Table 1.
Primers used for quantitative PCR.
Fig 1.
AM attenuated TGFß-induced phosphorylation of Smad2 and 3 in HaCaT cells.
(A) Representative scheme of the experimental approach followed for the treatment of HaCaT cells with AM, TGFß or both together. Alternatively, HaCaT cells were conditioned for 24 h treatment with AM and then treated with TGFß. In both cases, after treatment several signaling pathways were studied. (B), HaCaT cells were stimulated with AM, TGFß or both simultaneously for the indicated times. (C), HaCaT cells were stimulated for 24 h with AM and then treated with TGFß for the indicated times, non treated cells were used as the control. In (B) and (C), indicated proteins were detected by Western blot. Grb2 was used as a loading control. This experiment was repeated at least three times. A representative result is shown.
Fig 2.
AM treatment of HaCaT cells attenuated the expression of CDKN1A (p21) and CDKN2B (p15) proteins.
(A), HaCaT cells were stimulated with AM, TGFß or both simultaneously for the indicated times. (B), HaCaT cells were stimulated for 24 h with AM and then treated with TGFß for the indicated times, as a control non treated cells were used. Indicated proteins were detected by Western blot. Grb2 was used as a loading control. This experiment was performed at least three times. A representative result is shown.
Fig 3.
AM modified the genetic response induced by TGFß in HaCaT cells.
Several TGFß inducible genes were measured in HaCaT cells stimulated with TGFß or TGFß and AM. (A), isolated RNA from HaCaT stimulated with AM, TGFß, or both was analyzed by qPCR, represented as a ratio to GAPDH and represented as fold change of the untreated control sample. (B), isolated RNA from HaCaT stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to GAPDH and represented as fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s t-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.
Fig 4.
AM treatment induced the activation of several signalling pathways in human primary keratinocytes.
A similar experimental approach was followed to the one shown in Fig 1A. Human primary keratinocytes were stimulated with AM, TGFß or both simultaneously for the indicated times, non treated cells were used as the control. Indicated proteins were detected by Western blot. ZO-1 was used as a loading control. This experiment was repeated at least three times. A representative result is shown.
Fig 5.
AM modified the genetic response induced by TGFß in human primary keratinocytes.
Several TGFß inducible genes were measured in human primary keratinocytes in response to TGFß or in response to the combined treatment of TGFß and AM. Isolated RNA from primary keratinocytes stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to GAPDH and represented as a fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s t-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.
Fig 6.
AM attenuated cell cycle proliferation arrest of TGFß on HaCaT cells and induced the expression of c-Jun protein in HaCaT and in human primary keratinocytes.
Treatment of HaCaT cells with AM attenuates TGFß-induced cell cycle arrest in G1. (A), Cell cycle analysis of HaCaT cells in different conditions, treatment with AM, combined with serum starvation (SS) or TGFß is indicated. The histogram shows cells at G0/G1, S or G2/M stage respectively. AM induced the expression of c-Jun in HaCaT and human primary keratinocytes in clear synergy with TGFß. (B), HaCaT cells or, (D), human primary keratinocytes were stimulated with AM, TGFß or both simultaneously for the indicated times. Additionally, (C), HaCaT cells, or, (E), human primary keratinocytes, were stimulated for 24 h with AM and then treated with TGFß for the indicated times, as a control non treated cells were used. Indicated proteins were detected by Western blot. Grb2 or Zo-1 were used as loading controls where indicated. This experiment was performed at least three times. A representative result is shown.
Fig 7.
In HaCaT cells, AM induced motility and the expression of c-Jun at the migratory front.
Wound healing scratch assay was performed in HaCaT cells in the presence of AM, EGF or combinations of AM with different inhibitors. (A), cells forming a confluent epithelium were wounded and immediately treated as indicated for 21 h. Representative pictures were taken at the beginning of the treatment and 21 h later. (B), treatment of HaCaT cells with AM caused the cells to express c-Jun at the migratory front. Wound healing scratch assay was treated with AM, EGF or combinations of AM with different inhibitors. Cells were wounded and treated for 24 h, afterwards cells were fixed and immunostained for c-Jun. Images of c-Jun fluorescence were converted into pseudo-colour to show the intensity of c-Jun staining. Colour rainbow scale represents fluorescence intensity for c-Jun. Co-staining with phalloidin and Hoechst-33258 was used to show cells structure and nuclei, respectively. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope. This experiment was repeated at least three times. A representative result is shown. (C), HaCaT cells forming a confluent epithelium were treated with Mitomycin C, wounded and immediately treated for 21 h as indicated. Results were compared to non-treated cells. Representative pictures were taken at the beginning of the treatment and 21 h later. Scale Bars 100 μm.
Fig 8.
AM induced c-Jun expression is enhanced in the presence of TGFß.
Wound healing scratch assay was performed in HaCaT cells in the presence of AM, TGFß or a combination of both. (A), cells forming a confluent epithelium were wounded and immediately treated as indicated for 24 h. Representative pictures were taken at the beginning of the treatment and 24 h later. (B), wound healing scratch assay was treated with AM, EGF or combinations of AM with different inhibitors. Cells were wounded and treated for 24 h, afterwards cells were fixed and immunostained for c-Jun. Images of c-Jun fluorescence were converted into pseudo-colour to show the intensity of c-Jun staining. Colour rainbow scale represents fluorescence intensity for c-Jun. Co-staining with phalloidin and Hoechst-33258 was used to show the cell structure and nuclei, respectively. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope. This experiment was repeated at least three times. A representative result is shown. Scale Bars 100μm.
Fig 9.
Expression of c-Jun at the epidermal leading edge.
Histopathological study of AM induced epithelialisation from a patient´s wound that had been treated with AM. (A), microscopic section from wound border before AM treatment. (B), and (C), microscopic section of the wound border 5 and 15 days after AM treatment, respectively. Insets in Fig A to C show roughly the area where confocal microscopy images have been taken. (D), to (F), microscopic sections were also immune-stained against c-Jun (green) and F-Actin (red). Cell nuclei were revealed by Hoechst-33258 staining. (D), same as in A; (E), same as in (B) and (F), same as in (C). Arrows in (E) and (F) point to the epidermal leading edge. Several patients were analysed in this experiment, single patient data is shown for illustrative purposes. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope.