Journal: Neurology: Genetics

Article Title: Duplication and deletion upstream of LMNB1 in autosomal dominant adult-onset leukodystrophy

doi: 10.1212/NXG.0000000000000292

Figure Lengend Snippet: (A) Gene dosages for exons 3, 6, and 10 of LMNB1 were determined by TaqMan-based real-time PCR assay. The copy numbers of 3 exons of LMNB1 in patients 1–4 were increased by approximately 1.5-fold compared with control subjects, suggesting the presence of duplication of LMNB1 in these patients. (B) The copy number variations of regions upstream of LMNB1 including GRAMD3 , ALDH7A1 , and PHAX were determined by TaqMan-based real-time PCR assay. The copy numbers of ALDH7A1 and PHAX were decreased approximately by half in patients 5 and 6, suggesting the presence of the upstream deletion of LMNB 1. (C) The genomic regions of duplication (blue) and deletion (red) were analyzed using an Affymetrix CytoScan HD array and are shown on the basis of information obtained from the UCSC genome browser (assembly GRCh37/hg19). The regions of duplication were 153 kb in pedigree I, 220 kb in pedigree II, and 221 kb in pedigree III. The deletion upstream of LMNB1 in a previous report is shown by a dotted line. The positions of original enhancer A (Enh-A) and alternative enhancer B (Enh-B) for LMNB1 are indicated by arrowheads. ALDH7A1 = aldehyde dehydrogenase 7 family member A1; CNV = copy number variation; GRAMD3 = GRAM domain containing 3; LMNB1 = lamin B1; PHAX = phosphorylated adaptor for RNA export.

Article Snippet: CNV was analyzed by real-time PCR assay using TaqMan probes (Thermo Fischer Scientific, Waltham, MA) designed for exons 3 (Hs02537023_cn), 6 (Hs00696436_cn), and 10 (Hs00579415_cn) of LMNB1.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Neurology: Genetics

Article Title: Duplication and deletion upstream of LMNB1 in autosomal dominant adult-onset leukodystrophy

doi: 10.1212/NXG.0000000000000292

Figure Lengend Snippet: The relative mRNA expression level of LMNB1 in patients with LMNB1 duplication (n = 4) and control subjects (n = 7) was determined using RNA extracted from peripheral blood by quantitative RT-PCR assay. qRT-PCR was performed using primer pairs spanning exons 6 and 7 (A and B) or exons 9 and 10 (C and D) of LMNB1 . mRNA expression level of LMNB1 was normalized to those of ACTB (A and C) and TBP (B and D). The average value of control subjects was set to 1. Error bars indicate standard deviation. The statistical significance of difference was examined by the Mann–Whitney U test. LMNB1 = lamin B1; RT = reverse transcription.

Article Snippet: CNV was analyzed by real-time PCR assay using TaqMan probes (Thermo Fischer Scientific, Waltham, MA) designed for exons 3 (Hs02537023_cn), 6 (Hs00696436_cn), and 10 (Hs00579415_cn) of LMNB1.

Techniques: Expressing, Control, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY, Reverse Transcription

Journal: Neurology: Genetics

Article Title: Duplication and deletion upstream of LMNB1 in autosomal dominant adult-onset leukodystrophy

doi: 10.1212/NXG.0000000000000292

Figure Lengend Snippet: (A) Findings of MRI with T1WI (left panel), FLAIR (middle panel), and DWI (right panel) of patient 1 with LMNB1 duplication at the age of 56 years. Arrows point to the MCP lesion. (B) Findings of MRI with T1WI (left panel), FLAIR (middle panel), and DWI (right panel) of patient 5 with the upstream deletion of LMNB1 at the age of 50 years. ADLD = adult-onset demyelinating leukodystrophy; DWI = diffusion-weighted imaging; FLAIR = fluid-attenuated inversion recovery; MCP = middle cerebellar peduncle; LMNB1 = lamin B1.

Article Snippet: CNV was analyzed by real-time PCR assay using TaqMan probes (Thermo Fischer Scientific, Waltham, MA) designed for exons 3 (Hs02537023_cn), 6 (Hs00696436_cn), and 10 (Hs00579415_cn) of LMNB1.

Techniques: Diffusion-based Assay, Imaging

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Map of the lentiviral vector pLAS2w.Pneo. The full length of human LMNB1 cDNA (1,761 bp) is cloned into multiple cloning sites between the NheI and PmeI restriction sites on the vector, driven by the CMV promoter. The downstream neomycin (neo)-resistant gene is driven by the human PGK promoter.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Plasmid Preparation, Clone Assay

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Western blotting to detect the expression levels of LMNA and LMNB1 in cells. (A) The protein signal bands for LMNA, LMNB1, and GAPDH. (B, C) The relative expression levels of LMNB1 and LMNA (normalized to GAPDH expression) in different cell lines. The mean ± SEM of two independent experiments are shown (P = 0.0005 and P < 0.0001 for LMNB1 and LMNA, respectively; ANOVA). The results show that LMNB1-overexpressing EP156T (LMN-EP156T) cells express significantly higher amounts of LMNB1 than the parental and Mock EP156T cells. All three prostate cancer cell lines express high levels of LMNB1. All cells express LMNA, with PC-3 and DU145 expressing higher amounts of LMNA than the other cells.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Immunohistochemical (IHC) staining of LMNB1 expression. Staining results were shown for (A) parental EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells (positive control). Both LMN-EP156T and PC-3 cells expressed strong nuclear staining of LMNB1, which was higher than either parental EP156T or Mock EP156T cells.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Staining, Positive Control

