EMBO/soda-nel huggingface dataset:
TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability (2017)
Figure 5

Figure 5. PP2A activity is regulated by the Ppk18Gwl-Igo1 pathway to control differentiation and Taf12 phosphorylation, in response to nutrient availability. (A) Igo1 phosphorylation (P-Igo1) was followed by anti-MYC IB of protein extracts from WT, ppk18Η, and igo1-S64A strains, grown in rich conditions (R) or starved for 45 minutes (S). Igo1 migration was analyzed by electrophoresis of both a 12% SDS-polyacrylamide gel containing the Phos-tag™ molecule (right panel) and a standard 10% SDS-polyacrylamide gel (left panel). Arrowheads indicate the various phosphorylated isoforms of Igo1-MYC in each strain and condition. Shown is an IB representative of 2 independent experiments. (B,C) Expression of ste11+ (C) and mei2+ (D) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, ppk18Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni"s multiple comparison tests (n = 4). (D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, ppk18Η, and igo1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Tubulin IB is shown as a control for loading. (E) P-Taf12 was followed in WT, igoS64A, pab1Η, and pab1Η igoS64A strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the FLAG antibody in S. pombe.
TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability (2017)
Figure 6

Figure 6. The TORC2-Gad8AKT pathway induces Taf12 phosphorylation upon starvation. (A) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, tor1Η, and gad8Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. P-Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni"s multiple comparison tests (n = 3). (B) P-Taf12 was followed by anti-FLAG IB of protein extracts from sty1Η cells (left panel) or ssp2Η cells (right panel), grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The star (*) symbols label an unspecific band detected by the anti-FLAG antibody in S. pombe. Shown are IBs that are representative of 2 independent experiments. (C) TAP-tagged Gad8 was immuno-precipitated using IgG-sepharose beads (IP) from strains containing FLAG-tagged Taf12, grown either in rich medium (left panels) or starved for 45 minutes (right panels). TAP-tagged Gad8 was eluted from beads using the TEV protease, releasing a shorter form of Gad8 (CBP-Gad8). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of whole cell extracts (WCE), to detect Taf12 co-precipitation with Gad8. A strain which does not contain any TAP tag is shown as a negative control for the IP. Shown is an IB representative of two independent experiments. (A,C) The star (*) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S. pombe. (D) Endogenous Gad8-HA was affinity purified from starved cells, in order to activate its kinase activity (left and middle panels), and mixed with recombinant, purified GST-Taf12, GST-Taf12-5A, or GST-Fkh2 proteins (right panels) in a kinase assay buffer. Each assay was then analyzed by IB and probed with an anti-phospho-AKT substrate antibody.
TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability (2017)
Figure 7

Figure 7. Taf12 phosphorylation inhibits sexual differentiation downstream of TORC2. (A,B) Expression of ste11+ (A) and mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni"s multiple comparison tests (n = 6). (C,E) Zygotes and tetrads, which correspond to differentiated cells, were counted from cultures of homothallic cells grown to mid-log phase in rich medium (t0) and shifted to starvation medium for up to 24 hours (C) or from heterothallic cells mixed and incubated for 2 days on mating medium (E). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey"s multiple comparison tests (n = 6). (D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor1-1972A strains, grown in rich medium and shifted to starved conditions for 60 minutes. An anti-Tubulin IB is shown as a control for loading. Shown is an IB which is representative of 2 independent experiments. (F) Proposed model for the regulation and role of Taf12 phosphorylation during the early and late steps following nutrient starvation in S. pombe. Question marks represent unknown regulatory pathways. See Discussion for details.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf1

(a) Representative differential interference contrast microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3rd), late third instar larval (Late 3rd) and at puparium formation (white prepupal, WPP) stages. (b) Autophagy detected by formation of mCherry–Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown. (c) Midguts from control
Atg18KG03090/wild-type (Atg18/+), n = 14, and Atg18KG03090/Df(3L)6112 mutant (Atg18/Df), n = 11, animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (d) Wild-type, control (Atg18KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm2) at indicated stages; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage. (e) Differential interference contrast images of midgut cells from Atg2EP3697/wild-type control (Atg2/+) and Atg2EP3697/Df(3L)6091 mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm2) from e,
n = 12 (Atg2/+) and
n = 7 (Atg2/Df) animal intestines per genotype with 5 cells measured per intestine. (g–j) Representative TEM images of intestine cells 2 h after puparium formation. (g,i) Control Atg18KG03090/wild-type (Atg18/+, g) and control Atg2EP3697/wild-type (Atg2/+, i) cells with an enlarged image showing a double-membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18KG03090/Df(3L)6112mutant (Atg18/Df, h) and Atg2EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± s.d. Scale bars, 20 μm (a-c and e) and 1 μm (g–j).
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf2

