Journal: Viruses

Article Title: Therapeutic Potential of 5′-Methylschweinfurthin G in Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma

doi: 10.3390/v14091848

Figure Lengend Snippet: MeSG impairs MTT activity of human Merkel cell carcinoma cell lines. ( A ) Structures of the natural schweinfurthin G and the synthetic schweinfurthin analog MeSG, TTI-3114. ( B ) The human Merkel cell carcinoma cell lines MKL−1, MKL-2, MS-1, and MCC13 were incubated with increasing doses of MeSG (100 pM-100 μM) for 48 h. The impact of MeSG treatment on MTT activity was assessed by MTT assay. Data are displayed as a percentage of control and are representative of three independent experiments ( n = 3, mean ± SEM).

Article Snippet: MCC13 (RRID:CVCL_2583) and MKL-1 (RRID:CVCL_2600) cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Incubation, MTT Assay, Control

Journal: Viruses

Article Title: Therapeutic Potential of 5′-Methylschweinfurthin G in Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma

doi: 10.3390/v14091848

Figure Lengend Snippet: MeSG inhibits cell migration and invasion of MCC cell lines. VP-MCC (MS-1, MKL-1, MKL-2) and VN-MCC (MCC13) cells were seeded on a polycarbonate membrane (migration) or a polycarbonate membrane coated with a dried basement membrane matrix solution (invasion). Cells were allowed to migrate (24 h) or invade (48 h) toward 10% or 20% FBS in the presence or absence of 0, 3, 10, or 30 nM MeSG. Migrated or invaded cells on the bottom of the membrane were stained and quantified. At least three independent samples were analyzed (mean ± SEM).

Article Snippet: MCC13 (RRID:CVCL_2583) and MKL-1 (RRID:CVCL_2600) cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Migration, Membrane, Staining

Journal: Nature Communications

Article Title: High-throughput screening identifies Aurora kinase B as a critical therapeutic target for Merkel cell carcinoma

doi: 10.1038/s41467-025-56504-7

Figure Lengend Snippet: A RNAi druggable genome screen demonstrating mean viability reduction following knockdown in MKL-2 (VP-MCC) vs. MCC26 (VN-MCC) cells (n = 3). All viabilities > 100 set to 100. Please refer to Supplementary Fig. : knockdown of both INCENP (n = 3) and survivin (n = 5) reduce VP-MCC, but not VN-MCC viability. B AURKB knockdown reduces MKL-2 and MCC26 viability relative to negative control (NC) (n = 3). Mean viability from 3 different siRNAs, error bars represent SEM. C Representative Western Blot demonstrating AURKB knockdown with siRNA (si), but not NC in MCC cell lines (n = 3). Red: β-actin loading control, green: AURKB. D Western Blot quantification demonstrating efficient AURKB siRNA knockdown relative to NC in WAGA (p = 0.001; n = 4), MKL-1 (p = 0.00003; n = 3), MCC13 (p = 0.00009; n = 3), and UISO (p = 0.045; n = 3) cell lines as measured by one-tailed two-sample t-test with unequal variance. AURKB intensity was normalized to β-actin intensity from identical lane, and means from at least 3 independent experiments are shown, with error bars representing SD. E AURKB knockdown significantly reduced viability of WAGA (p = 0.0003; n = 4), MKL-1 (p = 0.0105; n = 3), MCC13 (p = 0.0004; n = 3), and UISO (p = 0.0094; n = 3) cells relative to NC as measured by one-sample t-test. Data points are means from at least 3 independent experiments, with error bars representing SD. A and B, n = number of siRNAs per gene; C, D and E, n = number of experimental replicates.

Article Snippet: Paraffin-embedded MCC13 (VN-MCC) tissue sections were deparaffinized, rehydrated, and treated with Bloxall solution (SP-6000, Vector Laboratories, Burlingame, CA, USA) at room temperature to block endogenous peroxidase activity, followed by heat-mediate antigen retrieval using sodium citrate buffer (Bio SB, Santa Barbara, CA, USA).

