Journal: BioMed Research International

Article Title: MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture

doi: 10.1155/2021/8463161

Figure Lengend Snippet: Morphology of melanocytes in 2D and 3D cell cultures. In passage 4, the culture of human melanocytes was represented by cells with spindle-shaped or dendritic cells (a). Cells in nonadhesive microwells formed dark compact spheroids both in control growth medium (b) and in the presence of Meso-Xanthin F199™ (c). Light phase-contrast microscopy.

Article Snippet: The study was performed using the primary culture of human melanocytes (104-05n, Cell Applications, Inc.).

Techniques: Control, Microscopy

Journal: BioMed Research International

Article Title: MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture

doi: 10.1155/2021/8463161

Figure Lengend Snippet: Descriptive primary analysis of the obtained proteomes among studied melanocyte cells cultivated in a monolayer and in spheroids before and after exposure to FX (a). Protein distribution among studied human melanocyte cultures. The total size of the significantly contributing proteome was 434 protein identifications, which is about 19% of the individual proteome of each sample. (b) Distribution of the symmetry coefficient dependent on the cumulative number of protein identifications. The maximum increase in the symmetry coefficient is observed with the accumulation of 80%-87% of the summarized size of the proteome. Most of the identified proteins among studied melanocyte samples make an insignificant contribution to the growth of the asymmetry of the distribution (up to 0.0025 units per protein).

Article Snippet: The study was performed using the primary culture of human melanocytes (104-05n, Cell Applications, Inc.).

Techniques:

Journal: BioMed Research International

Article Title: MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture

doi: 10.1155/2021/8463161

Figure Lengend Snippet: Heatmap of semiquantitative protein distribution in the shared proteome for M2319, Nm01, and Am02 melanocytes. The sample M2319 was employed as a baseline for normalization. Range correlation of proteins was performed using Spearman's test. The total size of the tested proteome involved 434 protein identifications. Samples Nm01 and Am02 are at the closest localization according to the results of cluster analysis, while M2319 is the most distal.

Article Snippet: The study was performed using the primary culture of human melanocytes (104-05n, Cell Applications, Inc.).

Techniques:

Journal: BioMed Research International

Article Title: MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture

doi: 10.1155/2021/8463161

Figure Lengend Snippet: The most significantly differed proteins exhibited in the primary culture of human melanocytes cultured under three-dimensional condition (spheroids) for 7 days and exposed to 50 μ M equivalent of FX (Am02) or treated with saline solution (Nm01).

Article Snippet: The study was performed using the primary culture of human melanocytes (104-05n, Cell Applications, Inc.).

Techniques: Cell Culture, Saline, Control

Journal: BioMed Research International

Article Title: MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture

doi: 10.1155/2021/8463161

Figure Lengend Snippet: Dynamic alterations of Rap1- and MAPK4/MAPK6-related proteins overlapped between analyzed melanocytes. Proteins regulating the activity of MAPK4 and MAPK6 kinases ( PRS6A , PRS6B , PRS7 , and PSMD2 ) are characterized by the maximum abundancy in the Am02 culture (after exposure to FX) and significantly decreased in Nm01 melanocytes. The RAP1B protein switching between different signaling cascades takes the most abundance in samples Nm01 and M2319 and is strongly inhibited in Am02 melanocytes after exposure to FX.

Article Snippet: The study was performed using the primary culture of human melanocytes (104-05n, Cell Applications, Inc.).

Techniques: Activity Assay

Journal: The American Journal of Pathology

Article Title: Slug Expression during Melanoma Progression

doi: 10.1016/j.ajpath.2012.02.014

Figure Lengend Snippet: Endogenous Slug expression is higher at both mRNA and protein levels in NHM than in primary (WM115) or metastatic (WC62) melanoma cell lines. A: RT-qPCR revealed significantly higher expression of Slug and E-cadherin mRNAs in NHM than in primary or metastatic melanoma cells, whereas Snail and N-cadherin expression were significantly lower. Error bars indicate SDs. *P < 0.01 compared to both primary and metastatic melanoma cells. B: Western blots confirmed that differences in mRNA levels were reflected in differences in protein expression in NHM versus primary and metastatic melanoma cell lines. Both mRNA and protein were extracted from confluent cell cultures to eliminate density-dependent variations in expression of Slug and its putative target genes. MITF isoforms include both melanocyte-specific (M) and ubiquitous (A) isoforms.

