GLUT1 is up-regulated in SIRT6-KO retina.

<p>a) GLUTl immunoreactivity in cross-section of WT and SIRT6-KO mice retina. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL) Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). GLUT1 protein (b) and mRNA levels (c) were determined by Western blot and RT-PCR respectively. β-actin was used as loading control. Data are mean ± SE (n = 6 eyes/group) **p<0.01</p

Retinal functional evaluation.

<p>Representative scotopic (A) and photopic (C) electroretinograms from WT and SIRT6-KO mice at different light intensities (dBs). Plots B and D depict average amplitudes of <i>a</i>-wave and <i>b</i>-wave. Note that the fold decrease of the scotopic <i>a</i>-wave amplitude (8) is greater than the fold decrease of the photopic <i>a</i>-wave amplitude (2,5). Data are mean ± SE (n =  4). **p<0.01, ***p<0.001.</p

Grm6 is down-regulated in SIRT6-KO retinas.

<p>Whole retina mRNA from WT and KO mice was used to profile the expression of several key genes of glutamate receptors involved in the synaptic transmission in an Affymetrix Mouse Gene 2.1 ST DNA microarray. a) Heatmap representing the hierarchical cluster analysis shows the differential expressed mRNAs between WT and SIRT6 KO retinas. The graphic depicts the expression levels of ionotropic AMPA glutamate receptors (Gria1–4), Glutamate receptor, ionotropic kainate (Grik1-2-4-5), Glutamate [NMDA] receptors (Grin1-2a-c) and metabotropic glutamate receptors (Grm1–8). The expression data for the hierarchical clustering image has been row normalized to a range of zero to one with blue representing the row minimum and red representing the row maximum. b) RNA was purified from SIRT6 WT and KO retinas, and Grm6 levels analyzed by RT-PCR. c) immunofluorescence was performed in SIRT6 WT and KO retinas with the indicated antibodies. PKC-alpha was used as a marker for ON bipolar cells. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). Data are mean ± SE (n = 4) **p<0.01 d) Representative fluorescent images of TUNEL analysis performed in WT and SIRT6 KO retinal sections. Apoptotic nuclei (bright green dots) labeled with fluorescein-dUTP were visualized by fluorescence microscopy. Data are mean ± SE (n  = 3) **p<0.01</p

TGFB1 is positively correlated with overall survival and the TS2/16 gene signature in melanoma.

<p>(A) Heatmap showing TCGA human skin cutaneous melanoma (SKCM) patient sample Pearson correlations for TGFB1 RNA-seq expression levels and tumor microenvironmental genes. Active TGF-β signaling (SERPINE1), a metagene for integrin β1 activity (ITGB1 metagene), the immune score (imm score) and Non-Synonymous Mutation rate (non-syn Mut) are included as well. (B) Gene set enrichment analysis (GSEA) for the stromal gene signature on TCGA SKCM patient samples with genes ranked based on Pearson correlation with TGFB1 expression level. (C) Kaplan-Meier curve displaying overall survival (OS) for TCGA SKCM skin/distant melanoma patients who were subdivided into a TGFB1^high and a TGFB1^low group. TGFB1^high > median (N = 63), TGFB1^low < median (N = 70). (D) GSEA analyses for T cell gene signature on TCGA SKCM patient samples with genes ranked based on Pearson correlation with TGFB1 expression level.</p

Neutralization of TGF-β reduces tumor infiltrating lymphocytes and A375 tumor growth.

<p><i>(</i>A) TGF-β signaling in the TME. IHC quantification on A375 NLS-mCerulean tumors treated with IgG control (white bars) or 1D11 (green bars) antibody for 3 weeks. Per field of view, cells negative for mCerulean (TME cells) but with nuclear pSMAD2/3 were counted and plotted as a fraction of total mCerulean^negative (TME) cells. N ≥ 4 animals per condition. (B-C) IHC quantification on A375 NLS-mCerulean tumors treated with IgG control (white bars) or 1D11 (green bars) antibody. The number of (B) CD3⁺CD4⁺, (C) CD3⁺CD8⁺ T cells was counted per field of view (FOV), and the average of ≥4 FOV in ≥4 tumors per condition was plotted in the bar graphs. (D) Mean tumor volume of A375 NLS-mCerulean tumors in <i>nu/nu</i> mice treated with IgG control antibody (dashed black line) or treated with 1D11 (solid green line). Treatments are indicated with an arrow. N ≥ 6 mice per group. (E) <i>In vitro</i> proliferation assay. A375 cells were treated with PBS or rTGF-β1 for 2 or 7 days. Proliferation was measured by calculating green fluorescent (living) cells and comparing it to a live control. N = 3 experiments performed in triplicate. (F) <i>In vitro</i> cell viability assay. A375 cells were treated with PBS or rTGF-β1 for 2 or 7 days. Viability was measured by calculating the red fluorescent (dead) cells and comparing it to a dead control. N = 3 experiments performed in triplicate. G) <i>In vitro</i> proliferation assay. A375 cells were treated with IgG control antibody or 1D11 antibody for 0, 1 or 2 days. After addition of cell titel 96 aquaous solution proliferation was assessed by measuring optical density at 490 nm and normalizing it to day 0. N = 3 experiments performed in triplicate. Error bars, SEM; Double-sided unpaired T-Test (A to C) or repeated measures two-way ANOVA (D-G): * P-value ≤ 0.05, n.s. P-value > 0.05.</p

SIRT6 is active in the mouse retina.