Techniques: Knockdown, Negative Control, Western Blot, Control, One-tailed Test

Journal: Nature Communications

Article Title: High-throughput screening identifies Aurora kinase B as a critical therapeutic target for Merkel cell carcinoma

doi: 10.1038/s41467-025-56504-7

Figure Lengend Snippet: A Representative Fluorescent Western Blot of total histone 3 expression and pH3-Ser10. MCC cells were treated with DMSO, 3 nM, or 30 nM AZD2811 for 24 h (72 h for MCC26). Images were cropped to improve clarity (n = 3). B Quantification of pH3-Ser10 protein expression normalized to total histone 3 relative to DMSO control treatment from three independent experiments, with error bars representing SEM. 30 nM of AZD2811 significantly reduced pH3-Ser10 in WAGA (3 nM: p = 0.002, 30 nM: p < 0.0001; n = 6), MKL-2 (3 nM: p = 0.097, 30 nM: p = 0.00072; n = 3), MKL-1 (3 nM: p = 0.002, 30 nM: p < 0.0001; n = 3), MCC13 (3 nM: p = 0.043, 30 nM: p = 0.011; n = 5), and MCC26 (3 nM: p = 0.026, 30 nM: p = 0.031; n = 3) cells, as measured by two-tailed, two-sample, unpaired t-test. C Representative flow cytometry propidium iodide (PI) intensity histogram traces following treatment with 30 nM AZD2811 demonstrating a G2/M cell cycle arrest at 24 h in WAGA, MKL-2, MKL-1, and MCC13 and increased polyploidy ( > G2) at 24 and 72 h in MCC26 (n = 3). D AZD2811 24-h treatment (30 nM) significantly increased the proportion of cells in G2 in WAGA (p = 0.022; n = 3), MKL-1 (p = 0.0008; n = 3), MKL-2 (p = 0.016; n = 3), MCC13 (p = 0.0056; n = 3), but not MCC26 cells (p = 0.149; n = 3) as measured by two-sample, one-tailed unpaired t-test. AZD2811 treatment (30 nM) significantly increased the proportion of polyploid cells ( > G2) in MCC26 at 24 h (p = 0.0006; n = 3) and 72 h (p = 0.00759; n = 3). Values are means from at least 3 independent experiments with error bars representing SEM. n = number of experimental replicates.

Article Snippet: Paraffin-embedded MCC13 (VN-MCC) tissue sections were deparaffinized, rehydrated, and treated with Bloxall solution (SP-6000, Vector Laboratories, Burlingame, CA, USA) at room temperature to block endogenous peroxidase activity, followed by heat-mediate antigen retrieval using sodium citrate buffer (Bio SB, Santa Barbara, CA, USA).

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Flow Cytometry, One-tailed Test

Journal: Nature Communications

Article Title: High-throughput screening identifies Aurora kinase B as a critical therapeutic target for Merkel cell carcinoma

doi: 10.1038/s41467-025-56504-7

Figure Lengend Snippet: A Schematic of xenograft tumor treatment protocol. Mice were subcutaneously injected with either MKL-1 or MCC13 cells (n = 10). Once the average tumor size reached 100 mm 3 animals were treated with intravenous AZD2811NP (25 mg/kg, twice weekly) or placebo nanoparticles for 4 weeks. B, C Rate of tumor growth, as calculated by linear regression, was significantly reduced by AZD2811NP treatment relative to placebo. P-values on the slope comparing vehicle and AZD2811NP were MKL-1 (p < 0.0001) and MCC13 (p < 0.0001). Data points represent average tumor volumes, with error bars representing SEM (n = 10). Please refer to Supplementary Fig. : AZD2811NP significantly slowed MKL-1 and MCC13 tumor growth relative to placebo during treatment period as measured by linear regression. Treatment with AZD2811NP did not reduce body weight in mice xenografted with MKL-1 (p = 0.71) or MCC13 (p = 0.9). D, E Kaplan Meier survival estimates showing that AZD2811NP extends survival of mice with MKL-1 (p < 0.0001) or MCC13 (p < 0.0001) xenograft tumors as calculated by Log-rank (Mantel-Cox) test in GraphPad Prism (n = 10). F, G Representative photographs of mice (MKL-1: day 28; MCC13: day 22) labeled with treatment. A , D , E , F , and G , n = number of animals; B and C , n = number of tumors.

Article Snippet: Paraffin-embedded MCC13 (VN-MCC) tissue sections were deparaffinized, rehydrated, and treated with Bloxall solution (SP-6000, Vector Laboratories, Burlingame, CA, USA) at room temperature to block endogenous peroxidase activity, followed by heat-mediate antigen retrieval using sodium citrate buffer (Bio SB, Santa Barbara, CA, USA).