Article Snippet: Cell Culture, Adenoviral Transduction, and Immunofluorescence Normal human melanocytes (NHM) of neonatal origin were obtained from the ATCC (Manassas, VA) and were maintained in Dermal Basal Medium supplemented with a melanocyte growth kit (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The American Journal of Pathology

Article Title: Slug Expression during Melanoma Progression

doi: 10.1016/j.ajpath.2012.02.014

Figure Lengend Snippet: NHM and melanoma cells transduced with Slug-expressing adenovirus (Ad-Slug) express less E-cadherin and more N-cadherin mRNA and protein than cells transduced with a control adenovirus (Ad-null). A: RT-qPCR revealed significantly lower E-cadherin expression in NHM transduced with Ad-Slug compared to Ad-Null-transduced cells. Neither Ad-Slug nor Ad-Null-transfected WC62 cells expressed detectable E-cadherin mRNA. N-cadherin levels were significantly increased in Ad-Slug-transduced NHM and melanoma cells compared to Ad-Null-transduced cells, although basal levels of expression were low and increases were modest. Note the difference in scale for the two graphs. Error bars indicate SEMs. *P < 0.03. B: Western blot results confirmed that Ad-Slug enhanced Slug protein expression in NHM and melanoma cells and that the differences seen in mRNA levels for E-cadherin in NHM and N-cadherin in melanoma cells were reflected in differences in protein expression. In addition, Ad-Slug transduction also enhanced expression of melanocyte-specific MITF (M) in NHM but not in melanoma cells. Melanoma cells expressed higher levels of the MITF isoform constitutively expressed in many tissues (A) and expression of this isoform was not altered by Ad-Slug transduction. Ad-Slug transduction did not alter levels of Snail expression in either cell type.

Article Snippet: Cell Culture, Adenoviral Transduction, and Immunofluorescence Normal human melanocytes (NHM) of neonatal origin were obtained from the ATCC (Manassas, VA) and were maintained in Dermal Basal Medium supplemented with a melanocyte growth kit (ATCC).

Techniques: Transduction, Expressing, Control, Quantitative RT-PCR, Transfection, Western Blot

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Western blot analysis of viral proteins in inoculated HeLa cells. Proteins were harvested from HeLa cells inoculated with the parental CVB3/0 or either of the chimeric strains by lysing the cell monolayers in Laemmli loading buffer. Lysates were prepared at the times shown (in hours p.i.) and electrophoresed in SDS–14% PAGE followed by electroblotting. Capsid protein 1D was detected with a polyclonal horse anti-CVB3 neutralizing antibody followed by peroxidase-conjugated rabbit anti-horse IgG. C, purified CVB3/0; TC, uninfected HeLa cell lysate. 1D, location of the detected capsid protein at 34 kDa is shown. Lane M, size markers (in kilodaltons).

Article Snippet: The blots were washed four times as described above, then the primary antibody was detected using peroxidase-conjugated rabbit anti-horse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at a dilution of 1:125,000.

Techniques: Western Blot, Purification

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Increase in CVB3-binding IgG1 in mice inoculated with CVB3-PL2-mIL4/46 compared to CVB3/0. Quantity of CVB3-binding IgG1 (A) or IgG2a (B) in individual mouse serum samples from mice inoculated 14 days previously with CVB3-PL2-mIL4/46 (46) or CVB3-0 (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.

Article Snippet: The blots were washed four times as described above, then the primary antibody was detected using peroxidase-conjugated rabbit anti-horse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at a dilution of 1:125,000.

