Additional file 1 of Caffeine activates HOG-signalling and inhibits pseudohyphal growth in Saccharomyces cerevisiae

Additional file 1: Figure S1. Growth of yeast cells (strain BY4743) with (+ caf; caffeine concentration 10 mM) and without (-caf) caffeine. Growth was followed at 30 °C by microcultivation in 350 μl synthetic YNB medium in a Bioscreen C (automated shaking and optical density readings), with optical density (OD) measurements every 20 min. Inoculation to cultures from a small number of cells taken from an overnight colony on synthetic YNB agar plates (without caffeine). The growth analysis was performed in two independent replicates (n = 2) with in each case 5 technical replicates. Typical growth curves are displayed

Additional file 2 of Caffeine activates HOG-signalling and inhibits pseudohyphal growth in Saccharomyces cerevisiae

Additional file 2: Figure S2. Western blot analysis showing Hog1 dual-phosphorylation in response to caffeine. Exponentially growing wild type cells (BY4743) in synthetic medium without caffeine were treated with media with different concentrations of caffeine (10, 20, 30, 40 mM) for 5 min. Negative control was basal growth medium without any caffeine addition, and positive control is treatment with medium containing 500 mM NaCl for 5 min. A) Hog1-p detected with dualphosphorylated-specific antibody. B) Loading control of total Hog1 detected by Hog1 specific antibodies (non-phosphorylated form). Molecular weight standard to the left-most lane in both cases. A typical result is shown from two independent replicates

Additional file 3 of Caffeine activates HOG-signalling and inhibits pseudohyphal growth in Saccharomyces cerevisiae

Additional file 3: Figure S3. Pseudohyphal growth in diploid cells was inhibited by caffeine. The diploid strain Σ1278 was cultured on solid SLAD media at 30 °C for 2 days. Upper panel control (water), lower panels: different concentration of caffeine (3, 5, 7, 10 mM). The edge of the colony (indicated by arrows) of the control shows filamentous extensions, while the edges of colonies grown in the presence of different concentrations of caffeine appeared smooth and even. A typical result is shown from two independent replicates, each containing two technical replicates for the control and 10 mM caffeine, while 3, 5 and 7 mM caffeine is a typical result from one run with two technical replicates. The original microscopic magnification for all images is 100×. However, in this supplementary figure selected portions of the images have been further enhanced (zoomed-in) to emphasis the colony edge-effects, resulting in another 10× magnification compared to the images presented in Figure 3 in the main text

Additional file 4 of Caffeine activates HOG-signalling and inhibits pseudohyphal growth in Saccharomyces cerevisiae

Additional file 4: Figure S4. Invasive growth under caffeine stress. Cells of the haploid Σ1278 strain were grown on YPD plates containing different concentrations of caffeine (3 and 10 mM). A typical result is shown from two independent replicates, each containing two technical replicates