Additional file 1: of Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action

Primers used for qRT-PCR. (DOC 51 kb

Chronological investigation of caspase activation after treatment with curcumin and light: A375 melanoma cells were incubated with 5 µg/ml curcumin and afterwards irradiated with visible light.

<p>Controls remained light-protected. After different periods of time (upper part: 0.5–4 hours; lower part: 1–24 hours) cells were lysed. Detection of inactive (black arrows) and active (white arrows) forms of caspase-8, caspase-9 and caspase-3 was carried out using the Western blot technique. β-actin-bands represent the loading control (dashed arrows).</p

DNA fragmentation in melanoma cells (A375 and G-361) after treatment with curcumin and light: After incubation with curcumin, cells were irradiated with UVA (gray bars), or visible light (black bars), or were light-protected (white bars).

<p>All values were referred to the untreated control as a 100% reference. Every bar represents the average of 4 simultaneously determined individual values; standard deviations are shown at the top. Significant increase of DNA fragmentation of the treated cells is marked with * (<i>P</i>≦0.05).</p

Mitochondrial redox activity in melanoma cells (A375 and G-361) after treatment with curcumin and light: After incubation with curcumin, cells were irradiated with UVA (gray bars), or visible light (black bars), or remained light-protected (white bars).

<p>All values were referred to the untreated control as a 100% reference. Every bar represents an average of 8 simultaneously determined individual values; standard deviations are shown at the top. Highly significant reduction of viability of the treated cells is marked with * (<i>P</i>≦0.001).</p

LDH release in melanoma cells (A375 and G-361) after treatment with curcumin and light: After incubation with curcumin, cells were irradiated with UVA (gray bars), or visible light (black bars), or were light-protected (white bars).

<p>Cells of the positive control were treated with 1% triton-X100 containing medium (dashed bars). All values were referred to the positive control as a 100% reference. Every bar represents the average of 8 simultaneously determined individual values; standard deviations are shown at the top.</p

mTOR signaling is deactivated during differentiation and only partially contributes to the control of proliferation.

<p>(a) Increasing densities of HaCaT were seeded to promote differentiation and harvested after 72h. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies. (b) NHK were serum-starved and differentiation was induced with 2mM CaCl₂ for the indicated time points. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies was performed. Below a densiometrical quantification of involucrin and P-S6 levels of n = 4–5 similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (* p ≤0.05, ***p ≤0.001). (c) Keratinocytes stem cells (KSC), transient amplifying (TA) and postmitotic (PM) cells were separated according to their ability to adhere to type IV collagen. Protein lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies, showing that mTORC1 signaling is mainly present in undifferentiated cells. (d) Normal human skin was stained with P-mTOR S2448 and Ki-67 antibody. Nuclei were stained with DAPI. Single color and overlay images are presented, which show that mTOR is activated in proliferation cells of the basal layer. Bars represent 100 μm. (e) HaCaT cells were reverse-transfected with siRNA targeting Akt, Raptor or control siRNA and seeded in 96 well plates. After 48h proliferation was quantified using the XTT-based assay. Graph presents mean ± SEM (n = 4–8). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (f) To control for the efficiency of knockdown, HaCaT cells were transfected as in (e) and seeded in 6 well plates. Protein lysates were harvested after 48h and a Western blot was performed with the indicated antibodies. To show the consequences of Akt and Raptor knockdown, phosphorylation of the downstream targets GSK-3 and S6 was also captured. (g) HaCaT cells were seeded in 96 well-plates in triplicates and after 24h 50 μM LY294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) were added. After another 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (h) To show efficiency of the used inhibitors, HaCatT cells were treated as in (g). Protein lysates were prepared and a Western blot was performed with the indicated antibodies.</p

Activation of mTORC1 signaling inhibits differentiation.

