Fig 1.
Effect of Golgi disrupting treatments on the Golgi apparatus of MDA-MB-231 cells.
Cells were left untreated (A; Control), or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant for 16 h (B; HA-ARF1-Q71L), or treated for 60 min either with 5 μg/ml Brefeldin A (C; BFA) or 10 μM Golgicide A (D; GCA). Cells were fixed, permeabilized, and immunolabeled with mouse monoclonal antibody to GM130, rabbit polyclonal antibody to Giantin, and sheep antibody to TGN46. Secondary antibodies were Alexa-594-conjugated donkey anti-mouse IgG (red channel), Alexa-488-conjugated donkey anti-rabbit IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Nuclei were stained with DAPI (gray channel). Stained cells were examined by fluorescence microscopy. Merging red, green, blue, and grey channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Bar, 10 μm.
Fig 2.
Effect of Actinomycin D and Vinblastine on the Golgi apparatus of MDA-MB-231 cells.
Cells were left untreated (A; Control), or treated for 60 min either with 10 ng/ml Actinomycin D (B; ActD) or 25 nM Vinblastine (C; VLB). Cells were fixed, permeabilized, and immunolabeled with mouse monoclonal antibody to GM130, rabbit polyclonal antibody to Giantin, and sheep antibody to TGN46. Secondary antibodies were Alexa-594-conjugated donkey anti-mouse IgG (red channel), Alexa-488-conjugated donkey anti-rabbit IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Nuclei were stained with DAPI (gray channel). Stained cells were examined by fluorescence microscopy. Merging red, green, blue, and grey channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Bar, 10 μm.
Fig 3.
Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the proliferation of MDA-MB-231 cells.
(A) Cells were left in normal culture medium containing 10% FBS, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant (ARF1-Q71L) for 16 h. Untransfected cells were either maintained in normal culture medium containing 10% FBS for 24 h (Serum), or serum-starved and either left untreated for additional 24 h (Control) or treated 24 h either with 0.4 ng/ml Actinomycin D (ActD) or 1 nM Vinblastine (VLB). Transfected cells were serum-starved, and either left without further treatment for additional 24 h (ARF1-Q71L), or treated either with ActD (ARF1-Q71L + ActD) or VLB (ARF1-Q71L + VLB), as in untransfected cells. (B) Untransfected cells were treated as in A, or serum-starved and treated for 24 h either with 0.2 μg/ml Brefeldin A alone (BFA), or in conjunction with ActD (BFA + ActD) or VLB (BFA + VLB). (C) Untransfected cells were treated as in A, or serum-starved and treated for 24 h either with 2 μM Golgicide A alone (GCA), or in conjunction with ActD (GCA + ActD) or VLB (GCA + VLB). In all conditions, cells were cultured during the last 24 h in the presence of [3H]-thymidine. Cells were harvested, and [3H]-thymidine incorporation was quantified with a scintillation counter. Bar represents the mean ± standard deviation (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not statistically significant.
Fig 4.
Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the migration of MDA-MB-231 cells.
(A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant (ARF1) for 16 h. Cultures of confluent cells were wounded with a sterile tip, cells were serum-starved, and either left untreated for additional 20 h (Control and ARF1), or treated 20 h either with 10 ng/ml Actinomycin D (ActD and ARF1 + ActD) or 25 nM Vinblastine (VLB and ARF1 + VLB). (B) Cultures of confluent cells were wounded as in A, cells were serum-starved, and either left untreated for additional 20 h (Control), or treated 20 h either with 5 μg/ml Brefeldin A (BFA), 10 ng/ml Actinomycin D (ActD) or 25 nM Vinblastine (VLB), or with BFA in conjunction either with ActD (BFA + ActD) or VLB (BFA + VLB). (C) Cultures of confluent cells were wounded as in A, cells were serum-starved, and either left untreated for additional 20 h (Control), or treated 20 h either with 10 μM Golgicide A (GCA), 10 ng/ml Actinomycin D (ActD) or 25 nM Vinblastine (VLB), or with GCA in conjunction either with ActD (GCA + ActD) or VLB (GCA + VLB). Images of the same regions were taken immediately after the wounding (0 h), and after 20-h of treatment (20 h). (B, D and F) Cell migration, under the treatments shown in A, C and E, was estimated as the area re-occupied by cells after the 20-h treatment. Bar represents the mean ± standard deviation (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001. Bar, 200 μm.
Fig 5.
Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the apoptosis of MDA-MB-231 cells.
(A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant (ARF1) for 16 h. Untransfected cells were left untreated for further 12 h (Control), or treated 12 h either with 10 ng/ml Actinomycin D (ActD) or 25 nM Vinblastine (VLB). Transfected cells were left untreated for further 12 h (ARF1), or treated 12 h either with 10 ng/ml Actinomycin D (ARF1 + ActD) or 25 nM Vinblastine (ARF1 + VLB). (B) Cells were left untreated for 12 h (Control), or treated 12 h either with 5 μg/ml Brefeldin A (BFA), 10 ng/ml Actinomycin D (ActD) or 25 nM Vinblastine (VLB), or with BFA in conjunction either with ActD (BFA + ActD) or VLB (BFA + VLB). (C) Cells were left untreated for 12 h (Control), or treated 12 h either with 10 μM Golgicide A (GCA), 10 ng/ml Actinomycin D (ActD) or 25 nM Vinblastine (VLB), or with GCA in conjunction either with ActD (GCA + ActD) or VLB (GCA + VLB). (A-C) Graphs depict the quantification of the number of cells decorated with Alexa-488-conjugated Annexin V. Bar represents the mean ± standard deviation (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not statistically significant.
Fig 6.
Effect of the expression of the constitutively-activated ARF1 mutant in conjunction with the treatment with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells.
(A-B) Cells were left untreated (Control), or transfected to transiently express for 16 h the HA-epitope-tagged constitutively-activated ARF1 mutant (ARF1-Q71L). Cells were left untreated for further 5 h (Control and ARF1-Q71L), or treated 5 h either with 10 ng/ml Actinomycin D (ActD; A) or 25 nM Vinblastine (VLB; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right, or to the HA-epitope to detect HA-epitope-tagged ARF1 mutant. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 as shown in A and B (C), and of the levels of phospho-AKT as shown in A and B (D). Bar represents the mean ± standard deviation (n = 3). ** P < 0.01; *** P < 0.001; ns, not statistically significant.
Fig 7.
Effect of the combined treatment of Brefeldin A with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells.
(A-B) Cells were left untreated for 5 h (Control), or treated 5 h either with 5 μg/ml Brefeldin A (BFA), 10 ng/ml Actinomycin D (ActD; A) or 25 nM Vinblastine (VLB; B), or with BFA in conjunction either with ActD (BFA + ActD; A) or VLB (BFA + VLB; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 shown as in A and B (C), and of the levels of phospho-AKT shown as in A and B (D). Bar represents the mean ± standard deviation (n = 3). ** P < 0.01; *** P < 0.001; ns, not statistically significant.
Fig 8.
Effect of the combined treatment of Golgicide A with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells.
(A-B) Cells were left untreated for 5 h (Control), or treated 5 h either with 10 μM Golgicide A (GCA), 10 ng/ml Actinomycin D (ActD; A) or 25 nM Vinblastine (VLB; B), or with GCA in conjunction either with ActD (GCA + ActD; A) or VLB (GCA + VLB; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 as shown in A and B (C), and of the levels of phospho-AKT as shown in A and B (D). Bar represents the mean ± standard deviation (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not statistically significant.