Techniques: Injection, Labeling

Journal: Nature communications

Article Title: Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.

doi: 10.1038/s41467-023-36818-0

Figure Lengend Snippet: Fig. 2 | UBE3A knockdown increases cofilin and decreases F-actin content and PIEZO2 function. a Top, representative whole-cell patch-clamp recordings of currents elicited by mechanical stimulation (−60 mV) in MCC13 cells transfected with scrambled, UBE3A, or PIEZO2 siRNAs. Bottom, current densities elicited by maximum displacement of siRNA-transfected cells. Bars are mean ± SD. Kruskal- Wallis (H = 18.76; p = 8.4−5) and Dunn’s multiple comparisons test. b Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells transfected as in (a). Bottom, mean/scatter-dot plot showing relative intensities of PIEZO2 protein nor- malized to PIEZO2 in the Sc. group. Lines are mean ± SD. Kruskal-Wallis (H = 12.78; p = 0.0017) and Dunn’s multiple comparisons test. c Top, currents elicited by mechanical stimulation (−60 mV) in cells transfected with UBE3A plasmid. Bottom, current densities elicited by maximum displacement of UBE3A transfected cells. Bars are mean ± SD. Two-tailed unpaired t-test with Welch’s correction (t = 3.9). d Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells transfected with UBE3A plasmid. Bottom, mean/scatter-dot plot showing relative intensities of PIEZO2 protein in UBE3A transfected cells normalized to PIEZO2 in the control group. Lines are mean ± SD. Two-tailed one-sample t-test (t = 4.4). e Top, currents elicited by mechanical stimulation (−60 mV) of latrunculin A (1 µM; 24 h)- treated MCC13 cells. Bottom, current densities elicited by maximum displacement. Bars are mean ± SD. Two-tailed unpaired t-test with Welch’s correction (t = 9.9). f Top, western blot (anti-PIEZO2) of the membrane fractions of MCC13 cells treated

Article Snippet: Protein concentrations were measured with the Bio-Rad protein assay or the PierceTM BCA Protein Assay Kit (ThermoFisher Scientific), and equivalent protein amounts were loaded in Mini-PROTEAN TGX Stain-Free Precast Gels (Bio-Rad).Mousemonoclonal anti-UBE3A (1:1,000; Sigma-AldrichCat# SAB1404508, RRID:AB_10740376), mouse monoclonal anti-actin for MCC13 cells (1:5,000; Bio-Rad Cat# MCA358GT, RRID:AB_323521), mouse monoclonal anti-actin for DRGs (1:1,000; Cytoskeleton Cat# AAN02, RRID:AB_2884962), goat polyclonal anti-mouse IgGH&L (HRP) (1:20,000; Abcam Cat# ab205719, RRID:AB_2755049), rabbit polyclonal anti-human PIEZO2 (1:1,000; abcepta Cat# AP16313b, RRID:AB_11136435), rabbit polyclonal anti-cofilin (1:1,000; abcepta Cat# AP53892, RRID:AB_2923306), rabbit monoclonal anti-cofilin for DRGs (1:1,000; Cell Signaling Technology Cat# 5175, RRID:AB_10622000), and goat anti-rabbit IgG HL-HRP conjugated (1:10,000; Bio-Rad Cat# 1706515, RRID:AB_2617112) antibodies were used for western blots.

Techniques: Knockdown, Patch Clamp, Transfection, Western Blot, Membrane, Plasmid Preparation, Two Tailed Test, Control

Journal: Cancers

Article Title: Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma

doi: 10.3390/cancers14041091

Figure Lengend Snippet: Growth of MCPyV + and MCPyV − MCC cell lines under different experimental conditions in OERCs.

Article Snippet: The other MCC cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC): MCC13 (Cat#10092302), MCC14/2 (Cat#10092303), MCC26 (Cat#10092304), MS-1 (Cat#09111802), and MKL-1 (Cat#09111801).

Techniques:

Journal: Cancers

Article Title: Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma

doi: 10.3390/cancers14041091

Figure Lengend Snippet: Histological analysis of organotypic epithelial raft cultures (OERCs) of MCC cell lines co-cultured with primary human keratinocytes (PHKs). MCPyV − MCC cell lines (MCC14/2, MCC26 and MCC13) grow in OERCs (indicated by arrows in the H&E sections) when co-cultured with PHKs at a ratio of 1 to 5. Except MCC13, these cell lines express NCAM, a typical MCC marker. MCPyV + MCC cell lines (MS-1, MKL-1 and WAGA) also proliferate in our 3-D culture model, as indicated by full arrows in the H&E sections, when co-cultured with PHKs. These cells express the LT of MCPyV (although the murine fibroblasts stain positive as well, likely due to cross-reactivity to the murine antibody) and the MCC marker CK20, as confirmed by IHC. Double IHC staining for MCPyV LT (brown) and CK20 (red) show co-localization of these signals. PHKs differentiate in a normal epithelium (pointed out by dashed arrows). All images were taken at 20× magnification and the bars equal 100 μm.

Article Snippet: The other MCC cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC): MCC13 (Cat#10092302), MCC14/2 (Cat#10092303), MCC26 (Cat#10092304), MS-1 (Cat#09111802), and MKL-1 (Cat#09111801).

Techniques: Cell Culture, Marker, Staining, Immunohistochemistry