B . The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin ( H & amp ; amp ; E ) stained sections ( scale bar 90 µm ) .

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B. The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&amp;E) stained sections (scale bar 90 µm).


C . Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret ' s diameter .

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C. Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter.


D . Average ± SD of minimum Feret ' s diameter in quadriceps ( p = 0 . 26 ) and tibialis anterior ( p = 0 . 33 ) muscles is not significantly different between summer and hibernation .

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D. Average ± SD of minimum Feret's diameter in quadriceps (p = 0.26) and tibialis anterior (p = 0.33) muscles is not significantly different between summer and hibernation.


A . Western blot of quadriceps muscle from summer active ( S ) and hibernating ( H ) squirrels using antibodies against the proteins indicated . An increased abundance of P − Foxo3a ( serine − 253 ) is accompanied by a decrease in P − Akt ( serine − 478 ) . Corresponding densitometry of P − Akt and P − Foxo3a as a function of total Akt and Foxo3a .

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A. Western blot of quadriceps muscle from summer active (S) and hibernating (H) squirrels using antibodies against the proteins indicated. An increased abundance of P−Foxo3a (serine−253) is accompanied by a decrease in P−Akt (serine−478). Corresponding densitometry of P−Akt and P−Foxo3a as a function of total Akt and Foxo3a.


B . Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation .

B. Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation.


C . Western blots analysis of mTOR downstream targets shows significant upregulation of P − P70 and P − 4E − BP1 during hibernation .

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C. Western blots analysis of mTOR downstream targets shows significant upregulation of P−P70 and P−4E−BP1 during hibernation.


A . Western blot analyses and densitometry of quadriceps muscles from non − hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1 . Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation .

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A. Western blot analyses and densitometry of quadriceps muscles from non−hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation.


B . Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation . Densitometric analysis of P ‐ Foxo3a as a function of Foxo3a .

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B. Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P‐Foxo3a as a function of Foxo3a.


A . The morphology of tibialis anterior muscle as revealed by haematoxylin - eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers ( scale bar 90 µm ) .

A.The morphology of tibialis anterior muscle as revealed by haematoxylin-eosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers (scale bar 90 µm).


B . Percentage distribution and mean minimum Feret ' s diameter in tibialis anterior muscle ( p = 0 . 02 ) .

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B. Percentage distribution and mean minimum Feret's diameter in tibialis anterior muscle (p = 0.02).


C . Increased levels of phosphorylated Akt ( S478 ) in skeletal muscles of sgk1 − / − mice .

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C. Increased levels of phosphorylated Akt (S478) in skeletal muscles of sgk1−/− mice.


D . sgk1 − / − mice exhibit decreased running distance after 36 days of exposure to a running wheel , ( p = 0 . 01 ; n = 6 animals per group ) .

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D. sgk1−/− mice exhibit decreased running distance after 36 days of exposure to a running wheel, (p = 0.01; n = 6 animals per group).


E . Isometric force from soleus muscles of WT and sgk1 - / - mice during repetitive electrical stimulations for 500 ms . Amplitudes of isometric force from soleus muscles at two stimulation frequencies . Specific force is plotted in all cases . Mean values ± SD from n = 9 ( sgk1 − / − ) and n = 10 ( WT ) independent animals ( p = 0 . 01 ) .

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E. Isometric force from soleus muscles of WT and sgk1-/- mice during repetitive electrical stimulations for 500 ms. Amplitudes of isometric force from soleus muscles at two stimulation frequencies. Specific force is plotted in all cases. Mean values ± SD from n = 9 (sgk1−/−) and n = 10 (WT) independent animals (p = 0.01).


A . H & amp ; amp ; E staining of tibialis anterior sections from 2 ‐ month old control and transgenic mice shows no differences ( scale bar 90 µm ) .

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A. H&amp;E staining of tibialis anterior sections from 2‐month old control and transgenic mice shows no differences (scale bar 90 µm).


B . Morphometric analyses of minimum Feret ' s diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size .

B. Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size.


C . Western blots and densitometric analysis of muscle from transgenic and control mice . Downstream targets of the mTOR signalling cascade ( p70S6K and 4EBP1 ) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice .

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C. Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice.


D . Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice ( n = 4 ) show an increase of phosphorylation at S315 and T32 .

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D. Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n = 4) show an increase of phosphorylation at S315 and T32.


A . H & amp ; amp ; E staining of tibialis anterior sections reveals no changes in muscle architecture ( scale bar 90 µm ) .

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A. H&amp;E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90 µm).


