Journal: International journal of cancer

Article Title: Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells.

doi: 10.1002/ijc.20865

Figure Lengend Snippet: FIGURE 4 – Effect on melanogenesis. (a) Color of cell pellets. 624Mel controls (5 x 106) and cells expressing T1796ABRAF RNAi or INK4A were pelleted, and a visible color difference was noted in cells expressing T1796ABRAF RNAi. (b) Melanin contents; 5 x 106 cells were used. Data are means 6 SE from 3 experiments performed in triplicate (p < 0.001, t-test). (c) Representative electron micrographs of cultured 624Mel controls and cells expressing T1796ABRAF RNAi or INK4A. Note the increased numbers of mature melanosomes in RNAi-expressing cells (arrows). Magni- fication x2,000. Similar changes were obtained using dissected xenograft tumors. (d) Expression of tyrosinase (TYR) and TRP-1 proteins. Western blot was probed with TYR, TRP-1 and a-tubulin antibodies. (e) Increased MITF in cells expressing T1796ABRAF RNAi, shown by Western blot- ting. (f) Model of separate regulation of proliferation and differentiation by mutant BRAF and p16INK4A in melanoma cells.

Article Snippet: Western blotting was performed as described.26 The following antibodies were used: BRAF (Upstate Biotechnology, Lake Placid, NY); p16INK4A, tyrosinase and cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-MEK, MEK-1 and phospho-pRB (Cell Signaling, Beverly, MA); TRP-1 and cyclin D3 (BD Biosciences, San Diego, CA); MITF (Novus Biologicals, Littleton, CO); tubulin (Sigma-Aldrich, St. Louis, MO); and horseradish peroxidase–conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis

Journal: Cosmetics

Article Title: Ethanolic Extract of Hippocampus abdominalis Exerts Anti-Melanogenic Effects in B16F10 Melanoma Cells and Zebrafish Larvae by Activating the ERK Signaling Pathway

doi: 10.3390/cosmetics7010001

Figure Lengend Snippet: Figure 4. EEHA inhibits the cAMP–CREB–MITF-tyrosinase axis in α-MSH-stimulated B16F10 melanoma cells. (A) B16F10 melanoma cells were seeded at a density of 5 × 104 cells/mL and pretreated with 1 mM IBMX for 10 min. The intracellular cAMP levels were subsequently measured 15 min after treatment with EEHA in the presence or absence of 500 ng/mL α-MSH (B). (C,D) In a parallel experiment, B16F10 melanoma cells were treated with EEHA at the indicated concentrations in the presence or absence of α-MSH. The expression of MITF and tyrosinase was detected by RT-PCR at 48 h (C), and Western blotting at 72 h (D). GAPDH and β-actin were used as the controls for normalizing the expression of MITF and tyrosinase and the relative density was calculated using ImageJ software. (E) The B16F10 melanoma cells were treated with EEHA at the indicated concentrations in the presence or absence of α-MSH for 72 h, following which the cell lysates were mixed with 5 mM l-DOPA in a 96-well microplate. After incubation at 37 ◦C for 30 min, the absorbance was measured at a wavelength of 405 nm. The results represent the average of data obtained from three independent experiments and are expressed as the mean ± SEM (*** p < 0.001 vs. the untreated group; ### p < 0.001, ## p < 0.01, and # p < 0.05 vs. the α-MSH-treated group).

Article Snippet: Antibodies against phospho (p)-CREB, MITF, tyrosinase, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Incubation

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet: Characterization of RPE (A–E) Immunostaining of iPSC derived RPE cultures for the marker expression during stages of retinal differentiation. Images showing the expression of RPE commitment marker MITF (A), melanocyte protein- PMEL17 (B), RPE maturation markers TYRP1, RPE65 and TYR (C-E)and tight junction marker ZO-1 (D and E). (F) Flow cytometry analysis of iPSC derived RPE cells for positive (MITF, RPE65, TYRP1) and negative (OCT4) markers. (G and H) Gene expression analysis using qPCR for selected markers represented as fold change compared to iPSC. All the positive markers (G) have higher expression and the negative markers (H) show a negligible expression in iPSC derived RPE. (I) ELISA based quantification of secreted PEDF from the in vivo culture supernatant from apical, basal and 2D RPE cultures. Images and graphs are representative of minimum three independent experiments. Scale bars 100 μm.

