Figure 1.
Fibroin and sericin induce motility in MDA-MB-231 cells.
A. Wound healing scratch assay was made on 24 hours serum-starved, confluent MDA-MB-231 cells by drawing a line across the bottom of the dish, following by treatment with either 0.4% fibroin or 0.05% sericin. The figure shows micrographs of the extent of closure obtained under control conditions compared to those with either fibroin or sericin after 25 hours treatment. Phase-contrast microscopy pictures were taken of the wounded area. B. Cell migration quantification of pictures in A was assessed by counting the number of cells in the central gap (see Materials and Methods). Cell migration was represented as number of cells filling the central gap. The experiment was performed independently at least three times. A representative result is shown.
Figure 2.
Fibroin and sericin induce phosphorylation of ERK1/2, JNK1/2 kinases and c-Jun expression and phosphorylation in MDA-MB-231 cells.
A. Total cell lysates from serum-deprived, sub-confluent MDA-MB-231 cells treated with either 0.4% fibroin or 0.05% sericin for different times were analyzed by Western blot for phospho-ERK1/2, c-Jun, phospho-c-Jun, phospho-JNK1/2 proteins. ß-Actin was used as a protein loading control.
Figure 3.
Fibroin and sericin induce the expression of PAI-1, a gene involved in motility.
A. RNAs from MDA-MB-231 cells treated with fibroin for indicated times were analyzed by qPCR for PAI-1 gene. B. RNAs from MDA-MB-231 cells treated with sericin for indicated times were analyzed by qPCR for PAI1 gene. Gene expression is represented as a ratio to GAPDH.
Figure 4.
Fibroin and sericin do not affect cell cycle distribution or proliferation of MDA-MB-231.
A. MDA-MB-231 cells were treated with either 0.05% sericin or 0.4% fibroin, or serum starved (indicated as SS) for 24 hours. Cell cycle distribution was measured using fluorescence activated cell sorting (FACS). Data are presented as percentage of cells at the different phases of cell cycle. B. Effect of fibroin on MDA-MB-231 cells proliferation was assessed by counting cells at the indicated days. The logarithm of the mean number of cells is plotted against time. C. Effect of sericin on MDA-MB-231 cells proliferation was assessed by counting cells at the indicated days. The logarithm of the mean number of cells is plotted against time.
Figure 5.
MEK, PI3K and JNK inhibitors prevent fibroin and sericin cells migration stimulation.
Wound healing scratch assay was made on 24 hours serum-starved confluent MDA-MB-231 cells that were treated with either 0.4% fibroin or 0.05% sericin for 25 hours, in the absence or presence of following inhibitors: SB431542, PD98059, LY294002, Y27632 and SP600125. A. Micrographs of the extent of closure obtained after 25 hours in the control sample or samples treated with fibroin supplemented or not with the indicated inhibitors. B. Micrographs of the extent of closure obtained after 25 hours in the control sample or samples treated with sericin supplemented or not with the indicated inhibitors. In all cases, the wounded area was examined by phase-contrast microscopy. C. Cell migration quantification of different samples in A and B was assessed by counting the number of cells in the central gap (see Materials and Methods). Cell migration was represented as number of cells filling the central gap. The experiment was performed independently at least three times. A representative result is shown.
Figure 6.
Fibroin and sericin induced c-Jun transcription and phosphorylation are prevented by JNK, PI3K or MEK inhibitors.
For A and B, serum-deprived, sub-confluent MDA-MB-231 cells were treated for different times with either 0.4% fibroin or 0.05% sericin in the absence or presence of the following inhibitors: SP600125, LY294002, SB431542 and Y-27632. A. Fibroin treated total cell lysates were analyzed by Western blot for phospho-ERK1/2, c-Jun, phospho-c-Jun, phospho-JNK1/2 proteins. ß-Actin was used as a protein loading control. B. Sericin treated total cell lysates were analyzed by Western blot for phospho-ERK1/2, c-Jun, phospho-c-Jun, phospho-JNK1/2 proteins. ß-Actin was used as a protein loading control. For C and D, serum-deprived, sub-confluent MDA-MB-231 cells were treated for different times with either 0.4% fibroin or 0.05% sericin in the absence or presence of the following inhibitors: SP600125 and PD98059. C. Fibroin treated total cell lysates were analyzed by Western blot for phospho-ERK1/2, c-Jun, phospho-c-Jun, phospho-JNK1/2 proteins. ß-Actin was used as a protein loading control. D. Sericin treated total cell lysates were analyzed by Western blot for phospho-ERK1/2, c-Jun, phospho-c-Jun, phospho-JNK1/2 proteins. ß-Actin was used as a protein loading control.
Figure 7.
c-Jun is directly involved in fibroin- and sericin-induced migration of the MDA-MB-231 cells.
Monolayer of confluent MDA-MB-231 cells, serum-deprived for 24 hours, was subjected to a wound healing scratch assay with fibroin and sericin treatments and inhibitors in the indicated combinations. After 25 hours the cells were fixed, permeabilized and immunostained against c-Jun (pseudo-color) and co-stained with phalloidin for F-Actin (red). Nuclei were revealed with Hoechst 33258 (blue). Representative images containing wound’s healing border (the leading edge) were taken with confocal microscope LSM 510 META from ZEISS. All images were acquired by confocal microscopy using the same settings for all the conditions, so c-Jun protein levels could be compared among different treatments. For clear visualization of c-Jun protein expression level, a pseudo-color code ranging from red (highest level expression) to blue (lowest expression level) was used. The arrows at the bottom indicate the direction of migration of the cells. A. Immunostaining of c-Jun at the leading edge of the MDA-MB-231 cells treated with either 0.4% fibroin or 0.05% of sericin. B. Immunostaining of c-Jun at the leading edge of the MDA-MB-231 cells treated with 0.4% fibroin or 0.4% fibroin with the following inhibitors: LY294002 (PI3K inhibitor) SP600125 (JNK inhibitor) and PD98059 (MEK inhibitor). C. Immunostaining of c-Jun at the leading edge of the MDA-MB-231 cells treated with 0.05% sericin or 0.05% sericin with the following inhibitors: LY294002 (PI3K inhibitor) SP600125 (JNK inhibitor) and PD98059 (MEK inhibitor). The experiment was performed independently at least three times. A representative result is shown.
Figure 8.
Fibroin and sericin induce phosphorylation of ERK1/2 and upregulation of c-Jun in keratinocytes.
Immunoblot of equal amounts of total cell lysates from serum-deprived HaCaT cells treated with either 0.4% fibroin or 0.05% sericin for the indicated times for the indicated proteins. ß-Actin was used as a protein loading control. Line between times 0 and 6 of the western blots indicates that two distant parts of the same gel were pasted together.