Techniques: Binding Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: bioRxiv

Article Title: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

doi: 10.1101/2021.06.06.447256

Figure Lengend Snippet: Fractional heme occupancy and activity of HO-2 as a function of free heme concentration. The heme occupancy of HO-2 (red curve; right y-axis) was simulated using a 1- site heme binding model governed by the following equation: [Heme] / {[Heme] + K D }, assuming a heme-HO-2 K D value of 3.6 nM. The fraction of maximal velocity, V/V max , (black curve; left y- axis) was simulated using a Michaelis-Menten model governed by the following equation: [Heme] / {[Heme] + K M }, assuming a heme K M value of 400 nM. The light blue shaded region represents the span of values reported for buffered free heme in cells and the black arrow indicates the concentration of intracellular free heme in which HO-1 is induced.

Article Snippet: The following primary and secondary antibodies were utilized: rabbit polyclonal heme oxygenase 2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Techniques: Activity Assay, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

doi: 10.1101/2021.06.06.447256

Figure Lengend Snippet: HO-2 silencing increases labile heme but does not affect total heme or bilirubin levels. ( a ) Representative immunoblot of HO-1 (HMOX1), HO-2 (HMOX2), and GAPDH expression in HEK293 cells treated with or without 350 µM 5-aminolevulinic acid (ALA) and scrambled (Ctrl siRNA) or targeted (HMOX2 siRNA) siRNA against HO-2 (Left). Immunoblot analysis from 6 independent trials demonstrates that siRNA against HMOX2 results in ∼80-90% silencing of HO- 2 protein expression (Right). ( b ) Median heme sensor eGFP/mKATE2 fluorescence ratio values derived from flow cytometry experiments from 9-13 replicates of HEK293 cultures grown in HD + SA media, regular media, or regular media supplemented with 350 µM ALA or control or targeted siRNA against HMOX2. Representative violin plots of eGFP/mKATE2 fluorescence ratio distributions from single cell analysis of HEK293 cultures are shown in Figure S8b . ( c ) Measurements of total heme in quintuplicate HEK293 cultures grown in regular media supplemented with control or targeted siRNA against HMOX2. ( d ) Measurements of total bilirubin in 3-6 replicates of HEK293 cultures grown in in HD + SA media, regular media, or regular media supplemented with 350 µM ALA or control or targeted siRNA against HMOX2. The statistical significance is indicated by asterisks using one-way ANOVA for multiple comparisons using Tukey’s range test. *p<0.05, **p<0.01,***p <0.001, ****p<0.0001, ns = not significant.

Article Snippet: The following primary and secondary antibodies were utilized: rabbit polyclonal heme oxygenase 2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Techniques: Western Blot, Expressing, Fluorescence, Derivative Assay, Flow Cytometry, Single-cell Analysis

Journal: bioRxiv

Article Title: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

doi: 10.1101/2021.06.06.447256

Figure Lengend Snippet: HO-2 overexpression depletes cytosolic and nuclear labile heme but not total heme, bilirubin levels, or mitochondrial labile heme. ( a ) Representative immunoblot of HO-1 (HMOX1), HO-2 (HMOX2), and GAPDH expression in untransfected (UT) or HO-2 overexpressing HEK293 cells treated with or without 10 µM hemin chloride. ( b ) Measurements of total heme in triplicate untransfected (-) or HO-2 overexpressing (OE) (+) HEK293 cultures grown in HD + SA or regular media. ( c ) Measurements of total bilirubin in triplicate untransfected (-) or HO-2 overexpressing (OE) (+) HEK293 cultures grown in regular media with or without 350 µM ALA. ( d ) Median cytosolic, nuclear or mitochondrial (mito)-targeted HS1 eGFP/mKATE2 fluorescence ratio values derived from flow cytometry experiments from triplicate untranfected (-) or HO-2 overexpressing (OE) (+) HEK293 cells grown in HD + SA or regular media. Representative violin plots of eGFP/mKATE2 fluorescence ratio distributions from single cell analysis of HEK293 cultures are shown in Figure S8c . The statistical significance is indicated by asterisks using one- way ANOVA for multiple comparisons using Tukey’s range test. *p<0.05, **p<0.01,***p <0.001, ****p<0.0001, ns = not significant.