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Immunostaining, Derivative Assay, Marker, Expressing, Flow Cytometry, Gene Expression, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet:

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Recombinant, Modification, Gentle, Saline, Knock-Out, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, cDNA Synthesis, Sequencing, Generated, Software, Suspension, Real-time Polymerase Chain Reaction, Microscopy

Journal: eLife

Article Title: Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes

doi: 10.7554/eLife.31657

Figure Lengend Snippet: List of antibodies tested and validated for t-CyCIF.

Article Snippet: MITF-488 , MITF , ‡ , Novus Biologicals , NB100-56561AF488 , 21D1418 , Alexa Fluor 488 , AB_838580.

Techniques:

Journal: Radiation Research

Article Title: UV-Radiation Response Proteins Reveal Undifferentiated Cutaneous Interfollicular Melanocytes with Hyperradiosensitivity to Differentiation at 0.05 Gy Radiotherapy Dose Fractions

doi: 10.1667/rr15078.1

Figure Lengend Snippet: FIG. 1. Immunohistochemistry for DNp63, eosin-PAS, MITF and Bcl-2 in skin biopsies. Row A: Before radiotherapy. Row B: One week of radiotherapy. Row C: Six and half weeks of radiotherapy. In the MITF staining, black arrows indicate examples of MITF-positive melanocytes and red arrows indicate MITF-negative melanocytes.

Article Snippet: Immunofluorescence Combinations of primary antibodies from different species were used for double-staining: MITF (1:100, monoclonal mouse, clone D5; Dako), MITF (1:50, polyclonal rabbit; Atlas Antibodies, Bromma, Sweden), DNp63 (1:200, monoclonal mouse, clone 4A4; Santa Cruz Biotechnology), DNp63 (1:100, polyclonal rabbit; Atlas Antibodies), Bcl-2 (1:20, monoclonal mouse, clone 124; Dako), p53 (1:100, monoclonal mouse, clone DO-7; Dako), Ki-67 (1:100, monoclonal mouse, clone MIB-1; Dako), SOX10 (1:100, polyclonal goat, N-20; Santa Cruz Biotechnology), PAX3 (1:100, polyclonal rabbit; Invitrogen), p21 (1:100, monoclonal mouse, clone EA10; Abcamt, Cambridge, MA), c-KIT (1:100, polyclonal rabbit; Dako) and DCT (1:500, monoclonal mouse, clone C-9; Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Staining

Journal: Radiation Research

Article Title: UV-Radiation Response Proteins Reveal Undifferentiated Cutaneous Interfollicular Melanocytes with Hyperradiosensitivity to Differentiation at 0.05 Gy Radiotherapy Dose Fractions

doi: 10.1667/rr15078.1

Figure Lengend Snippet: FIG. 2. Mean number of stained cells per mm in the basal layer for various molecular markers at 1 week of radiotherapy. Panel A: DNp63-negative cells. Panel B: Morphological characterization via eosin-PAS staining. Panel C: MITF-positive cells and MITF-negative cells morphologically characterized as melanocytes. Panel D: Bcl-2-positive cells and Bcl-2-negative cells morphologically characterized as melanocytes. Error bars represent standard error. Error bars for MITF and Bcl-2-negative cells are hidden within the symbols.

Article Snippet: Immunofluorescence Combinations of primary antibodies from different species were used for double-staining: MITF (1:100, monoclonal mouse, clone D5; Dako), MITF (1:50, polyclonal rabbit; Atlas Antibodies, Bromma, Sweden), DNp63 (1:200, monoclonal mouse, clone 4A4; Santa Cruz Biotechnology), DNp63 (1:100, polyclonal rabbit; Atlas Antibodies), Bcl-2 (1:20, monoclonal mouse, clone 124; Dako), p53 (1:100, monoclonal mouse, clone DO-7; Dako), Ki-67 (1:100, monoclonal mouse, clone MIB-1; Dako), SOX10 (1:100, polyclonal goat, N-20; Santa Cruz Biotechnology), PAX3 (1:100, polyclonal rabbit; Invitrogen), p21 (1:100, monoclonal mouse, clone EA10; Abcamt, Cambridge, MA), c-KIT (1:100, polyclonal rabbit; Dako) and DCT (1:500, monoclonal mouse, clone C-9; Santa Cruz Biotechnology).