B . Morphometric analysis demonstrates a decrease in muscle fiber size of the wild − type control mice compared with sgk1tg transgenic littermates ( p = 0 . 0015 ) .

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B. Morphometric analysis demonstrates a decrease in muscle fiber size of the wild−type control mice compared with sgk1tg transgenic littermates (p = 0.0015).


C . Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg .

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C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.


A . Transfection of constitutively active Sgk1 ( CA ) into immobilized tibialis anterior muscle ( green ) reveals increased fiber size diameter when compared to control , EGFP only transfected muscle fibers of immobilized tibialis anterior muscles ( 100 µm ) . Laminin γ − 1 staining ( red ) outlines the basement membrane and blue staining marks nuclei ( DAPI ) .

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A. Transfection of constitutively active Sgk1 (CA) into immobilized tibialis anterior muscle (green) reveals increased fiber size diameter when compared to control, EGFP only transfected muscle fibers of immobilized tibialis anterior muscles (100 µm). Laminin γ−1 staining (red) outlines the basement membrane and blue staining marks nuclei (DAPI).


B . Representation of the minimum Feret ' s diameter average ± SD ( control and immobilized ; n = 9 - 10 per group ; 800 - 950 fibers were measured ) ( * p = 0 . 029 , * p = 0 . 034 , * p = 0 . 047 , p = 0 . 43 , respectively ) .

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B. Representation of the minimum Feret's diameter average ± SD (control and immobilized; n = 9-10 per group; 800-950 fibers were measured) (*p = 0.029, *p = 0.034, *p = 0.047, p = 0.43, respectively).


C . Percentage distribution of the minimum Feret ' s diameter of tibialis anterior muscle immobilized and transfected with eGFP ( GFP ) , wild − type Sgk1 ( WT ) , kinase dead Sgk1 ( KD ) and constitutively active Sgk1 ( CA ) compared to non − immobilized control TA muscle ( black dotted line ) .

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C. Percentage distribution of the minimum Feret's diameter of tibialis anterior muscle immobilized and transfected with eGFP (GFP), wild−type Sgk1 (WT), kinase dead Sgk1 (KD) and constitutively active Sgk1 (CA) compared to non−immobilized control TA muscle (black dotted line).


A - F Immunostaining of primary HL with H2A . B ( brown , indicated by black arrows ) with a nuclear counterstain ( blue ) . Human testis ( positive control ) ( A ) , Human tonsils ( negative control ) ( B ) , Nodular sclerosis classical cHL , double staining with CD15 ( pink ) to identify HRS cells and H2A . B ( brown ) ( C ) , Mixed cellularity cHL ( D ) , Lymphocyte rich cHL ( E ) , Nodular sclerosis HL ( F ) . Scale bar for panels A , B , D - F is 50 µm ; scale bar for panel C is 15 µm .

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A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm.


A Relative mRNA expression levels of H2A . B in L1236 , L428 , HDLM2 HL cell lines and Germinal centre B lymphocytes ( GC ) compared to the Ramos B lymphocyte cell line . The results are presented as the mean ± SD of three biological replicates .

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A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates.


D H2A . B interacts with Pol I . Western blot showing co - immunoprecipitation of RPA194 with H2A . B . The Pol I subunit RPA194 was immunoprecipitated from L1236 ( left panel ) and L428 ( right panel ) nuclear extracts . The pull - down by RPA194 antibody ( IP ) , pull - down by empty beads or IgG isotype control , and 1 % input protein were loaded for Western blot detection using anti - H2A . B and anti - RPA194 antibodies .

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D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.


E H2A . B interacts with RPA194 in live cells . Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells ( left panel ) , and L428 cells ( right panel ) . The samples were prepared using pull - down with streptavidin beads from the cell lysate of the empty vector , BirA * - H2A . B and H2A . B - BirA * transduced cells , which were preincubated with 50 µM biotin for 24 h . The positive control was loaded as 1 % input .

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E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells (left panel), and L428 cells (right panel). The samples were prepared using pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin for 24 h. The positive control was loaded as 1% input.


B Cell proliferation assay in the presence ( shNTC ) or during depletion of H2A . B ( shH2A . B ) . The results are presented as mean ± SD for five biological replicates . The significance of the difference was determined by Student ' s t test and denoted as ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.


E Genome browser screen shots of gene examples ( RPs RPLP1 and RPL23A , and HDAC5 and TAF10 ) that contain H2A . B at their promoters identified by the CUT & RUN peak calling and whose expression decreased significantly following H2A . B knockdown in the RNA - seq analysis . Green and purple indicates L1236 and L428cells , respectively .

