Journal: The American journal of pathology

Article Title: Slug expression during melanoma progression.

doi: 10.1016/j.ajpath.2012.02.014

Figure Lengend Snippet: Figure 3. 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 expres- sion of Slug and its putative target genes. MITF isoforms include both melanocyte-specific (M) and ubiquitous (A) isoforms.

Article Snippet: 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: Figure 4. 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 adeno- virus (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-trans- duced NHM and melanoma cells compared to Ad- Null-transduced cells, although basal levels of ex- pression 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 ex- pression 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 re- flected in differences in protein expression. In addi- tion, 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: 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, Virus, Quantitative RT-PCR, Transfection, Western Blot

Journal: Scientific Reports

Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

doi: 10.1038/srep05885

Figure Lengend Snippet: (A–B) Flow cytometric histograms showing fluorescent intensity of cells incubated with purified IgG/IgE antibodies plus secondary FITC-labelled goat anti-human IgG/IgE (open) and cells with secondary antibody only (filled) against the number of cells. (A) Flow cytometric assessment of anti-CSPG4 IgG 1 /IgE antibodies shows specific binding to native CSPG4 antigen present on the cell surface of A375 melanoma cells and no binding above background to primary melanocytes. (B) Fc regions of anti-CSPG4 IgG 1 and 102.1F10 IgG 4 isotypes demonstrate effector-binding to U937 monocytic cell line, expressing human Fcγ receptors. The IgE antibody isotypes also bind similarly to RBL SX38 mast cells, expressing human FcεR1 receptor. (C) Immunofluorescence staining of A375 cells confirms specific binding of anti-CSPG4 IgG 1 /IgE and no background binding with isotype control hapten specific NIP-IgG 1 /IgE detected by goat anti-human IgG/IgE-FITC. (D) Grass pollen allergen specificity is confirmed by anti-human sandwich ELISA, showing specific binding of recombinant 102.1F10 IgG 4 /IgE and original patient serum to the ELISA plate-bound Phl p 7 allergen and no binding above background with unspecific human myeloma IgG 4 and MOv18 IgE isotype controls.

Article Snippet: Primary human epidermal melanocytes (PCS-200-012, ATCC) were grownin Dermal Cell Basal Medium (PCS-200-030, ATCC) and supplemented with the Melanocyte Growth Kit (PCS-200-041, ATCC).

Techniques: Incubation, Purification, Binding Assay, Expressing, Immunofluorescence, Staining, Control, Sandwich ELISA, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Antibiotics

Article Title: Evaluation of Biotechnological Active Peptides Secreted by Saccharomyces cerevisiae with Potential Skin Benefits

doi: 10.3390/antibiotics13090881

Figure Lengend Snippet: Cytotoxicity of the peptides (2–10 kDa) in melanoma cell line MNT1 and normal dermal fibroblasts, keratinocytes, and melanocytes. Cells were exposed to increasing concentrations of the peptide for 48 h and viability was evaluated with the MTS assay. Bars represent the average ± standard deviation.

Article Snippet: The medium for melanocytes and keratinocytes was Dermal cells basal media (PCS-200-030, ATCC) supplemented with melanocytes growth kit (PCS-200-041, ATCC) or keratinocytes growth kit (PCS-200-040, ATCC), respectively.

Techniques: MTS Assay, Standard Deviation

Journal: Human Mutation

Article Title: A Macrophage/Monocyte‐Related Four‐Gene Signature for Prognostic Assessment of Uveal Melanoma: BTBD6 , C2CD4B , CCL24 , and S100A4

doi: 10.1155/humu/4978880

Figure Lengend Snippet: Role of four prognostically relevant genes. (A) Quantitative reverse transcription PCR for the calculated levels of four prognostically relevant genes in human epidermal melanocytes. (B) Validation of the knockdown efficiency of C2CD4B. (C, D) Scratch and Transwell assays exploring the migration and invasion of A375 cells in vitro. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, p > 0.05.

Article Snippet: Melanoma cell line A375 (CRL‐1619) and human epidermal melanocytes (PCS‐200‐013) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia) and cultured as follows.

