Journal: Biomolecules & Therapeutics

Article Title: Inhibition of Melanosome Transport by Inducing Exon Skipping in Melanophilin

doi: 10.4062/biomolther.2022.167

Figure Lengend Snippet: List of OPNA, primer sequences

Article Snippet: Quantitative real-time PCR was performed using a FastStart Essential DNA Probes Master kit (Roche, Mannheim, Germany), Universal ProbeLibrary (Roche), and TaqMan probe (Thermo Scientific, Mm00453498_m1).

Techniques: Sequencing

Journal: Biomolecules & Therapeutics

Article Title: Inhibition of Melanosome Transport by Inducing Exon Skipping in Melanophilin

doi: 10.4062/biomolther.2022.167

Figure Lengend Snippet: Effects of OPNA on the Mlph exon in melan-a cells. RT-PCR on melan-a melanocytes showed exon skipping with 1 μM OPNA for 48 h. Exon 1 is 231 bp, exon 2 is 134 bp, and exon 3 is 222 bp in size. The control band is composed of 587-bp Mlph from exon 1 to exon 3. Effects of siMlph and OPNAs on Mlph mRNA expression level in melan-a melanocytes. As indicated, cells were treated with siMlph (20 nM) or OPNAs (1 μM) for 48 h. Level of Mlph mRNA was measured in melan-a melanocytes using real-time PCR. Values are the mean expression normalized to that of β-actin and relative to the control from five independent experiments (n=5). ** p <0.01 (Student’s t test).

Article Snippet: Quantitative real-time PCR was performed using a FastStart Essential DNA Probes Master kit (Roche, Mannheim, Germany), Universal ProbeLibrary (Roche), and TaqMan probe (Thermo Scientific, Mm00453498_m1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Biomolecules & Therapeutics

Article Title: Inhibition of Melanosome Transport by Inducing Exon Skipping in Melanophilin

doi: 10.4062/biomolther.2022.167

Figure Lengend Snippet: Effects of siMlph and OPNAs on Mlph protein expression level in melan-a melanocytes. As indicated, cells were treated with siMlph (20 nM) or OPNAs (1 μM) for 48 h. (A) Expression level of Mlph protein in melan-a melanocytes was measured by western blot. (B) Relative expression level of Mlph proteins shown in . Values are the mean expression normalized to that of β-actin and relative to the control from five independent experiments (n=5). *** p <0.001, ** p <0.01 (Student’s t test).

Article Snippet: Quantitative real-time PCR was performed using a FastStart Essential DNA Probes Master kit (Roche, Mannheim, Germany), Universal ProbeLibrary (Roche), and TaqMan probe (Thermo Scientific, Mm00453498_m1).

Techniques: Expressing, Western Blot, Control

Journal: Genomics Data

Article Title: Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial

doi: 10.1016/j.gdata.2015.08.029

Figure Lengend Snippet: Real-time PCR confirms differences in gene expression in colonic epithelium and stroma. Quantitative real-time RT-PCR was done on both biopsies (taken at two separate visits) from 10 study participants for four candidate genes. The histogram bars from the same individual are located next to each other, i.e., samples 1 and 2 are from the same person but taken at two separate visits. Total RNA was reverse transcribed and then amplified by PCR and normalized to β-glucuronidase (GUSB) expression. Blue bars: epithelial tissue. Red bars: stromal tissue. Upper panel shows expression of: (i) ABCC3, an epithelial gene involved in drug and lipid metabolism; and (ii) MLPH (melanophilin), an epithelial gene involved in protein binding and protein transport. Lower panel shows expression of: (iii) ANXA1 (annexin A1), a stromal gene involved in cell migration and cell adhesion; and (iv) ITGA8 (integrin α8), a stromal gene involved in extracellular matrix formation.

Article Snippet: Relative gene expression was examined by TaqMan PCR using 2 × TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, California) and TaqMan Gene Expression Assays for two epithelial genes (ABCC3, MLPH; assay identifiers Hs00358677_m1 and Hs00983106_m1) and two stromal genes (ITGα8 and ANXA1; assay identifiers Hs00943530_m1 and Hs00945401_m1).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Quantitative RT-PCR, Reverse Transcription, Amplification, Expressing, Protein Binding, Migration