Article Snippet: The following primary and secondary antibodies were utilized: rabbit polyclonal heme oxygenase 2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Techniques: Over Expression, Western Blot, Expressing, Fluorescence, Derivative Assay, Flow Cytometry, Single-cell Analysis

Journal: bioRxiv

Article Title: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

doi: 10.1101/2021.06.06.447256

Figure Lengend Snippet: Heme sequestration and buffering requires the HO-2 heme binding pocket but not catalytic activity, heme regulatory motifs (HRMs), or ER membrane-tethering. ( a ) Schematic of HO-2 and residues of interest, including heme binding ligand H45, a residue that accommodates heme binding within the heme binding pocket, G159, Cys residues within Cys-Pro (CP; stars) dipeptides in the HRMs, C265 and C282, and the ER membrane-spanning region spanning residues 288-316 (blue). ( b ) Median cytosolic HS1 eGFP/mKATE2 fluorescence ratio values derived from flow cytometry experiments from triplicate HEK293 cells that were untransfected (UT) or overexpressing WT or the indicated mutant HO-2 alleles. Cells were cultured in HD + SA, treated with or without 50 µM hemin chloride, or regular media, treated with or without 350 µM ALA. Representative violin plots of eGFP/mKATE2 fluorescence ratio distributions from single cell analysis of HEK293 cultures are shown in Figure S9 . ( c ) Representative immunoblot demonstrating overexpression of the indicated HO-2 (HMOX2) alleles relative to untransfected (UT) cells. ( d ) Measurements of total heme in triplicate cultures of HEK293 cells that were untransfected (UT) or overexpressing WT or mutant HO-2 grown in regular media. The statistical significance is indicated by asterisks using one-way ANOVA for multiple comparisons using Tukey’s range test. *p<0.05, **p<0.01,***p <0.001, ****p<0.0001, ns = not significant.

Article Snippet: The following primary and secondary antibodies were utilized: rabbit polyclonal heme oxygenase 2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Techniques: Binding Assay, Activity Assay, Fluorescence, Derivative Assay, Flow Cytometry, Mutagenesis, Cell Culture, Single-cell Analysis, Western Blot, Over Expression

Journal: bioRxiv

Article Title: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

doi: 10.1101/2021.06.06.447256

Figure Lengend Snippet: The effects of HO-2 depletion or overexpression on HS1 heme occupancy are consistent with labile heme being oxidized. ( a ) Simulation of HS1 heme occupancy as a function of HO-2 expression assuming the equilibrium model depicted in , with [HS1] = 10 nM, and heme being oxidized and present at 1 nM (black), 5 nM (blue), or 10 nM (grey). ( b ) Simulation of HS1 heme occupancy as a function of HO-2 expression assuming the equilibrium model depicted in , with [HS1] = 10 nM, and heme being reduced and present at 1 nM (black), 5 nM (blue), or 10 nM (grey). See text for details and Supporting Information for derivations of the equilibrium model. See also Figure S4 , which depict simulations for 100 nM and 1 µM [HS1] and 0.1, 0.5, and 1.0 equivalents of heme relative to [HS1] in oxidized or reduced states.

Article Snippet: The following primary and secondary antibodies were utilized: rabbit polyclonal heme oxygenase 2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Techniques: Over Expression, Expressing

Journal: Clinical Cancer Research

Article Title: Anti-EGFR Antibody–Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth

