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

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: (A) 2.5 μg FLAG-EAF2 and 2.5 μg GFP-FOXA1 were transfected into HEK 293 cells separately or in combination, and then whole cell lysates were prepared for co-immunoprecipitation with anti-GFP or anti-FLAG antibody-conjugated agarose beads. FLAG-EAF2 and GFP-FOXA1 could be pulled down by each other. (B) Co-immunoprecipitation of endogenous EAF2 and FOXA1 was performed using nuclear extracts prepared from LNCaP cells with rabbit anti-FOXA1 antibody or control rabbit IgG. Co-precipitated EAF2 was detected by Western blot using a monoclonal mouse anti-EAF2 antibody. (C) 2.5 μg Myc-tagged full-length EAF2, EAF2 deletion mutants 5 μg Myc-EAF2 (amino acids (aa) 1-113), 5 μg Myc-EAF2 (aa 1-161), or 2.5 μg Myc-EAF2 (aa 113-260) were co-transfected with 2.5 μg GFP-FOXA1 into HEK 293 cells. Co-immunoprecipitation was performed with anti-Myc antibody and GFP-FOXA1 was detected with anti-GFP antibody.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: (A) Two different siRNAs targeting EAF2 were used separately at 100 pmol each to knockdown EAF2 expression in LNCaP cells. Endogenous protein levels of FOXA1, AR and EAF2 were detected by Western blot using anti-FOXA1 anti-AR, and anti-EAF2 antibodies. The protein level of FOXA1 increased upon EAF2 knockdown. (B) Total RNA was isolated from LNCaP cells after knockdown of EAF2. Relative mRNA levels of FOXA1 normalized against GAPDH were determined using real-time PCR. (C) HEK293 cells were co-transfected with 0.2 μg FOXA1 and increasing amounts of GFP-EAF2. Expression levels of FOXA1and EAF2 were determined by Western blot. GAPDH protein was detected as a loading control.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Transfection

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: LNCaP cells were cultured in the presence of indicated siRNAs for 24 hours and then treated with 1 nM R1881 for 48 h. Subsequently, total RNA was extracted and mRNA levels of EAF2 (A), FOXA1 (B), AR (C), PSA (D) and TMPRSS2 (E) were determined by real-time PCR. (F) LNCaP cells were transfected with siRNA targeting the indicated proteins followed by transfection with 0.5 μg PSA-promoter driven fire-fly luciferase vector and 0.05 μg pCMV-Renilla in the presence or absence of the androgen analog R1881 (1 nM). Luciferase assays were performed as described in the Materials and Methods.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: Effect of EAF2 and/or FOXA1 transfection on LNCaP cell colony formation. Left panel shows the colonies formed by LNCaP cells transfected with indicated expression vectors. Right panel shows the quantitative analysis of the colonies of the left panel. Transfection and quantitative analysis of colonies were conducted as described in Materials and Methods.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Transfection, Expressing