction monitoring (SRM) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22. Bars represent the mean ratio of endogenous phosphopeptide versus spiked-in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM (n=3.). Asterisks indicate significant difference level compared to t = 0 (student t-test, *>0.05, **>0.01 and ***>0.001). The color of each bar corresponds to the different time points (0 minutes = dark blue, 5 minutes = red, 10 minutes = green, 20 minutes = purple, 30 minutes = light blue). Above each graph is the protein name and the phosphorylated residue (in brackets) is indicated and below the corresponding phosphopeptide is shown with the Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation site indicated by "[+80]".


Figure 3A ) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col - 0 and in mkkk7 . Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t = 0 , 10 and 30 min post induction . The p44 / 42 antibody was used to detect phosphorylated MAPKs . Position of the individual phosphorylated MAPKs is indicated at the right . Equal loading of proteins is shown with an α - Actin antibody as a loading control ( bottom panel ) . Three biological replicates were done with identical resultsB ) MPK6 phosphorylation is specifically enhanced in mkkk7 . Comparison of phosphopeptide abundances from selected MAP kinases in Col - 0 ( blue ) and mkkk7 ( red ) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring ( SRM ) mass spectrometry . Phosphopeptide corresponding to MKKK7 are only detectable in Col - 0 seedlings and are non - detectable ( ND ) in mkkk7 . Bars represent means of measured peptide areas ( sum of all transition areas ) for three biological replicates , with error bars ± SEM ( n = 3 ) . Asterisks indicate significant difference between Col - 0 and mkkk7 at individual time points ( student t - test , * > 0 . 05 , * * > 0 . 01 and * * * > 0 . 001 ) . ND indicates integration of an area without transitions significantly above background . Above each graph the protein name and the phosphorylated residue ( in brackets ) is indicated as well as the corresponding phosphopeptide sequence . Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation is indicated by " [ + 80 ] " .

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

Figure 3A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical resultsB) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by "[+80]".


Figure 4A ) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment . Protoplasts were isolated from 4 week old plants and transfected with pWRKY29 : fLUC ( WRKY29 ) or pFRK1 : fLUC ( FRK1 ) constructs together with 35S : rLUC , as indicated in the graph . Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated . The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample , as fold induction . All measurements were normalized to the rLUC activity . Bars represent means ± STDEV ( n = 2 ) . Experiment was repeated 6 times with similar results . B ) WRKY29 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 and mkkk7 were treated with 1 µM flg22 for t = 0 , 1 , 2 , and 4h . WRKY29 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value and error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - test ) . C ) FRK1 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 and mkkk7 were treated with 1 µM flg22 fort = 0 , 1 , 2 , and 4h . FRK1 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value and error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - test ) .

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Figure 4A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results.B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


Figure 5A ) Transient co - expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22 - induced WRKY29 gene expression . Protoplasts were transfected with pWRKY29 : fLUC , 35S : rLUC and indicated overexpression constructs of MKKK7 ( OE - MKKK7 , OE - MKKK7AA or OE - MKKK7DD ) as indicated on the horizontal axis . Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs . All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample , shown as fold induction on the vertical axis . Results shown are means ± STDEV ( n = 2 ) . At least two biological replicates were done with similar

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Figure 5A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results


Figure 6A ) Four - week - old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored . Data represents mean values ± SEM ( n = 20 ; * * * , p < 0 . 001 ; paired t - test ) . Three biological experiments were done showing similar results . B ) Quantification of bacterial growth in Arabidopsis lines Col - 0 , mkkk7 and p35S : MKKK7 - GFP in the mkkk7 background . Four to five - week - old plants were pressure - infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re - isolated on selective media . The number of colony forming units ( cfu / cm2 ) was determined at t = 0 , 2 and 3days post inoculation ( dpi ) . Data represents mean values ± SEM ( n = 6 ; * * , p < 0 . 01 ; paired t - test ) . Experiments were done at least twice with similar results . C ) Disease symptom development in Pst - infected lines with estradiol inducible constructs of ind - MKKK7AA L8 , ind - MKKK7AA L10 , ind - MKKK7DD L1 and ind - MKKK7DD L3 . Two independent transgenic lines for each construct were grown under short - day conditions and disease symptoms were scored 3 dpi . Data represents mean values ± SEM ( n = 20 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; paired t - test ) . The vertical axis represents the percentage disease symptoms . Experiments were done at least twice with similar results .