doi: 10.1158/1078-0432.CCR-23-3110

Figure Lengend Snippet: Stochastic conjugation of cetuximab to CDK inhibitor and ADC internalization in live breast cancer cells. A, Flow cytometric evaluation of surface EGFR expression (TNBC: MDA-MB-468, HCC1143, HCC1806, MDA-MB-231, HCC1937, SUM149, and CAL51; HER2+: SKBR3; ER+: MCF7, T47D; nontumorigenic epithelial cell model: MCF10A; immune cell model: human B lymphocytes RPMI8866, RPMI8226, and monocytic cell line U937; human primary melanocyte: melanocyte). B, EGFR mRNA expression from the Cancer Cell Line Encyclopedia database showed a positive correlation with surface EGFR measured by flow cytometry in A (Spearman’s rank coefficient, r = 0.723). A high level of correlation was found between EGFR and cyclin E ( r = 0.738), but not with cyclin A or CDK2. Nonsignificant P values are marked as NS. C, Top, Schematic diagram of stochastic ADC conjugation by antibody reduction with TCEP and then conjugation to SNS-032 via MC-Val–Ala-PAB. Middle, HIC analysis confirmed an average DAR of 4.4. Bottom, SEC trace indicates negligible ADC aggregation and minimal free linker–payload (less than 0.8%). D, Surface plasmon resonance analysis demonstrated similar binding affinity ( K D ) for cetuximab (0.73 nmol/L) and ADC (1.28 nmol/L). Isotype IgG1 and isotype ADC showed no measurable binding. E, Monitoring internalization of Fabfluor-pH-labeled cetuximab, ADC, or isotype control (10 nmol/L) by Incucyte live-cell imaging. Phase and red fluorescence time-course images were captured for 24 hours. Images of internalized antibody display in cytosolic, low pH lysosomal vesicle-associated red fluorescence in cells. Scale bar, 0.2 mm. F, Cells were seeded in Matrigel for 5 days, allowing the formation of spheroids. Fabfluor-pH-labeled antibodies or ADC (10 nmol/L) were introduced in the Matrigel and showed rapid internalization in EGFR-high MDA-MB-468 and MDA-MB-231, whereas EGFR-low CAL51 displayed little red fluorescence signals. A low level of internalization was observed for isotype or isotype-ADC controls. Scale bar, 0.5 mm. P values determined by two-tailed unpaired t test of three independent experiments compared with isotype control.

Article Snippet: Human primary epidermal melanocytes were cultured in Melanocyte Growth Medium (Cell Applications Inc.).

Techniques: Conjugation Assay, Expressing, Flow Cytometry, SPR Assay, Binding Assay, Labeling, Control, Live Cell Imaging, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Systematic analysis uncovers SYK dependency in NF1 LoF melanoma cells

doi: 10.1101/2022.03.06.483170

Figure Lengend Snippet: (a) A simplified schematic illustration of the function of NF1 in the context of RAS-mediated activation of ERK and PI3K/mTOR signaling in response to upstream growth factors. The nominal targets for kinase inhibitors tested in this study are highlighted. (b) Dose-dependent changes in normalized growth rates (a.k.a. DIP rates) calculated by dividing the 7-day average net growth rate for inhibitor-treated cells to that measured for vehicle (DMSO)-treated cells across cell lines with different NF1/BRAF/NRAS mutation status or primary epidermal melanocytes. The average net growth rates for each condition were calculated from measurements of live cell count (across two replicates) at four timepoints (including 1, 3, 5, and 7 days). Normalized growth rates < 0 indicate a net cell loss (i.e., inhibitor-induced cytotoxicity), a value of 0 represent no change in viable cell number (i.e., cytostasis), a value > 0 indicates a net cell gain, and a value of 1 represents no effect as cells grow in the presence of inhibitor at the same rate as in the DMSO condition. (c, d) Inhibitor maximal effect (E max ), defined as the normalized growth rate evaluated at the highest tested concentration, of different inhibitors across NF1 LoF melanoma cell lines (c) and between NF1 LoF cell lines and NF1 WT cells, including two BRAF V600E melanoma cell lines and primary melanocytes (d) . Statistical significance was determined by two-sided, paired-sample t test (c) or two-sided, two-sample t test (d) . (e) Mice bearing xenografts of COLO792 cells (top) and WM3918 cells (bottom) were treated with MTX-216 or vehicle for a period of 20 and 11 days, respectively, to determine the effect on tumor growth. Tumor volume data represent mean values ± s.e.m across 4 mice per group (in case of COLO792) or 5 mice per group (in case of WM3918). Statistical significance was determined by two-way ANOVA.

Article Snippet: Melanoma cell lines used in this study were obtained from the following sources: LOXIMVI (from DCTD Tumor Repository, National Cancer Institute), COLO858 and COLO792 (from ECACC), MeWo and A375 (from ATCC), SKMEL113 (from Memorial Sloan-Kettering Cancer Center), WM3918 (from Rockland Immunochemicals), human epidermal primary melanocytes (NHEM-Ad; from Lonza), YUHEF, YURED, YUHOIN, and YUTICA (all from Yale University Dermatology Cell Culture Facility).

