Journal: Human Molecular Genetics

Article Title: Fibroblast phenotype in male carriers of FMR1 premutation alleles

doi: 10.1093/hmg/ddp497

Figure Lengend Snippet: Immunofluorescence microscopy reveals that cells from a fixed skin biopsy and from cultured fibroblasts derived from patient F4 with severe FXTAS, clinical stage 5, (A and B, lower panels), have decreased expression of lamin A/C protein compared with those of an age-matched control subject, C2 (A and B, upper panels). (A) Tissue biopsy of subject F4 displays a lack of the typical ring-like distribution of lamin A/C protein (green) and abnormal morphology of the nuclei counterstained with DAPI (blue), in comparison to C2 (magnification ×400). (B) Cultured skin fibroblasts were stained with anti-lamin A/C (green) and anti-beta tubulin (red) antibodies to define the contour of the cells. Control fibroblasts derived from C2 display normal ring-like lamin A/C architecture (upper panel); whereas fibroblasts derived from F4 (lower panel) exhibit distorted lamin A/C organization and overall decreased staining (magnification ×800). Arrows indicate uneven distribution of lamin A/C; arrowheads indicate non-fusiform fibroblasts and abnormal morphology of the nuclei. (C) Dot plot representing the percentage of skin fibroblasts with complete lamin A/C rings in cultured fibroblasts derived from five premutation carriers with no clinical signs of FXTAS (asymptomatic; Asymp Pre) and six subjects with FXTAS (black dots, premutation carriers) compared with six age-matched controls (white dots) described in Table 1. Horizontal lines represent the mean value per phenotypic group.

Article Snippet: The membranes were subsequently blocked with BLOTTO (5% non-fat dry milk in 100 m m Tris–HCl, 0.9% NaCl, 0.1% polyoxyethylene (20) sorbitan monolaurate, pH 7.5) followed by overnight incubation with primary antibodies: mouse monoclonal anti-lamin A/C (BD Biosciences), rabbit polyclonal anti-HSP70 (SPA-812, Stressgen), mouse monoclonal anti-HSP27 (SPA-800, Stressgen), mouse monoclonal anti-αB-crystallin (SPA-222, Stressgen) or mouse monoclonal anti-GAPDH (GTX29484, GeneTex, Inc.), diluted in BLOTTO.

Techniques: Immunofluorescence, Microscopy, Cell Culture, Derivative Assay, Expressing, Staining

Journal: Human Molecular Genetics

Article Title: Fibroblast phenotype in male carriers of FMR1 premutation alleles

doi: 10.1093/hmg/ddp497

Figure Lengend Snippet: Reduction of the number of cultured MEFs with normal lamin A/C rings derived from knock-in (Premutation, Pre) mice with ∼200 CGG repeats. Left: immunocytochemical localization of lamin C in mouse embryonic fibroblasts (MEFs). Upper panel: wt mouse fibroblast nuclei. Lower panel: Pre mouse fibroblasts display marked lamin A/C disruption with loss of the normal ring-like architecture (green); nuclei counterstained with DAPI, blue (×630 magnification). Right: mean number of MEFs with normal nuclear lamin A/C organization are decreased in KI (dark gray bar), compared with an age-matched wt (open bar).

Article Snippet: The membranes were subsequently blocked with BLOTTO (5% non-fat dry milk in 100 m m Tris–HCl, 0.9% NaCl, 0.1% polyoxyethylene (20) sorbitan monolaurate, pH 7.5) followed by overnight incubation with primary antibodies: mouse monoclonal anti-lamin A/C (BD Biosciences), rabbit polyclonal anti-HSP70 (SPA-812, Stressgen), mouse monoclonal anti-HSP27 (SPA-800, Stressgen), mouse monoclonal anti-αB-crystallin (SPA-222, Stressgen) or mouse monoclonal anti-GAPDH (GTX29484, GeneTex, Inc.), diluted in BLOTTO.

Techniques: Cell Culture, Derivative Assay, Knock-In

Journal: Molecular Biology of the Cell

Article Title: Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites

doi: 10.1091/mbc.E10-12-0938

Figure Lengend Snippet: PCNA nuclear focus formation and monoubiquitination in response to UV damage. (A) UV-induced colocalization of PCNA with Rev3 in the low-passage human fibroblast cell line GM08402 as revealed by ICC. Although Rev3 was found to be variably distributed in cytoplasm and the nucleus (left), a UV treatment (12 J/m 2 for 4 h, middle) caused a nearly complete translocation of Rev3 to the nucleus, regardless of the cell cycle stage. The staining patterns of Rev3 and PCNA in untreated S-phase (PCNA-positive) cells do not appear to overlap (left). However, after UV treatment, the discrete distribution of Rev3 appears to match that of PCNA (middle). This nuclear focus colocalization is further revealed following NP40 preextraction before fixation (0.4% on ice for 40 min, right). (B) NF1604 whole-cell lysates and nuclear fractions were loaded based on equal culture volume and analyzed by Western blotting to reveal the induction of a slow-migrating anti-PCNA immunoreactive band following UV exposure, indicative of monoubiquintinated PCNA. Cells were incubated for 12 h following UV irradiation at the indicated doses. Anti–lamin B immunoreactivity was used as a loading control in a parallel Western blot.

Article Snippet: Antibodies used include mouse anti-PCNA (1:5000, overnight, clone Ab-1; Calbiochem, La Jolla, CA) and mouse anti–lamin B (1:5000, overnight; Oncogene Science, Cambridge, MA).

Techniques: Translocation Assay, Staining, Western Blot, Incubation, Irradiation

Journal: Frontiers in Pharmacology

Article Title: Modulation of Keap1/Nrf2/ARE Signaling Pathway by Curcuma- and Garlic-Derived Hybrids

doi: 10.3389/fphar.2019.01597

Figure Lengend Snippet: Nrf2-pathway activation by hybrids: nuclear translocation and targets induction. (A) Nuclear cellular extracts of SH-SY5Y cells were treated for 3 hours with compounds at 5 µM, 500 nM, and 50 nM or with 20, 10, and 5 µM dimethyl fumarate (DMF). Nrf2 protein content in the nucleus was determined by Western blot. Anti-lamin A/C was used as a protein loading control. Results are shown as ratio Nrf2/lamin A/C ± SEM; *p < 0.05, **p < 0.01 and ****p < 0.0001 versus CTR; Dunnett’s multiple comparison test (F ratio = 6.797, n≥3). (B–C) RNA from total cellular extracts of SH-SY5Y cells, treated for 24 hours with 5 μM compounds or 20 μM DMF, were analyzed for NQO1 (B) and HO-1 (C) mRNA expression by RT-qPCR. GAPDH was used as housekeeping gene. Results are shown as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 versus CTR; Dunnett’s multiple comparison test (B, n≥3, F ratio = 10.44; C, n≥3, F ratio = 13.95). (D–E) Cellular extracts of SH-SY5Y cells treated for 24 hours with compounds at 5 μM or 20 μM DMF were analyzed for NQO1 (D) and HO-1 (E) protein levels by Western blot. Anti-actin was used as protein loading control. Results are shown as ratio (% of CTR) ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus CTR; Dunnett’s multiple comparison test (D, n ≥ 3, F ratio = 5.144; E, n≥3, F ratio = 17.26).

Article Snippet: Mouse monoclonal anti-human β-actin (612656) and mouse anti-human lamin A/C (612162) antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Activation Assay, Translocation Assay, Western Blot, Expressing, Quantitative RT-PCR