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Figure 6A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results.B) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results.C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.


Figure 7A ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col - 0 and mkkk7 . B ) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col - 0 and two independent inducible MKKK7AA transgenic lines . C ) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col - 0 and two independent inducible MKKK7DD transgenic lines . Graphs represent means with error bars ± SEM ( n = 24 ) . The vertical axis represents the relative increase in ROS production ( photon counts ) after PAMP treatment . At least three biological replicate experiments were done with similar results .

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Figure 7A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7.B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.


Figure 1 ( A ) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15 - days old ( upper panel ) and 1 - month old animals ( lower panel ) . ( B ) Body weight comparison over time of Ndufs3 smKO male mice ( filled blue squares ; n = 14 ) and age matched wild - type ( WTFlx ; n = 15 ) males ( empty blue squares ) . Ndufs3 smKO females ( filled red circles ) showed similar results ( n = 10 for smKO , and n = 12 for WTFlx group ) . P values were calculated by Student ' s t test ; ( C ) Representative image of a smKO male Ndfu3s3 - smKO mouse and a WTFlx littermate of 6 - month old showing decreased body weight . ( D ) Survival curve of Ndufs3 smKO female mice ( red line ) and males ( blue line ) . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0010 for males ( n = 19 ) , and p = 0 . 0004 for females ( n = 10 ) . ( E ) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild - type littermates from 1 and 2 months old male mice . P values were calculated by Student ' s t test . P values were calculated by Student ' s t test to determine the level of statistical difference . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 . ( F ) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice , starting at 2 months ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t test to determine the level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 . ( G ) Rotarod performed by Ndufs3 smKO and wild - type male mice . ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t test to determine the level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 .

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

Figure 1(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel).(B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test;(C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight.(D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10).(E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. P values were calculated by Student's t test to determine the level of statistical difference. ; *p<0.05, **p<0.01 and ***p<0.0001.(F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.(G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.


Figure 2 ( A ) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age . Muscle weight from Ndufs3 smKO males ( black bar ) and wild - type mice ( white bar ) from 6 , and 8 months of age ( n = 5 each group ) . Bars represent means ± standard error ( SEM ) . ( B ) Representative H & E , COX , and SDH staining of quadriceps from WTFlx and Ndufs3 - Mlc1f smKO at 8 months old . ( C ) Cross - sectional area ( CSA ) of quadriceps muscle fibers . A minimum of 100 fibers / sample were analyzed ( n = 3 / genotype ) . The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months ( n = 3 mice for WTFlx and n = 4 mice for Ndufs3 smKO ) . P values were calculated by Student ' s t test . ( D ) Serum lactate levels of WTFlx and Ndufs3 - smKO animals at different ages . Ndufs3 smKO male mice ( filled black squares ) showed increased levels compared to matched WTFlx males ( empty black squares ) . Error bars represent SEM . ( E ) BN - PAGE In - Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild - type and Ndufs3 - Mlc1f smKO animals at 2 and 4 months . ( F - G ) Western blots of muscle homogenates ( quadriceps ) of WTFlx and Ndufs3 - Mlc1f smKO animals at different ages ( 2 and 4 months , respectively ) using antibodies against PGC - 1α , COX1 ( complex IV subunit ) , NDUFS3 , VDAC1 , SDHA ( complex II subunit ) , and β - actin . ( H ) Quantification of the western blots in panels F and G . Bars represent means ± SEM of n = 4 for each group . P values were determined by Student ' s t test .

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

Figure 2(A) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age. Muscle weight from Ndufs3 smKO males (black bar) and wild-type mice (white bar) from 6, and 8 months of age (n=5 each group). Bars represent means ± standard error (SEM).(B) Representative H&E, COX, and SDH staining of quadriceps from WTFlx and Ndufs3-Mlc1f smKO at 8 months old.(C) Cross-sectional area (CSA) of quadriceps muscle fibers. A minimum of 100 fibers/sample were analyzed (n =3/genotype). The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months (n =3 mice for WTFlx and n=4 mice for Ndufs3 smKO). P values were calculated by Student's t test.(D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM.(E) BN-PAGE In-Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild-type and Ndufs3-Mlc1f smKO animals at 2 and 4 months.(F-G) Western blots of muscle homogenates (quadriceps) of WTFlx and Ndufs3-Mlc1f smKO animals at different ages (2 and 4 months, respectively) using antibodies against PGC-1α, COX1 (complex IV subunit), NDUFS3, VDAC1, SDHA (complex II subunit), and β-actin. (H) Quantification of the western blots in panels F and G. Bars represent means ±SEM of n=4 for each group. P values were determined by Student's t test.


