Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 1: GFP-Rab17 localization in B16 cells. B16 cells were transiently transfected with GFP-Rab17 for 24 h. Panel D was imaged using the Delta Vision microscope, panel A and B were imaged via confocal microscopy. Brightness and contrast was adjusted for each colour channel individually in photoshop. Scale bars on full-size images represent 10 μm and scale bars on the zoom images represent 5 μm. A) Transfected cells were incubated with mouse Tfn-Alexa-546 (Tfn-546). Left to right: GFP-Rab17 (green), Tfn-536 (red) and an overlay of the coloured images also including DAPI (blue). White arrows indicate peripheral GFP-Rab17 that does not colocalize with Tfn, yellow arrows indicate GFP-Rab17 colocalization with Tfn. B) Transfected cells were stained with the HMB45 Ab. Left to right: The distribution of GFP-Rab17 (green), the HMB45 melanosome marker (red), and a merge of the coloured images. Yellow indicates colocalization between Rab17 and the melanosome. C) Pearson’s correlation coefficients for colocalization between GFP-Rab17 and melanosome marker (HMB45) (white bar) or GFP-Rab17 and Golgi marker GM130 Ab in B16 cells (black bar). Error bars represent ±SD from 6 to 12 individual cells. D) Transfected cells were stimulated with FSK and stained with the TRP1 Ab to the Tyrp1 protein. FSK was used to increase the number of cells with a high number of mature, Tyrp1-positive melanosomes. From left to right: The distribution of the GFP-Rab17 fusion protein (green), Tyrp1 (red), a merge of the coloured images and a differential interference contrast (DIC) image of melanin distribution. The yellow arrow indicates colocalization between GFP-Rab17, Tyrp1 and melanin-filled melanosomes. The white arrow indicates colocalization between GFP-Rab17 and a melanin-filled melanosome. Manual counting using IMAGEJ cell counter revealed approximately 66% of the dark melanosomes in DIC colocalized with GFP-Rab17.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Transfection, Microscopy, Confocal Microscopy, Incubation, Staining, Marker

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 2: GFP-Rab17 localizes to the RE and melanosomes in primary melanocytes. Scale bars represent 10 μm. QF863 primary melanocytes were transiently transfected with GFP-Rab17 for 24 h. A) Cells were incubated with human Tfn-Alexa647 before fixing. From left to right: The distribution of GFP-Rab17 (green), Tfn-647 (red) and a merge of the coloured images also including DAPI (blue). Arrow indicates colocalization of GFP-Rab17 with the RE. B) Cells were fixed, permeabilized and stained with the HMB45 Ab. From left to right: The distribution of the GFP-Rab17 fusion protein (green), HMB45 (red) and a merge of the coloured images. This image is the tip of a melanocyte dendrite.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Transfection, Incubation, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 3: Rab17 expression in melanocytic cells and regula- tion by MITF. The level of Rab17 mRNA was determined by Q-RT-PCR and normalized to B2M expression. A) Two differenti- ated melanocyte strains (QF1177 and QF1185) were stimulated with 20 μM FSK or dimethyl sulphoxide (DMSO) (control) for 12 h in the absence of cholera toxin (CT). Rab17 mRNA was expressed relative to the control. Error bars indicate ±SEM (n = 3). B) West- ern blot of QF863 melanocytes stimulated with 10 μM FSK or DMSO (control) for 4 days in the absence of CT. Bands were cropped from the same blot at the same exposure. This result was replicated in the QF1177 and QF1185 strains. C) Rab17 mRNA in MM96L cells or differentiated QF1160 melanocytes transfected with negative control or MITF siRNA for 48 h. Rab17 mRNA was expressed as fold relative to the appropriate control cells (negative siRNA). Error bars represent ±SEM (n = 3). D) Western blot of differentiated QF1177 melanocytes transfected with negative control or MITF siRNA for 48 h. This result was replicated in the QF1185 strain. E) Rab17 mRNA in A06MLC cells transfected with either negative control or MITF siRNA (Ambion) as indicated. Twenty-four hours after transfection, cells were stimulated with normal media plus 0.25% BSA (control), 10 nM NDP-MSH in the presence of 100 μM IBMX (MSH) or 10 μM FSK for a further 24 h. A06MLC cells were used as they were known to be responsive to both FSK and MSH (37). Rab17 mRNA was expressed as fold over the control cells. Error bars indicate ±SEM (n = 3–4). MITF siRNA transfected cells (control, MSH and FSK) are not significantly different to the negative siRNA (control).

