Journal: The Journal of biological chemistry

Article Title: OCA7 is a melanosome membrane protein that defines pigmentation by regulating early stages of melanosome biogenesis.

doi: 10.1016/j.jbc.2022.102669

Figure Lengend Snippet: Figure 1. OCA7 localizes to melanosomes. A, spinning disc confocal fluorescence microscopy analysis of live primary human melanocytes coexpressing OCA7-EGFP with mCherry-Rab32 or RFP-SKL. The scale bars indicate 10 μm for unmagnified images and 1 μm for magnified insets. B, Pearson correlation coefficient (PCC) for images from (A). Error bars represent the 95% confidence interval (mCherry-Rab32, n = 55 cells; RFP-SKL, n = 34 cells). C, colocalization quantification as determined by PCC for images of MNT1 cells expressing OCA7-EGFP with organelle markers from Fig. S1, showing median with error bars representing the 95% confidence interval. n = 50, n = 30, n = 16, n = 45, n = 24, n = 13, n = 29, n = 33, n = 31, n = 33, n = 25, and n = 32 cells for TPC2-iRFP, mCherry-Rab32, mCherry-Rab38, mCherry-CD63, tyrosinase-mCherry, tyrosinase-P1-mCherry, mCherry-Rab27a, mCherry-Rab7a, mCherry-Rab11a, mCherry- Rab5a, BFP2-KDEL, and RFP-SKL, respectively. D, live cell Airyscan super-resolution and brightfield images of MNT1 melanocytes expressing OCA7-EGFP showing a cohort of OCA7 localizes to membranes surrounding melanin pigment. The scale bars indicate 10 μm for unmagnified image and 1 μm for magnified inset. EGFP, enhanced GFP; iRFP, infrared fluorescent protein; OCA7, oculocutaneous albinism type 7; RFP, red fluorescent protein.

Article Snippet: To generate TPC2BioID2-HA, TPC2 was amplified from pEGFP-N3-TPC2 (Addgene #80153) and inserted into MCS-Linker-BioID2-HA (gifted from Kyle Roux, Addgene #74224) linearized by inverse PCR.

Techniques: Microscopy, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro

doi: 10.3892/etm.2012.692

Figure Lengend Snippet: Effect of CD133 downregulation by As-CD133 on cancer cell viability. (A) Reduced viability of B16F10, MCF7 and INER51 cells subsequent to transfection with As-CD133. Images were captured with a confocal fluorescent microscope under visible light (left and right) and ultraviolet light (center). Viable cells express the GFP reporter as a control for transfection. (B) Cell viability analysis of cancer cells treated with As-CD133 is shown. B16F10, MCF7 and INER51 cells were transfected with various concentrations (0.1–0.6 μg) of As-CD133 or with pEGFP-N3 as a control. Cell viability was evaluated by the MTT assay. Values are means of the average cell viability from three independent experiments ± SD (P<0.05). (C) Immunocytochemical analysis of downregulation of the CD133 protein in B16F10 cells after transfection with As-CD133. C+, stained with Ab-CD133; C-, lacking Ab-CD133; T, transfected with As-CD133 and stained with Ab-CD133. (D) RT-PCR analysis of the downregulation of CD133 mRNA in B16F10 cells subsequent to transfection with As-CD133. Amplification was performed with CD133-1 and CD133-2 primers, or with G3PDH primers as a control. T, transfected with the As-CD133; N, not transfected, negative control. RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: The resulting product was cloned into the vector pEGFP-N3 (Gene Therapy Systems, Inc., San Diego, CA, USA).

Techniques: Transfection, Microscopy, MTT Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro

doi: 10.3892/etm.2012.692

Figure Lengend Snippet: Analysis of B16F10 cell death induced by As-CD133. (A) Analysis of DNA integrity following transfection with various concentrations of As-CD133 by orange acridine staining visualized under a UV microscope. (B) Multiplex RT-PCR analysis of the expression of apoptotic genes (ICE, CMYC and P53), an anti-apoptotic gene (BCL2) and a constitutively expressed gene (G3PDH) as a control. M, marker. Lanes: N1, untransfected B16F10 cells; T, B16F10 cells transfected with As-CD133; N2, cells transfected with pEGFP-N3; +, positive control kit; -,negative control kit. RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: The resulting product was cloned into the vector pEGFP-N3 (Gene Therapy Systems, Inc., San Diego, CA, USA).

Techniques: Transfection, Staining, Microscopy, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Positive Control, Negative Control