Figure 3 ( C ) The KO - GFP mouse was visibly smaller and weaker than a KO - NDUFS3 mouse , at 7 months . ( D ) Females body weight injected with rAAV9 - Ndufs3 ( heretofore referred to as KO - NDUFS3 ) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9 - eGFP injected mice ( KO - GFP ) starting at 5 months . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group . ( E ) Males body weight of KO - NDUFS3 mice were similar to WTFlx mice , and significantly different from KO - GFP from 4 months onwards . Statistical analysis was performed by One - way ANOVA followed by a Tukey post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group . ( F ) Treadmill performance was significantly improved in the KO - NDUFS3 mice when compared to the KO - GFP , from 2 months onwards . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group . ( G ) Four limb strength was evaluated by the grid hang test . KO - NDUFS3 mice performed significantly better starting at 3 months , when compared to the KO - GFP mice . F ) and G ) Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group . ( H ) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO - NDUFS3 mice . Quadriceps weight was significantly increased in KO - NDUFS3 mice when compared to KO - GFP injected . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . ns - not significant . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group . ( I ) Survival curve of Ndufs3 smKO mice injected with rAVV9 - Ndufs3 ( blue line ) and with rAVV9 - eGFP ( green line ) . WTFlx mice are depicted in black . rAAV9 - Ndufs3 injections extended the survival of the smKO mice .

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

Figure 3(C) The KO-GFP mouse was visibly smaller and weaker than a KO-NDUFS3 mouse, at 7 months.(D) Females body weight injected with rAAV9-Ndufs3 (heretofore referred to as KO-NDUFS3) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9-eGFP injected mice (KO-GFP) starting at 5 months. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(E) Males body weight of KO-NDUFS3 mice were similar to WTFlx mice, and significantly different from KO-GFP from 4 months onwards. Statistical analysis was performed by One-way ANOVA followed by a Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(F) Treadmill performance was significantly improved in the KO-NDUFS3 mice when compared to the KO-GFP, from 2 months onwards. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(G) Four limb strength was evaluated by the grid hang test. KO-NDUFS3 mice performed significantly better starting at 3 months, when compared to the KO-GFP mice. F) and G) Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.(I) Survival curve of Ndufs3 smKO mice injected with rAVV9-Ndufs3 (blue line) and with rAVV9-eGFP (green line). WTFlx mice are depicted in black. rAAV9-Ndufs3 injections extended the survival of the smKO mice.


Figure 4 ( A ) Western blots of 6 months old quadriceps of WTFlx , KO and KO - NDUFS3 injected mice at 15 - 18 days old . GFP expression was observed only in the KO - GFP mice . Protein loading was obtained with stain - free gel technology . ( B ) Quantification of NDUFS3 , COXI and SDHA expression levels in female mice showed in A . ( C ) Western blot of BN - PAGE of quadriceps of 6 - month - old mice . We analyzed : CI ( NDUFA9 , NDUFB8 ) , CIV ( COX1 ) , CII ( SDHA ) . ( D ) Quantification of the BN - PAGE western blots from female mice shown in panel C . ( E ) BN - PAGE in - gel activity of CI and CIV in quadriceps of 6 - month - old mice . ( F ) Quantification of BN - PAGE in - gel activities in females . ( G ) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1 / 18S . Inset shows the appearance of the quadriceps , reflecting normalization of mitochondrial levels after replacement of NDUFS3 . ( H ) Spectrophotometric CI / citrate synthase activity ratio was measured in quadriceps homogenates of 6 - month - old males and females . ( I ) Plasma lactate levels were determined as described in methods . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test .