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Negative Control, Western Blot

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 4: B16 Rab17 siRNA treatment: imaging of morphology and melanosome distribution. B16 cells were transiently transfected with negative control siRNA, Rab17 siRNA (17.1 and 17.2) or Rab27a siRNA. Some negative control cells were also treated with 10 nM NDP-MSH (MSH) for 24 h. Transfected cells were fixed after 48 h, permeabilized and stained with HMB45 Ab, Texas-red phalloidin and DAPI. All cells were imaged via confocal microscopy. Scale bars represent 20 μm. Arrows indicate accumulation of mature melanosomes. A) Upper panel: Merged images are of HMB45 Ab staining (melanosome – green) and DAPI (blue). Lower panel: Transmitted light images. The outline of the Rab27a siRNA cells was drawn with coloured lines using Texas-red phalloidin staining as a guide. B) Q-RT-PCR analysis of mouse Rab17 mRNA in B16 cells transiently transfected with negative control siRNA or Rab17 siRNA. Rab17 mRNA was normalized to GAPDH expression then expressed as fold relative to the control cells. Error bars indicate ±SEM (n = 3). C) Rab17 western blot of B16 cells transiently transfected with negative control or Rab17 siRNA. This blot is representative of three independent experiments. D) Rab27a western blot of B16 cells transiently transfected with negative control siRNA or Rab27a siRNA. This result is representative of three independent experiments.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Imaging, Transfection, Negative Control, Staining, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 5: Representation of melanosome marker intensity and distribution, quantification of melanosome localization and numbers per cell. B16 cells were transiently transfected with negative control siRNA, Rab17.1 siRNA or Rab27a siRNA for 48 h as indicated. A) HMB45 fluorescence intensity is represented by the raised surfaces and colour indicates proximity to the cell membrane or nucleus. B) Quantification of melanosome localization in B16 siRNA knockdown cells. The boundary of individual cells is defined by actin staining, the nucleus is defined by DAPI staining and the intensity of the HMB45 Ab staining is used to determine melanosome distribution. The intensity ratio is calculated by the average intensity of HMB45 pixels proximal to the cell boundary divided by the average intensity of pixels further from the cell boundary (closer to the nucleus). Therefore, a cell with a relatively high intensity ratio has a relatively high proportion of melanosomes near the periphery of the cell. Error bars represent ±SEM (n ≥9 individual cells). C) Quantification of the numbers of HMB45-positive melanosomes per cell in negative siRNA- or Rab17 siRNA-treated cells as indicated. Error bars represent ±SEM (n ≥18 cells per treatment).

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Marker, Transfection, Negative Control, Membrane, Knockdown, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

doi: 10.1111/j.1600-0854.2011.01172.x

Figure Lengend Snippet: Figure 6: B16 Rab17 knockdown and dendrite length, melanosome velocity, melanin content and melanosome imaging by EM. A) B16 dendrite length (distance in micrometers of the longest dendrite from the nucleus) was quantified using IMAGEJ. Lengths were normalized to the control cells, error bars represent ±SEM (n = 4). B) Melanosome velocity in live B16 cells transfected with negative control siRNA or Rab17 siRNA imaged via bright-field time-lapse microscopy. Error bars represent ±SEM of melanosome velocity from 2 to 3 cells, with at least four individual melanosomes tracked per cell. Movements >0.1 μm/second but <0.3 μm/second are thought to be actin movements and movements >0.3 μm/second are thought be microtubule movements (38). C) Total melanin content in B16 cells was measured 3 days after transient siRNA transfection. Values were normalized to the negative control. Error bars indicate ±SEM (n ≥4). D) Representative digital photo of cell pellets of transient siRNA knockdown B16 cells, or cells treated with 10 nM NDP-MSH (MSH). E) Transmission electron micrographs of control, Rab17.1 knockdown or Rab27a knockdown cells. Control B16 cells show low numbers of melanosomes near the cell body and in dendrites, while Rab17 knockdown cells show an increase in melanosomes which were mainly in peripheral regions of the cell body and in the dendrites. Rab27a knockdown cells showed an even larger increase in melanosomes, which were concentrated throughout the cell body/perinuclear region. Yellow arrows indicate mature, stage IV melanosomes and black arrows indicate stage II–III melanosomes. N = Nucleus, G = Golgi, mc = mitochondria. Scale bars = 2 μm.

Article Snippet: Anti-Rab17 rabbit polyclonal antibody was raised against glutathione S-transferase (GST)-Rab17 and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) as described previously (62).

Techniques: Knockdown, Imaging, Control, Transfection, Negative Control, Time-lapse Microscopy, Transmission Assay