<p>a) H3K56 acetylation is shown by immunofluoescence. b) Representative Western blot showing protein levels of SIRT6 and the acetylation levels of H3K56 and H3K9 in chromatin preparations from WT and KO mice retinas. Total H3 was used for normalization. c) Quantification of the intensity of bands was determined by using the ImageJ and is represented as arbitrary units. Data are mean ± SE (n = 6 eyes/group). **p<0.01, ***p<0.001</p

TS2/16 attenuates tumor growth by indirectly increasing apoptosis.

<p>(A-B) IHC quantification on A375 NLS-mCerulean tumors treated with IgG control (white bars) or TS2/16 (blue bars) antibody. The number of (A) CD3⁺CD4⁺, (B) CD3⁺CD8⁺ T cells was counted per field of view (FOV), and the average of ≥5 FOV in ≥3 tumors per condition was plotted in bar graphs. D10: tumors were treated once and were harvested 10 days after injection (2 days after treatment). D28: Tumors were treated 3x and were harvested 28 days after injection (5 days after last treatment). (C) Mean tumor volume of A375 tumors in <i>nu/nu</i> mice left untreated (dashed line), treated with TS2/16 (blue line) or Paclitaxel (grey line). Treatments are indicated with an arrow. N ≥ 5 mice per group. (D) A375 tumor growth measurements in <i>nu/nu</i> mice. Each curve represents the growth of a single tumor. Treatments are indicated with an arrow: TS2/16 or IgG (blue), paclitaxel (grey). Mice were sacrificed when tumors reached > 130 mm³, cured mice are indicated by the number on the right. N ≥ 8 tumors per group. (E) Mean tumor volume of A375 tumors shown in D. Mice were left untreated (dashed line), treated with paclitaxel/IgG/Paclitaxel (grey line) or treated with paclitaxel/TS2/16/paclitaxel (blue line). Treatments are indicated with an arrow: TS2/16 or IgG (blue), paclitaxel (grey). Mice were sacrificed when tumors reached > 130 mm³. N ≥ 8 mice per group. (F-G) IHC quantification on A375 NLS-mCerulean tumors treated with IgG control (white bars) or TS2/16 (blue bars) antibody for 5 weeks. The number of KI67⁺ cells (proliferation, F) or Cl. Casp3⁺ cells (apoptosis,G) per FOV was measured. N ≥ 10 FOV per condition. (H) Kaplan Meier survival curves of <i>nu/nu</i> and NSG mice respectively injected with A375 tumor cells and treated with indicated treatment regimen: IgG (IgG control antibody), P (paclitaxel), TS (TS2/16). Mice were sacrificed when tumors reached >130 mm³. N ≥ 10 mice per group. Survival analyses with Bonferroni post-hoc: in both <i>nu/nu</i> and NSG mice IgG/P vs TS/P and P/IgG/P vs P/TS/P was significant (P-value≤ 0.05). Error bars, SEM; Two-sided unpaired T-tests (A—B) or 1-way ANOVA with Bonferroni post-hoc test comparing treatments to control (F—G): * P-value ≤ 0.05, n.s. P-value > 0.05.</p

TGFB1 correlations for various tumor types.

<p>Heatmap displaying correlations between TGFB1 expression and active ITGB1 (ITGB1 metagene) and TGF-β activity (SERPINE1/PAI1) for various tumor types (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175300#pone.0175300.s010" target="_blank">S4 Table</a>). The heatmap also includes rows with mean mutation rates (mean mut. rate), TGFB1 Kaplan-Meier treatment outcome log-rank p-values (Treatment outcome), and GSEA Normalized Enrichment Score (NES) for T-cell (T cell genes) and stromal (Stromal genes) gene sets for each of the tumor types.</p

Additional file 1: of AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer

S2 Antibody target specificity is unaffected by sequence of primary antibody application. Merged (right) and single color (left) confocal microscopy images at × 60 of an untreated primary TNBC tumor stained in an alternate sequence: pan-AKT ➔ H3K9me2 ➔ HES1 (c.f. standard sequence of H3K9me2 ➔ pan-AKT ➔ HES1) demonstrating consistent cytoplasmic pan-AKT (green) and HES1 (red) staining and nuclear H3K9me2 (yellow) staining in an example QCC (white arrows). (PDF 3624 kb

Additional file 2: of AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer

S1 Determination of fluorescence intensity thresholds and staining reproducibility. For each marker (HES1, H3K9me2, pan-AKT) the proportion of cells at a specific fluorescence intensity level was different between sequential sections from control tumor 4, stained simultaneously (S1A and S1B, respectively). QCC percentage (red bars) and QCC density (box and whisker plots) in control tumors 1–4 increased proportionally at 25%, 33%, and 50% thresholds (S1C, S1D, and S1E, respectively). (PDF 2666 kb