Techniques: Reverse Transcription, Biomarker Discovery, Knockdown, Migration, In Vitro

Journal: Blood Advances

Article Title: Melanocortin/MC5R axis regulates the proliferation of hematopoietic stem cells in mice after ionizing radiation injury

doi: 10.1182/bloodadvances.2022009249

Figure Lengend Snippet: MC5R deficiency aggravates myelosuppression after radiation injury. (A) Enzyme-linked immunosorbent assay of α-MSH level in the BM supernatant of WT mice at the indicated time after 5.0 Gy total body irradiation (TBI) (n = 5). Unirradiated WT mice were used as controls (n = 5). (B) qRT-PCR analysis of MC5R expression in LSKs sorted from the BM of WT mice at the indicated time after 5.0 Gy TBI (n = 3). The relative expression of MC5R was compared with that at 0 hour. (C-E) The counts of (C) WBC, (D) RBC, and (E) PLT in the PB of WT and MC5R –/– mice at the indicated time after 5.0 Gy TBI (n = 9). (F) Total number of BM cells in WT and MC5R –/– mice at the indicated time after 5.0 Gy TBI (n = 6). (G) Representative flow cytometric plots showing the percentages of MPs, LSKs, LT-HSCs, ST-HSCs, and MPPs in the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI. (H) The numbers of MPs and LSKs in the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI (n = 6). (I) The number of LT-HSCs, ST-HSCs, and MPPs in the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI (n = 6). (J) The number of MEPs, CMPs, GMPs, and CLPs in the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI (n = 6). (K) Flow cytometric analysis of the percentages of T cells, B cells, and myeloid cells in the BM of WT and MC5R -/- mice 15 days after 5.0 Gy TBI (n = 6). (A,B) one-way ANOVA with Tukey multiple comparisons test; (C-F,H-K) unpaired t test (two-tailed). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. Con, control; IR, irradiation.

Article Snippet: A total of 1 × 10 3 Lineage – Sca1 + c-Kit + cells (LSKs) sorted from the BM of WT and MC5R –/– mice were cultured in StemSpan SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with different concentrations of α-MSH (0, 5, 50, or 500 nM; MedChemExpress, Princeton, NJ) for 7 days.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Quantitative RT-PCR, Expressing, Two Tailed Test, Control

Journal: Blood Advances

Article Title: Melanocortin/MC5R axis regulates the proliferation of hematopoietic stem cells in mice after ionizing radiation injury

doi: 10.1182/bloodadvances.2022009249

Figure Lengend Snippet: Loss of MC5R compromises proliferation and reconstitution ability of HSCs. (A-C) Flow cytometric analysis of the cell cycle distribution of (B) LSKs and (C) LT-HSCs in the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI (n = 6). Representative flow cytometric plots are shown (A). (D) Flow cytometric analysis of the percentage of BrdU + cells in LSKs and LT-HSCs from the BM of WT and MC5R –/– mice 15 days after 5.0 Gy TBI (n = 6). Representative flow cytometric plots (left). (E-G) A total of 5 × 10 3 LSKs obtained from the BM of WT and MC5R –/– mice (CD45.2) were mixed with 5 × 10 5 CD45.1 BM cells and transplanted into lethally irradiated CD45.1 recipient mice. (E) The scheme of competitive BMT. (F) Flow cytometric analysis of the percentage of donor-derived cells in the PB of recipient mice 16 weeks after transplantation (n = 6). Representative flow cytometric plots (left). (G) Chimerism levels of BM, LSKs, and LT-HSCs in recipient mice at 16 weeks after transplantation (n = 6). (H) LSKs sorted from the BM of WT and MC5R –/– mice were cultured in the presence of different concentrations of α-MSH (0, 5, 50, or 500 nM). The total cell counts in the medium were counted 7 days after culture (n = 6). (I,J) A total of 1 × 10 6 CD45.1 BM cells were transplanted into lethally irradiated WT or MC5R –/– recipient mice (CD45.2). (I) The scheme of reciprocal BMT. (J) Flow cytometric analysis of the percentage of donor-derived cells in the PB of recipient mice at 16 weeks after transplantation (n = 6). (K,L) A total of 2 × 10 5 LSKs obtained from the BM of WT and MC5R –/– mice were labeled with CFSE and transplanted into lethally irradiated WT recipient mice. The percentage of CFSE + cells in the BM of recipient mice 16 hours after transplantation was detected using flow cytometry. (K) The scheme of homing assay. (L) The homing efficiency of WT and MC5R –/– LSKs (n = 6). (B-D,F-H, J,L) unpaired t test (two-tailed). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. CFSE, 5(6)-carboxyfluorescein diacetate succinimidyl ester.