Techniques: Staining

Journal: Radiation Research

Article Title: UV-Radiation Response Proteins Reveal Undifferentiated Cutaneous Interfollicular Melanocytes with Hyperradiosensitivity to Differentiation at 0.05 Gy Radiotherapy Dose Fractions

doi: 10.1667/rr15078.1

Figure Lengend Snippet: FIG. 5. Double-staining of epidermal skin biopsies illustrating the response of melanocytes to daily fractionated treatments of 1.1 Gy. Cells in the melanocyte lineage are exclusively negative for DNp63. Each double-staining was merged with DAPI. Panel A: Double-staining for DNp63 and MITF. Before radiotherapy, the left arrows indicate an MITF-negative melanocyte and right arrows indicate a MITF-positive melanocyte in the basal layer. During radiotherapy, the arrows indicate MITF-positive melanocytes. Panel B: Double-staining for DNp63 and Bcl-2. Before radiotherapy, the left arrows indicate a Bcl-2-negative melanocyte and right arrows indicate a Bcl-2-positive melanocyte in the basal layer. During radiotherapy, the arrows indicate a Bcl-2-positive melanocyte. Panel C: Double-staining for MITF and Bcl-2. Arrows indicate Bcl-2 cells co-expressing MITF and vice versa before and during radiotherapy. Panel D: Double-staining for MITF and SOX10. Before radiotherapy, the left arrows indicate a MITF-negative/SOX10-positive melanocyte in unexposed skin. During radiotherapy, the arrows indicate a MITF-positive/SOX10-positive melanocyte. Panel E: Double-staining for DNp63 and PAX3. The arrows indicate DNp63-negative/PAX3-positive melanocytes before and during radiotherapy. Panel F: Double-staining for MITF and PAX3. Before radiotherapy, the arrows indicate a MITF-negative/PAX3- positive melanocyte. During radiotherapy, the arrows indicate melanocytes that co-express MITF and PAX3. Panel G: Double-staining for PAX3 and SOX10. Before radiotherapy, the arrows indicate a PAX3-negative/ SOX10-positive melanocyte. During radiotherapy, the left arrows indicate a PAX3-positive/SOX10-negative melanocyte and right arrows indicate a PAX3-negative/SOX10-positive melanocyte. Panel H: Double-staining for DNp63 and Ki-67. The arrows indicate Ki67-negative/DNp63-negative melanocytes before and during radiotherapy. Panel I: Double-staining for DNp63 and p53. The arrows indicate p53-negative/DNp63-negative melanocytes before and during radiotherapy. Panel J: Double-staining for p21 and DNp63. The arrows indicate nuclear p21-negative/DNp63-negative melanocytes before and during radiotherapy. Panel K: Double-staining for c-KIT and DNp63. The arrows indicate DNp63-negative/c-KIT-positive melanocytes before and during radiotherapy. Panel L: Double-staining for DCT and MITF. The arrows indicate DCT-positive/MITF-positive melanocytes before and during radiotherapy.

Article Snippet: Immunofluorescence Combinations of primary antibodies from different species were used for double-staining: MITF (1:100, monoclonal mouse, clone D5; Dako), MITF (1:50, polyclonal rabbit; Atlas Antibodies, Bromma, Sweden), DNp63 (1:200, monoclonal mouse, clone 4A4; Santa Cruz Biotechnology), DNp63 (1:100, polyclonal rabbit; Atlas Antibodies), Bcl-2 (1:20, monoclonal mouse, clone 124; Dako), p53 (1:100, monoclonal mouse, clone DO-7; Dako), Ki-67 (1:100, monoclonal mouse, clone MIB-1; Dako), SOX10 (1:100, polyclonal goat, N-20; Santa Cruz Biotechnology), PAX3 (1:100, polyclonal rabbit; Invitrogen), p21 (1:100, monoclonal mouse, clone EA10; Abcamt, Cambridge, MA), c-KIT (1:100, polyclonal rabbit; Dako) and DCT (1:500, monoclonal mouse, clone C-9; Santa Cruz Biotechnology).

Techniques: Double Staining, Expressing