Additional file 2: Figure S1. of MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

MicroRNA-195 modulates PHB1 expression in melanoma cells. SK-MEL-5 melanoma cells were transfected with either miR-control/mir-195. miR-195 mimics transfection resulting in a reduction of PHB1 (P ≤ 0.01) (a) mRNA and (b) protein levels compared to miR-control. For RT-qPCR experiments, ACT-B mRNA was used as an endogenous control and the data were analyzed using the 2 (−∆∆Ct) method; for immunoblotting ACT-B was also used as loading control. Protein quantification (fold-change based on the control) is indicated above the blots. In (c) and (d), miR-195 levels 48 and 72 h after transfection, respectively. RNU48 was used as an endogenous control and the data were analyzed using the 2 (−∆∆Ct) method. **P ≤ 0.01. (PNG 74 kb

Hyphae expansion at 120 h after pre-injection of different chemicals.

<p>PBS buffer (A). c-PTIO (B). EGTA(C). imidazole (D). L-NAME (E). Na₂WO₄ (F). (A-F: Bar = 122.5 μm)</p

Effect of different chemicals on hyphae expansion area in compatible combinations.

<p>Each value was shown as mean ± SD (N = 3)</p

Additional file 6: Figure S5. of MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

MicroRNA 195 and PLX-4032 effects in UACC-62 melanoma cells. UACC-62 cells were transfected with either miR-control/miR-195 (25 nM). After 24 h, cells were treated with 1 or 10 μM vemurafenib (PLX-4032) for 48 hs and cell death was determined by flow cytometry after propidium iodide staining. Statistical analysis was carried out using Two-Way ANOVA followed by Bonferroni post-test and are reported by mean ± SD. Representative data of three independent experiments are reported. ***P ≤ 0.001. (JPEG 222 kb

Changes of NO in L10 leaf after <i>P</i>. <i>triticina</i> infection.

<p>NO distributions in L10 leaves at 4, 12, 24, 48, 72 h after inoculation with <i>P</i>. <i>triticina</i> race 260 (A)—(E). Enlargements of A—E (a)—(e). Observations of <i>P</i>. <i>triticina</i> on the surfaces of a—e (F)—(J). Transmitted light photographs of F—J (f)—(j). At 72 h after inoculation with <i>P</i>. <i>triticina</i> race 165 (K). Transmitted light photograph of e (e'). Observations of HR and <i>P</i>. <i>triticina</i> at 72 h after inoculation with <i>P</i>. <i>triticina</i> race 260 (L). Enlargement of L, with a- haustorium mother cell; b- HR cells (l). (A—E, K: Bar = 150 μm; a—e, F—J, f—j, e': Bar = 50 μm; L: Bar = 122.5 μm; l: Bar = 61.25 μm)</p

Effect of different chemical pre-injections on HR.

<p>Each value was shown as mean ± SD (N = 3)</p

Effect of L-NAME or Na₂WO₄ pre-injection on NO and HR.

<p>At 48 h after inoculation (L-NAME pre-injection) (A). At 72 h after inoculation (L-NAME pre-injection) (B). Development of HR and <i>P</i>. <i>triticina</i> at 96 h after inoculation (L-NAME pre-injection) (C). Development of HR and <i>P</i>. <i>triticina</i> at 120 h after inoculation (L-NAME pre-injection) (D). At 48 h after inoculation (Na₂WO₄ pre-injection) (E). At 72 h after inoculation (Na₂WO₄ pre-injection) (F). Development of HR and <i>P</i>. <i>triticina</i> at 96 h after inoculation (Na₂WO₄ pre-injection) (G). Development of HR and <i>P</i>. <i>triticina</i> at 120 h after inoculation (Na₂WO₄ pre-injection) (H). Transmitted light photograph of A (a). Transmitted light photograph of B (b). Transmitted light photograph of E (e). Transmitted light photograph of F (f). haustorium mother cell (c). (A, B, a, b, E, F, e, f: Bar = 50 μm; C, D, G, H: Bar = 37.5 μm)</p

Additional file 7: Figure S6. of MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

Drug-induced cell death is accentuated by miR-195. This panel shows the cell cycle profile of SK-MEL-5 melanoma cells transfected with either miRNA-control/miR-195 (10 nM) (24 h) and treated with cisplatin (CIS-2.5 and 5 μM) or temozolomide (TMZ-50 and 250 μM) for 48 h (total time 72 h). The percentage of the cell population distributed in each cell cycle phase is indicated: G0/G1 = blue, S = green, and G2/M = pink. (a)-MicroRNA-195 alone increased cell death (cells accumulated at sub G0/G1). (b-e) Treatment with drugs induces mainly arrest of SK-MEL-5 cells in G2/M whereas the cytotoxic effects of cisplatin and temozolomide were higher when combined with miR-195 transfection, inducing cell death (sub G0/G1 cells population). Cell cycle distribution of propidium iodide (PI)-labeled cells was analyzed using FlowJo Cytometric software. Representative examples of at least three independent experiments are reported. (JPEG 314 kb

Changes of NOS and NR activities in different combinations.

<p>NOS activity (A). Results were expressed as unit activity per milligram protein (U mgprot^-1). NR activity (B). Each value was shown as mean ± SD (N = 3).</p

Changes of NO fluorescence area in different combinations.

<p>Each value was shown as mean ± SD (N = 3).</p