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E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.


B Differential exon usage of the CD44 gene . C . Differential exon usage of the RPL17 gene .

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B Differential exon usage of the CD44 gene. C. Differential exon usage of the RPL17 gene.


D , E Two examples show increased accessibility at the promoter peak ( NXF1 ) ( D ) and decreased accessibility at the peak across the intron - exon boundary ( KDM4A ) ( E ) , respectively .

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D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.


Module - trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples . Gene modules ( y axis ) are denoted by an arbitrary color name . Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype / disease stage ( x axis ) and p values . A value of 1 ( red ) quantifies the strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) the strongest negative correlation ( genes are downregulated ) , and 0 ( white ) no correlation .

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Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.


Microarray analysis was performed following doxycycline - regulated specific AR - V7 depletion ( using a tet - plKO backbone ) in 22Rv1 PC cells compared to doxycycline - treated shGFP controls . The genes that were significantly regulated by shAR - V7 ( in either direction , p value < 0 . 05 ) were distributed among the gene modules defined by WGCNA in panel A . Upregulated genes ( red ) are those in which expression decreased following AR - V7 depletion and conversely downregulated genes ( blue ) are those that increased following AR - V7 depletion .

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Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.


Hornberg et al . , 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases , grouped by their relative levels of AR - VS , mainly AR - V7 . ( High - levels of AR - V7 ( top quartile ) or lower levels of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as the mean ± s . e . m . . Non - parametric Mann - Whitney test was performed ( two - tailed ) . Note that BUB1B expression was not measured in these microarrays . * * Significant at a p value < 0 . 05 . N ( AR - V7 low ) = 20 ; N ( AR - V7 high ) = 10 .

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Hornberg et al., 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases, grouped by their relative levels of AR-VS, mainly AR-V7. (High-levels of AR-V7 (top quartile) or lower levels of AR-V7 (quartiles 1-3). Data are plotted as the mean ± s.e.m.. Non-parametric Mann-Whitney test was performed (two-tailed). Note that BUB1B expression was not measured in these microarrays. ** Significant at a p value < 0.05. N (AR-V7 low) = 20; N (AR-V7 high) = 10.


The Kaplan - Meier curves for Disease - Free Survival ( DFS ) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset ( 465 samples ) ( upper graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of the seven genes were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0009 ; for death : p - value = 0 . 026 . An independent dataset was analyzed ( Prostate Adenocarcinoma MSKCC , Cancer Cell 2010 , 123 samples ) ( lower graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0007 ; for death : p - value = 0 . 00546 .

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The Kaplan-Meier curves for Disease-Free Survival (DFS) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset (465 samples) (upper graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of the seven genes were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0009; for death: p-value = 0.026. An independent dataset was analyzed (Prostate Adenocarcinoma MSKCC, Cancer Cell 2010, 123 samples) (lower graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0007; for death: p-value = 0.00546.


Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls , using shRNA against the coding region for each gene ( shRNA # 2 ) . Cell number was measured using a non - perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System . Data shown are mean ± s . e . m . of 8 to 12 replicates normalized to their shGFP control . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed .

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Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.


Representative images of 22Rv1 stably depleted of BUB1B , or control ( shGFP ) are shown .

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Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.


22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription . The assay was conducted in 2 % CSS to measure AR ligand - independent transcriptional activity . Data represent two independent experiments performed in triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The expression of FKBP5 and UBE2C determined by RT - qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes . Cells were cultured in 2 % CSS . Data represent two independent experiments performed in duplicate or triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The CRPC cell line 22Rv1 was cultured in 2 % CSS media and was treated for 72 hours with vehicle ( DMSO ) , doxorubicin ( DOX ) , N9 - isopropylolomoucine ( N - 9 ) , or the combination of DOX and N - 9 at different concentrations . Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each . The data were analyzed using Compusyn software , and a normalized isobologram was built . The table shows the Combination Index ( CI ) for the different drug combinations . CI = 1 represents additivity , CI < 1 synergism , and CI > 1 antagonistic effects .

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The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.