<p>HaCaT cells (a) or NHK cells (b) were serum starved overnight and treated with increasing doses of MHY1485 or DMSO for 30 min If indicated cells were pre-treated with 100 nM Rapamycin for 30 min. Cells were harvested and protein lysates were analyzed by Western blotting with the indicated antibodies, showing that MHY1485 induces mTORC1 signaling. (c) Increasing numbers of HaCaT cells were seeded and driven into differentiation by post-confluent growth in the presence of the indicated concentrations of MHY1485. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below a quantification of 6–8 similar Western blots is shown. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01). (d-g) MHY1485 or vehicle control was topically applied to the dorsal skin of mice for 12 consecutive days with increasing doses. (d) DSFT (Double Skin Fold Thickness) was measured before the first treatment (day1) and repeated every day. Data shown are mean values from one experiment, with n = 3 mice per treatment. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01, *** p ≤0.001, **** p ≤0.0001). (e) Representative images of H&E-stained sections from dorsal skin of a mouse of control and MHY 1485 treated groups (scale bar, 100 μM). (f) Evaluation of histological features, including number of epidermal layers and epidermal thickness in μM. Data shown are mean values of five measurements per mouse ± SEM. Statistical significance was calculated with Mann-Whitney test (**p ≤0.01). (g) Involucrin staining of vehicle control or MHY1485 treated mice. Overview images and close-ups are shown. MHY1485 induces in vivo a psoriasis like phenotype and interferes with proper differentiation (h) Hypothetical model how mTOR serves as a switch to determine the fate of keratinocytes.</p

mTORC1 and its downstream mediator are strongly activated in psoriatic lesions.

<p>Punch biopsies from lesional psoriatic skin (a,c,e,g) of 20 patients and five healthy donors (b,d,f,h) were stained with antibodies for specific for Rheb, Raptor, P-PRAS40 and P-4E-BP. Nuclei were stained with DAPI. Representative overlay images from one patient and one healthy donor are shown. Bars represent 100 μm.</p

Inhibition of mTORC1 signaling promotes differentiation.

<p>(a) Increasing cell numbers of HaCaT cells were seeded and after 24h 100 nM Rapamycin, 250 nM Torin or solvent control (DMSO) were added and differentiation was allowed to proceed for 72h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Quantification of 3–6 similar blots is depicted below. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison, comparing different treatments with the corresponding cell density in the control group (*p ≤0.05, ***p ≤0,001, ns: non-significant). (b) In NHK cells differentiation was induced with 2mM CaCl₂ in the presence of 100nM Rapamycin, 250 nM Torin or solvent control for 48h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Below a quantification of six similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05). (c) NHK cells were treated with 2 mM CaCl₂ and 100nM Rapamycin or DMSO control. RNA was isolated and quantitative RT-PCR was performed to measure expression of the indicated differentiation markers. Graph presents mean ± SEM (n = 5–8). Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison. (*p ≤0.05, ****p ≤0.0001, ns: non-significant). (d+e) HaCaT (d) or NHK (e) cells were transfected with siRNA specific for Raptor (Rap), mTOR or an siRNA control (si contr) and differentiation was induced either by post-confluent growth (d) or with 2mM CaCl₂ (e) for 48h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below each blot a quantification of IVL and P-S6 band relative to actin bands of 4–6 similar blots is shown. Statistical significance was calculated with 2-way ANOVA and Bonferroni multiple comparison, comparing Raptor or mTOR knockdown with the corresponding differentiation state in the control group (*p ≤0.05, ** p ≤0,01, ****p ≤0,0001).</p

Psoriatic cytokines induce mTOR signaling.

<p>Starved HaCaT (a) or NHK cells (b) were treated for 30 min with 20 ng/ml of the indicated cytokines. Serum starved HaCaT (c) or NHK cells (d) were treated for 30 min with the indicated inhibitors (1 μM Wortmannin, 50 μM LY294002, 50 μM U0126 or 100 nM Rapamycin), followed by a 30 min stimulation with IL-1 β or TNF- α (20ng/ml). Cell lysates were analyzed by Western blotting with antibodies for the indicated proteins. (e) HaCaT cells were seeded in triplicates in 96 well plates, starved overnight and treated with 50 μM LY 294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) as well as 20 ng/ml IL-1 β or TNF- α. After 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, ****p ≤0.0001). This shows that mTORC1 does not play a major role in controlling keratinocyte proliferation.</p