Techniques: Activation Assay, Mutagenesis, Cell Counting, Concentration Assay

Journal: International Wound Journal

Article Title: Bacterial invasion into the epidermis of rats with sodium lauryl sulphate‐irritated skin increases damage and induces incontinence‐associated dermatitis

doi: 10.1111/iwj.13864

Figure Lengend Snippet: Representative images of immunohistochemistry for the neutrophil marker myeloperoxidase (MPO; magnification: ×20). Brown colour indicates cells with positive expression. No expression of MPO was detected in untreated (A) and sodium lauryl sulphate (B) tissues. Infiltrating inflammatory cells in the dermis of rats treated with cultured urine filtrate (CUF) (C) and CUFB (D) stain positive for MPO (yellow arrows). (a‐d) Magnified images of boxed areas in A‐D. Scale bar = 100 μm. (E) Quantification of average number of MPO positive cells per 0.1 mm 2 in CUF and CUFB treated rats. No significant differences were found ( P = .328)

Article Snippet: Primary antibodies were as follows: goat myeloperoxidase (MPO) polyclonal antibody (sc‐34 159; 1:50 dilution) purchased from Santa Cruz Biotechnology (Santa Cruz, California); mouse monoclonal anti‐major histocompatibility complex (MHC) class II (OX‐6, PA1‐73119, 1:200 dilution) and biotin‐conjugated (SP) polyclonal rabbit anti‐Pseudomonas antibody (PA1‐73119, 1∶300 dilutions) purchased from Thermo Fischer Scientific (Waltham, Massachusetts); Secondary antibodies were purchased as follows: Biotin‐SP (long spacer) AffiniPure Donkey Anti‐Mouse IgG (H + L; 715‐065‐150; dilutions 1:1000) from Jackson ImmunoResearch Laboratories and rat anti‐goat Histofine® Simple Stain MAX PO Universal immunoperoxidase polymer (HI1902) from Nichirei Biosciences (Tokyo, Japan).

Techniques: Immunohistochemistry, Marker, Expressing, Cell Culture, Staining

Journal: International Wound Journal

Article Title: Bacterial invasion into the epidermis of rats with sodium lauryl sulphate‐irritated skin increases damage and induces incontinence‐associated dermatitis

doi: 10.1111/iwj.13864

Figure Lengend Snippet: Representative images of Immunohistochemistry staining for the macrophage marker MHC‐II (magnification: ×20). Brown colour indicates cells with positive expression. Untreated (A) and sodium lauryl sulphate (B) tissues showed a negative result with no expression of MHC‐II. MHC‐II expression was detected in the dermis of rats treated with cultured urine filtrate (CUF) (C) and CUFB (D), as shown by the yellow arrows, with larger MHC‐II positively stained areas observed in (D). (a‐d) Magnified images of boxed areas in A‐D. Scale bar = 50 μm. (E) Quantification of average number of myeloperoxidase positive cells per 0.1 mm 2 in treated rats. Significant differences were observed between the CUF and CUFB groups ( P = .011)

Article Snippet: Primary antibodies were as follows: goat myeloperoxidase (MPO) polyclonal antibody (sc‐34 159; 1:50 dilution) purchased from Santa Cruz Biotechnology (Santa Cruz, California); mouse monoclonal anti‐major histocompatibility complex (MHC) class II (OX‐6, PA1‐73119, 1:200 dilution) and biotin‐conjugated (SP) polyclonal rabbit anti‐Pseudomonas antibody (PA1‐73119, 1∶300 dilutions) purchased from Thermo Fischer Scientific (Waltham, Massachusetts); Secondary antibodies were purchased as follows: Biotin‐SP (long spacer) AffiniPure Donkey Anti‐Mouse IgG (H + L; 715‐065‐150; dilutions 1:1000) from Jackson ImmunoResearch Laboratories and rat anti‐goat Histofine® Simple Stain MAX PO Universal immunoperoxidase polymer (HI1902) from Nichirei Biosciences (Tokyo, Japan).

Techniques: Immunohistochemistry, Staining, Marker, Expressing, Cell Culture