(a) Midguts dissected from animals expressing
Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a,
n = 12 animal intestines per genotype with 1–5 cells measured per intestine. (c) Midguts expressing mCherry–Atg8a in all cells, and expressing
Atg18IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown. (d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d,
n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1–5 cells measured per intestine. (f) Midguts dissected from animals expressing mCherry–Atg8a in all cells, and expressing Atg1IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown. (g–j) Representative cut views from three-dimensional reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early third instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm3) from g–j, n = 5 animal intestines per genotype with 1–5 cells measured per intestine per stage. Black columns: early third instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf3

(a) Midguts dissected from animals at puparium formation that contain
Vps34Δm22 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm2) from a,
n = 14 animal intestines per genotype with 1–5 cells measured per intestine. (c) Midguts dissected from animals expressing Atg8aIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines per genotype with 1–5 cells measured per intestine. (e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e,
n = 14 animal intestines per genotype with 1–5 cells measured per intestine. (g–i) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1; g, GFP in nucleus and cytoplasm), both Atg1 +Atg8aIR (h), or both Atg1 +Atg7IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g’ and i’) DIC images of midguts expressing mCherry–Atg8a in all cells, Atg1 (g’), and Atg1+Atg7IR (i’) in GFP-marked clones of cells. mCherry–Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d. Scale bars, 20 μm.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf4

(a) Midguts dissected from animals at puparium formation that contain Atg7d4 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm2) from a,
n = 8 animal intestines per genotype with 1–5 cells measured per intestine. (c) Midguts expressing mCherry–Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown. (d) Gastric caeca expressing GFP–Atg8a in all cells from control (Atg7d30/d14) and Atg7 mutant (Atg7d77/d14) animals. Representative images are shown. (e) Quantification of GFP–Atg8a puncta number from d, n = 31 animal intestines per genotype with puncta quantified in image fields per intestine. (f) Midguts expressing GFP–Atg5 in enterocytes (larger nuclei) with an
Atg7d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown. (g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g,h) Control (Atg7d30/d14; g) and Atg7 mutant (Atg7d77/d14; h) cells both contain autophagic structures. (h, right) Enlarged Atg7 mutant cell image of a double-membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm (a,c,d and f) and 1 μm (g,h).
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf5

(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype. (b,c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf6

(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a;
n = 16 animal intestines per genotype with 1–5 cells measured per intestine. (c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1–5 cells measured per intestine. (e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry–Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (f) Midguts expressing GFP–Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown. (g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h,
n = 6 animal intestines per genotype with 1–5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf7

(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b),
n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of
β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag–Uba1, 135K; Flag–Atg7, 81K; His–Atg8a, 29K; Ub, 9K. (c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7d77/d14) and Atg8 (Atg8KG07569) mutant animals blotted with anti-Atg8 and anti-Tubulin,
n = 3 independent biological experiments. Representative images are shown. (d) Midguts from control Atg310/wild-type (Atg3/+) and Atg310/Df(3L)cat mutant (Atg3/Df) animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (e) Cell size quantification (μm2) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines per genotype with 5 cells measured per intestine. (f) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1) and Atg3IR only in the DsRed-marked cell clone and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (g) Midguts dissected from animals expressing Atg4DN specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 10 animal intestines per genotype with 1–5 cells measured per intestine. (i) Midguts dissected from animals expressing mCherry–Atg8a in all cells, and expressing UbIR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown. (j) Midguts dissected from animals expressing UbIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (k) Cell size quantification (μm2) from (j), n = 12 animal intestines per genotype with 1–5 cells measured per intestine. (l,m) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 (Atg1) mis-expression in either a wild-type GFP-positive cell clone (l) or a Uba1H33 loss-of-function MARCM mutant cell clone (GFP-positive; m) and analysed by fluorescence and differential interference contrast microscopy. Control wild-type and heterozygous Uba1H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm2) from l and m, n = 22 (control), n = 16 (Atg1) and n = 6 (Atg1 +Uba1H33) animal intestines per genotype with 1–5 cells measured per intestine. Quantification data for control cells (l,m) were pooled because they are genetically identical. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.
Uba1 functions in Atg7- and Atg3-independent autophagy (2013)
figf8

(a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. (c) Midguts dissected at puparium formation that express Uba1IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown. (d–f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation.
Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a–c) and 1 μm (d–f).
Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 2

Figure 2. JNK signature in breast cancer cells is linked to ECM, stem cell and wound healing gene networks and is enriched in basal-like breast cancer.

A Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red.

B Biological functions regulated by the JNK pathway in MDA231-LM2 cells determined by gene ontology using DAVID analysis. False discovery rate (FDR) < 0.1. BP, biological process; CC, cellular compartment; BH, Benjamini-Hochberg.

C, D GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK and (D) under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score.

E GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells (Lim et al, 2010); b, up in fetal mammary stem cells (Spike et al, 2012); c, up in adult mammary stem cells (Spike et al, 2012) and d, up in mammary repopulating units (Stingl et al, 2006). *P < 0.05, **P < 0.01, ***P < 0.001. For panels C-E, P values were determined by random permutation test (Subramanian et al, 2005).

F Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi, as analyzed in panel A.

G Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes (Chia et al, 2012; Curtis et al, 2012). ****P < 0.0001. P value was determined by a Fisher's exact test. JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.