Article Snippet: A total of 1 × 10 3 Lineage – Sca1 + c-Kit + cells (LSKs) sorted from the BM of WT and MC5R –/– mice were cultured in StemSpan SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with different concentrations of α-MSH (0, 5, 50, or 500 nM; MedChemExpress, Princeton, NJ) for 7 days.

Techniques: Irradiation, Derivative Assay, Transplantation Assay, Cell Culture, Labeling, Flow Cytometry, Two Tailed Test

Journal: Blood Advances

Article Title: Melanocortin/MC5R axis regulates the proliferation of hematopoietic stem cells in mice after ionizing radiation injury

doi: 10.1182/bloodadvances.2022009249

Figure Lengend Snippet: Melanocortin/MC5R axis promotes hematopoietic regeneration in irradiated mice. (A,B) Flow cytometric analysis of the expression of (A) p-AKT and (B) p-ERK1/2 in LT-HSCs from the BM of WT mice treated with saline or α-MSH, along with vehicle, AKT inhibitor MK-2206, or ERK1/2 inhibitor PD325901 (n = 6). (C) Cell cycle analysis of LT-HSCs from the BM of WT mice treated with saline or α-MSH along with vehicle, MK-2206, or PD325901 (n = 6). (D,E) The number of (D) MPs, LSKs, and (E) LT-HSCs from the BM of WT mice treated with saline or α-MSH, along with vehicle, MK-2206, or PD325901, 15 days after 5.0 Gy TBI (n = 6). (F-H) The counts of (F) WBC, (G) RBC, and (H) PLT in the PB of WT mice treated with saline or α-MSH, along with vehicle, MK-2206, or PD325901, at the indicated time after 5.0 Gy TBI (n = 10). (I) The survival rates of WT mice treated with saline or α-MSH, along with vehicle, MK-2206, or PD325901, after 7.5 Gy TBI (n = 10). (A-H) One-way ANOVA with Tukey multiple comparisons test; (I) Log-rank test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; # P < .05; ## P < .01; † P < .05; †† P < .01. NS, not significant.

Article Snippet: A total of 1 × 10 3 Lineage – Sca1 + c-Kit + cells (LSKs) sorted from the BM of WT and MC5R –/– mice were cultured in StemSpan SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with different concentrations of α-MSH (0, 5, 50, or 500 nM; MedChemExpress, Princeton, NJ) for 7 days.

Techniques: Irradiation, Expressing, Saline, Cell Cycle Assay

Journal: bioRxiv

Article Title: POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death

doi: 10.1101/2025.11.26.690880

Figure Lengend Snippet: WT POLQ and variants E2406K, and T2161I reduce the number of double strand brakes in UV treated cells. MCF7 cells melanocytes were co-transfected with either a scrambled control siRNA oligo or an siRNA oligo to POLQ and either a siRNA resistant WT, L2538R, E2406K, or T2161I POLQ rescue plasmid. Cells were allowed to grow for 48-72 hrs. and then treated with either 5J ultra-violet radiation or not and allowed to recover for 24 hrs. Neutral comet assays were performed using Comet slides (n ≥ 50 cells) and analyzed using CometScore software. Error bars represent standard deviation, and significance was determined using One-way ANOVA (**** p < 0.0001).

Article Snippet: MCF7 (ATCC HTB-22) or dermal melanocyte (ATCC CRL-4059) cells were dispensed into 96-well plates and grown to 80-90% confluency.

Techniques: Transfection, Control, Plasmid Preparation, Software, Standard Deviation