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

Figure 4(A) Western blots of 6 months old quadriceps of WTFlx, KO and KO-NDUFS3 injected mice at 15-18 days old. GFP expression was observed only in the KO-GFP mice. Protein loading was obtained with stain-free gel technology. (B) Quantification of NDUFS3, COXI and SDHA expression levels in female mice showed in A.(C) Western blot of BN-PAGE of quadriceps of 6-month-old mice. We analyzed: CI (NDUFA9, NDUFB8), CIV (COX1), CII (SDHA). (D) Quantification of the BN-PAGE western blots from female mice shown in panel C.(E) BN-PAGE in-gel activity of CI and CIV in quadriceps of 6-month-old mice. (F) Quantification of BN-PAGE in-gel activities in females.(G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3.(H) Spectrophotometric CI/citrate synthase activity ratio was measured in quadriceps homogenates of 6-month-old males and females.(I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.


Figure 5 ( B ) The smKO mice group that received retro - orbital injections at 2 months was tested prior to the rAAV9 - Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill . The KO - NDUFS3 gradually recovered over time . Individual KO - NDUFS3 mice are color coded . Green triangles represent the animals injected with GFP whereas white triangles represent non - injected mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . ( C ) The hang test was performed in symptomatic mice which had a significant decrease in four - limb muscle strength and coordination , at 2 months . The KO - NDUFS3 mice showed a significant recovery 3 . 5 months after injection . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . ( D ) KO - NDUFS3 muscle weight was similar to WTFlx mice , and it was significantly increased in gastrocnemius when compared to KO - GFP mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . ( E ) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6 - month - old animals . The expression of NDUFB8 ( representative of assembled CI ) was recovered in the KO - NDUFS3 mice and COX1 expression was similar to WTFlx animals . ( F ) COX and COX / SDH activity staining showed that KO - NDUFS3 quadriceps were reverted to levels found in wild - type , at 6 months . n - indicates the number of animals used per group , scale : 100 µm .

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

Figure 5(B) The smKO mice group that received retro-orbital injections at 2 months was tested prior to the rAAV9-Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill. The KO-NDUFS3 gradually recovered over time. Individual KO-NDUFS3 mice are color coded. Green triangles represent the animals injected with GFP whereas white triangles represent non-injected mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(C) The hang test was performed in symptomatic mice which had a significant decrease in four-limb muscle strength and coordination, at 2 months. The KO-NDUFS3 mice showed a significant recovery 3.5 months after injection. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(D) KO-NDUFS3 muscle weight was similar to WTFlx mice, and it was significantly increased in gastrocnemius when compared to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.(E) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6-month-old animals. The expression of NDUFB8 (representative of assembled CI) was recovered in the KO-NDUFS3 mice and COX1 expression was similar to WTFlx animals.(F) COX and COX/SDH activity staining showed that KO-NDUFS3 quadriceps were reverted to levels found in wild-type, at 6 months. n-indicates the number of animals used per group, scale: 100 µm.


Figure 6 ( A ) Western blots showed NDUFS3 expression in KO - NDUFS3 and GFP expression in the KO - GFP quadriceps samples . PGC - 1α expression was normalized in the KO - NDUFS3 mice which was abnormally elevated in the KO - GFP . The same was observed for CIV ( COX1 ) , CIII ( Core 2 ) and CII ( SDHA ) . ( B ) Quantification of western blots showed in A . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM . ( C ) BN - PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO - NDUFS3 injected mice . CIV and CII were similar to wild - type in the KO - NDUFS3 samples , at 6 months . ( D ) Quantification of BN - PAGE western blots Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM . ( E ) BN - PAGE In - gel CI and IV activities of KO - NDUFS3 mice were also reverted to levels similar to the WTFlx samples ( F ) Quantification of in - gel activities . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM . ( G ) Complex I / Citrate synthase activity of KO - NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO - GFP mice . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM . ( H ) Total mtDNA levels were determined by qPCR . MtDNA levels in KO - NDUFS3 mice were decreased when compared to KO - GFP mice and not different from the levels in WTFlx mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM . ( I ) The serum lactate levels were similar to WTFlx in KO - NDUFS3 samples and significantly decreased when compared to KO - GFP levels . The individual KO - NDUFS3 animals are color coded . Green triangles represent the animals injected with rAAV9 - eGFP whereas black triangles represent animals which did not receive an rAAV9 - eGFP injection . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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

Figure 6(A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months.(D) Quantification of BN-PAGE western blots Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.(I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.