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with wogonin at a concentration of 50 μM ( A ) or the indicated concentrations ( B–C ). ( A ) Nuclei (blue) and F-actin (magenta) were analyzed by confocal microscopy. Bright field images show the melanosome (black pigment) distribution. Scale bar = 10 μm. ( B ) MLPH, Rab27A, and myosin Va levels were determined by immunoblotting with anti-MLPH, Rab27A, and myosin Va antibodies, respectively. ( C ) The expression of Mlph mRNA was quantified using qPCR.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Concentration Assay, Confocal Microscopy, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 2 days with or without 50 μM wogonin. Following replacement of the medium, B16F10 cells were cultured for an additional 24 h with or without wogonin (50 μM) in combination with MG132 (120 nM) or leupeptin (20 μM). ( A–B ) MLPH levels were determined by immunoblotting with an anti-MLPH antibody. ( C ) Bright field images show the melanosome distribution. Scale bar = 50 μm.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with or without 50 μM wogonin. Following replacement of wogonin-free medium, B16F10 cells were cultured for an additional 24 h. ( A ) Bright field images show the melanosome distribution. Scale bar = 50 μm. ( B ) The MLPH level was determined by immunoblotting with an anti-MLPH antibody.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with 50 μM wogonin, wogonoside, or norwogonin. ( A ) Bright field images show the melanosome distribution. Scale bar = 10 μm. ( B ) The results are expressed as the percentage of cells showing perinuclear melanosome aggregation. ( C ) MLPH levels were determined by immunoblotting with an anti-MLPH antibody.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: ( A ) The structures of three mono- O -methylated flavones. ( B–C ) B16F10 cells were cultured for 3 days with 50 μM wogonin or each of the two wogonin analogs. The results are expressed as the percentage of cells showing perinuclear melanosome aggregation. MLPH levels were determined by immunoblotting with an anti-MLPH antibody. ( D ) B16F10 cells were cultured for 24 h with 50 μM of each flavone. The melanin content in each culture was quantified.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Methylation, Cell Culture, Western Blot

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 7. Immunohistochemical analysis of molecular apocrine markers derived from trancriptomics data within the apocrine cyst and carcinoma dataset. Representative staining of FFPE of apocrine cysts and IACs with antibodies against SLC2A10 (Glucose transporter type 10, sections A and B) - cytoplasmic immunoreactivity; SLC7A8 (L-type amino acid transporter 2, sections C and D) - mainly cytoplasmic and membranous immunereactivity; MLPH (Melanophilin, sections E and F) - cytoplasmic and luminal membranous immunoreactivity; FOXA1 (Hepatocyte nuclear factor 3-alpha, sections G and H) – nuclear staining; XBP1 (X-box-binding protein 1, sections I and J) – mainly nuclear and cytoplasmic immunereactivity; BLVRA (Biliverdin reductase A, sections K and L) - cytoplasmic immunoreactivity which combined with rare nuclear positivity; RHOB (Rho-related GTP-binding protein RhoB, sections M and N) - cytoplasmic immunoreactivity; TSC22D3 (TSC22 domain family protein 3, sections O and P) - mainly nuclear and cytoplasmic immunereactivity; ABCA12 (ATP-binding cassette sub-family A member 12, sections Q and R) - cytoplasmic immunoreactivity and SIDT1 (SID1 transmembrane family member 1, sections I and G) - cytoplasmic immunoreactivity. Magnification: x10. Representative areas are shown in higher magnification (x20) and examples of positive staining are indicated by red arrows. doi:10.1371/journal.pone.0112024.g007

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Immunohistochemical staining, Derivative Assay, Staining, Binding Assay

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 8. Protein expression profile of transcriptome-derived apocrine markers among breast cancer subtypes. The diagram presents immunohistochemical profiles of four breast tumor subtypes, TNBC, Luminal A Luminal B and HER2, reacted with antibodies against ten transcriptome-derived apocrine markers: XBP1, TSC22D3, ABCA12, SIDT1, FOXA1, RHOB, BLVRA, SLC2A10, SLC7A8 and MLPH. The rows indicate the expression of particular protein in every core in breast cancer TMA (BRC1501, 1502 and 1503; Pantomics INC, USA): red box – positive staining, white box – negative staining grey box – parameter was not determined. Corresponding frequencies of positives are shown on the right side of the diagram. Samples were considered as positive if 10% or more of the cells showed a clear positive staining with the antibodies. IDC = invasive ductal carcinoma; TNBC = triple negative breast cancer. Tumors have been stratified as specified in Materials and Methods. doi:10.1371/journal.pone.0112024.g008

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Negative Staining

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 9. MLPH is expressed by non-malignant apocrine cells but is lost in IACs. Representative images of FFPE sections immunostained with antibody against MLPH. (A) normal ducts, (B) benign apocrine cysts (mainly luminal membranous immunoreactivity), (C) sclerosing adenosis with apocrine differentiation, (D) IAC showing positive immunostaining in pseudo-glands structures (cytoplasmic and luminal membranous immunoreactivity) and (E) IAC with negative immunostaining. Magnification: x20. doi:10.1371/journal.pone.0112024.g009

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Immunostaining