The non - tumorigenic prostate epithelial cell line RWPE1 , the AR - null PC cell line PC3 , the androgen dependent cell line LNCaP , and the CRPC cell lines C4 - 2B and 22Rv1 were treated for 72 hours with vehicle ( DMSO ) , DOX ( 100 ng / mL [ 184 nM ] ) , N - 9 ( 200 ng / mL [ 613 nM ] ) , or the combination of DOX ( 100 ng / mL [ 184 nM ] ) and N - 9 ( 200 ng / mL [ 613 nM ] ) . C4 - 2B and 22Rv1 cells were kept in 10 % CSS media , and the other cell lines in 10 % FBS . Cell confluence was monitored using the Incucyte Zoom System . Data represent two independent experiments , with four to six replicates each , showing the mean ± s . e . m . , and normalized to vehicle controls ( Kruskal - Wallis test , P value < 0 . 0001 , two - tailed ) . * Significant at a p value < 0 . 05 , * * p value < 0 . 01 , * * * p value < 0 . 001 .

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The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.


The non - tumorigenic prostate cell line RWPE - 1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle ( DMSO ) or the combination of DOX and N - 9 at 100 ng / mL and N - 9 200 ng / mL , respectively . Cell confluence was monitored using the Incucyte Zoom System .

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The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.


Sashimi plots of representative SRSF3 - dependent exons . C2C12 cells were treated with control siRNA ( siCont ) , Srsf3 siRNA ( siSrsf3 ) , Hnrnpk siRNA ( siHnrnpk ) , or both Srsf3 and Hnrnpk siRNAs ( siSrsf3 + siHnrnpk ) , and RNA - seq was performed . Lines indicate junction - spanning reads . Gene structures are shown below the Sashimi plots .

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Sashimi plots of representative SRSF3-dependent exons. C2C12 cells were treated with control siRNA (siCont), Srsf3 siRNA (siSrsf3), Hnrnpk siRNA (siHnrnpk), or both Srsf3 and Hnrnpk siRNAs (siSrsf3+siHnrnpk), and RNA-seq was performed. Lines indicate junction-spanning reads. Gene structures are shown below the Sashimi plots.


Volcano plot showing fold changes versus P - values of the normalized MASCOT scores ( see Materials and Methods ) of identified proteins between 3XFLAG - SRSF3 - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) . Volcano plot showing fold changes versus P - values of the normalized MASCOT scores of identified proteins between 3XFLAG - HNRNPK - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) .

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Volcano plot showing fold changes versus P-values of the normalized MASCOT scores (see Materials and Methods) of identified proteins between 3XFLAG-SRSF3-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).   Volcano plot showing fold changes versus P-values of the normalized MASCOT scores of identified proteins between 3XFLAG-HNRNPK-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).


mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA . * * p < 0 . 01 by CuffDiff ( Trapnell et all . , 2010 ) .

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mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA. **p < 0.01 by CuffDiff (Trapnell et all., 2010).


Western blots showing protein expression levels of CPSF6 and β - actin ( ACTB ) . Whole cell lysates were harvested 42 h after siRNA transfection .

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Western blots showing protein expression levels of CPSF6 and β-actin (ACTB). Whole cell lysates were harvested 42 h after siRNA transfection.


SRSF3 - responsive alternative 3 ′ ends of the Med1 gene . ( Top panel ) Disorder analysis for MED1 using PONDR VL - XT ( deep blue ) and IUPred2 ( light blue ) . The scores in the ordinate indicate disordered tendencies between 0 and 1 ( a score of more than 0 . 5 indicates disordered ) . ( Middle panel ) Protein and gene structures of MED1 . The protein structure was segmented into individual exonic regions . The SRSF3 - responsive last exon is indicated in pink . ( Bottom panel ) Sashimi plots of RNA - seqs from Srsf3 - and / or Hnrnpk - silenced cells .

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SRSF3-responsive alternative 3′ ends of the Med1 gene. (Top panel) Disorder analysis for MED1 using PONDR VL-XT (deep blue) and IUPred2 (light blue). The scores in the ordinate indicate disordered tendencies between 0 and 1 (a score of more than 0.5 indicates disordered). (Middle panel) Protein and gene structures of MED1. The protein structure was segmented into individual exonic regions. The SRSF3-responsive last exon is indicated in pink. (Bottom panel) Sashimi plots of RNA-seqs from Srsf3- and/or Hnrnpk-silenced cells.


Immunofluorescence images of MED1 in C2C12 cells treated with siCont ( C ) , siSrsf3 ( S ) , siHnrnpk ( K ) , or siSrsf3 + siHnrnpk ( S + K ) . A white dotted contour outlines the nucleus . Box plots ( right ) show quantification of nuclear ( NUC ) and cytoplasmic ( CYT ) localization of MED1 . The average intensity of one nucleus or a cytoplasmic segment is individually plotted .

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Immunofluorescence images of MED1 in C2C12 cells treated with siCont (C), siSrsf3 (S), siHnrnpk (K), or siSrsf3+siHnrnpk (S+K). A white dotted contour outlines the nucleus. Box plots (right) show quantification of nuclear (NUC) and cytoplasmic (CYT) localization of MED1. The average intensity of one nucleus or a cytoplasmic segment is individually plotted.


The intrinsically disordered region ( IDR ) in MED4 . Disorder analysis , protein and gene structures , and Sashimi plots are shown in ( B ) . Splicing of these genes was not affected by the siRNA treatment .

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The intrinsically disordered region (IDR) in MED4. Disorder analysis, protein and gene structures, and Sashimi plots are shown in (B). Splicing of these genes was not affected by the siRNA treatment.


Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs . Box plots show quantification of nuclear localization of MED4 . Each dot represents one nucleus .

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Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs. Box plots show quantification of nuclear localization of MED4. Each dot represents one nucleus.


Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta , including both MED1 and MED4 .

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Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta, including both MED1 and MED4.


The number of MED1 puncta ( left ) and MED4 puncta ( right ) per nucleus in cells treated with the indicated siRNA .

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The number of MED1 puncta (left) and MED4 puncta (right) per nucleus in cells treated with the indicated siRNA.


MED1 signals on MED4 puncta normalized to nuclear MED1 signals . MED1 signal intensity on each MED4 punctum was individually plotted .

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MED1 signals on MED4 puncta normalized to nuclear MED1 signals. MED1 signal intensity on each MED4 punctum was individually plotted.


B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS ( top right ) and OS ( bottom right ) of breast cancer patients in the METABRIC dataset . Genes located at the HER2 amplicon were nominated according to hazard - ratio levels ( all P - values < 0 . 01 ) . The HER2 co - amplification percentage of the indicated genes is represented as bar graphs ( left ) .

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B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard-ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left).


C The frequency of CDK12 amplification in HER2 - amplified - and HER2 - nonamplified breast cancer . The bar graphs indicate the frequency of HER2 and CDK12 co - amplified cases among the patients with HER2 gene amplification ( left ) . Tables show the percentage of CDK12 amplification in the indicated cohorts ( right ) . Amp , amplification ; non - amp , non - amplification .

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C The frequency of CDK12 amplification in HER2-amplified- and HER2-nonamplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification.


D Scatter plot showing the correlation between CDK12 expression and its copy number alteration ( CNA ) in METABRIC ( left ) . The r value was calculated as the Pearson ' s correlation coefficient . Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC ( right ) . P - values were calculated using one - way ANOVA , followed by post - hoc LSD test .

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D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.


E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC ( top ) and KM plotter ( bottom ) using the Kaplan - Meier method with the log - rank test .

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E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


A 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 -

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A 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-sensitive


Subcellular localization of β - catenin in the indicated stable cell lines were analysed by cellular fractionation ( B )

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Subcellular 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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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 .

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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 .

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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 .

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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.


a , 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 .

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a, 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 .

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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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c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP-DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.


a , COS7 cells stably expressing yellow fluorescent protein ( YFP ) - ATG5 were transfected with RFP - SEC61b ; and teal fluorescent protein ( TFP ) - mito , and immunostained for TOMM20 .

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a, 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 .

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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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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.


a , COS7 cells expressing GFP - SEC61b ; and STX17 were immunostained for TOMM20 .

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a, COS7 cells expressing GFP-SEC61b; and STX17 were immunostained for TOMM20.


b , HeLa cells expressing GFP - ATG14 were immunostained for STX17 .

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b, HeLa cells expressing GFP-ATG14 were immunostained for STX17.


c , Immunoblot of subcellular fractions of HEK293 cells .

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 .

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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 .

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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 .

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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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g, Pull-down of ATG14 complex from HeLa cells expressing Myc-STX17 or Myc. Scale bars, 5 µm (a, b) and 2 µm (d).


a , 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 .

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a, 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 .

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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 .

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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 .

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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 .

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

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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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).


PS 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 .

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

PS 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 .

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

(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.


PS 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 -

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

PS 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-serine


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) the corresponding molecular signatures of PSD activity .

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


( 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 .

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(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 .

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

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

(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).


( 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 .

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

(A) 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 .

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

(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 .

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

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 .

(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.


SFA / 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 .

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

SFA/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.


A 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 .

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

A 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 .

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

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.


A 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 .

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

A 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 )

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

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)


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 , 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 .

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

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, 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 .

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

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 .

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.


A , 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 .

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

A, 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 ) .

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

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

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.


A 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 .

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

A 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 .

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

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.


B , 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 .

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

B, 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 .

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

D, 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.