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: MTS assay to measure the rate of cell growth. A total of 6.5 × 103 cells in 0.1 ml of complete medium were seeded onto each well of 96 well-plates in triplicate for each cell line. Five repeated plates were prepared for measurements at 5 time points. At each time point, one plate was used for this assay. MTS reagent (20 μl) was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at 490 nm is shown as the mean ± SEM of three independent experiments. LMNB1-overexpressing EP156T (LMN-EP156T) cells show significantly faster growth compared to the other two cell lines (P < 0.01; ANOVA).

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: MTS Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Matrigel-coated transwell assay (37°C for 48 h, 40 × magnification). A total of 5 × 104 cells of each cell line in 0.1 ml medium + 0.1% FBS were seeded onto an insert well (pore size, 8 µm) for a 24-well plate pre-coated with Matrigel. The insert wells were placed on receiver wells with 0.65 ml of medium + 10% FBS per well. After incubation at 37°C for 48 h, the cells on the apical side of the insert wells were removed with swabs. Only cells that had passed the membrane could be visualized after staining in 20% methanol with 0.25% crystal violet for 10 min. The results are shown for (A) EP156T, (B) Mock EP156T, and (C) LMN-EP156T cells. (D) The number of cells that penetrated the wells are shown as the mean ± SEM of three independent experiments. Statistical significance was determined by ANOVA (P < 0.0001). Significantly more LMNB1-overexpressing EP156T (LMN-EP156T) cells passed through the 8-µm pores than parental and Mock EP156T cells.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Transwell Assay, Incubation, Staining

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Colony-formation assay. Results are shown for (A) EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells. A total of 5 × 103 cells of each cell line were mixed in 0.3% soft agar with complete growth medium, and plated onto a culture dish (diameter, 3.5 cm). Numerous colonies can be seen in the plates inoculated with PC-3 after 2 weeks of incubation (magnification, 100 ×) at 37°C. However, no colonies have formed in the plates inoculated with the other cell lines. White arrows indicate PC-3 cell colonies.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Colony Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Immunohistochemical (IHC) staining of LMNB1 cells in archival paraffin blocks of prostate cancer tissues with graded Gleason scores (GS). (A-E) Representative block sections graded as GS 3+3, 3+4, 4+3, 4+4, and ≥ 4+5, respectively. Each row shows the same block section at different magnifications, as indicated at the top of each column. (F) LMNB1 IHC scores in tumors (N = 143) classified according to GS. The IHC staining of 143 archival paraffin blocks was evaluated by a qualified pathologist (Dr. Sun, CD) using a scoring system based on the percentage (0: no staining; 1: 1%-25%; 2: 26%-50%; 3: 51%-75%; 4: 76%-100%) and intensity (0: negative; 1: weak; 2: moderate; 3: intense) of IHC staining. An immune-reactivity score (range, 0-12) was obtained by multiplying the percentage and intensity scores. There is a significant trend, showing that the higher the GS, the higher the LMNB1 IHC score (trend analysis by ANOVA; P < 0.0001). The box plots show the median and interquartile range, and the whiskers indicate the minimum and maximum scores in each GS category. The medians of the GS 4+4 and GS ≥ 4+5 groups reach 100%.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Immunohistochemical staining, Immunohistochemistry, Blocking Assay, Staining

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Xenograft mouse model showing evident subcutaneous tumors in the right chest 7 weeks after the injection of PC-3 cells. (A-D) Four of the five mice injected with PC-3 cells formed subcutaneous tumors (red arrows). The tumor sizes range between 13 mm and 19 mm in diameter. No tumors were seen in mice injected with EP156T, Mock EP156T, or LMNB1-overexpressing EP156T cells (photos not shown).

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Injection

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Hematoxylin and eosin-stained tissue sections from xenografted mice. (A) An enlarged and metastasized lymph node from the PC-3-injected mouse. (B-E) Lung sections from mice injected with PC-3, EP156T, Mock EP156T, and LMNB1-overexpressing EP156T cells, respectively. Each row shows the same section with increasing magnification. Red arrows indicate PC-3 metastatic cell nests with clear nucleoli in either the lymph node (A) or lung (B). There are no subcutaneous growths or metastatic lesions in mice injected with the parental, Mock, or LMNB1-overexpressing EP156T cells.

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Staining, Injection

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Clinicopathological characteristics of patients with prostate cancer

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques:

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Univariable and multivariable Cox-regression analyses of biochemical recurrence in the radical prostatectomy group

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques:

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Univariable and multivariable Cox-regression analyses of disease-free survival in the TCGA cohort

Article Snippet: Briefly, the tissue sections were blocked in the blocking solution for 5 min and then sequentially probed with primary antibodies against LMNB1 (1:300; TA349381, OriGene) for 30 min and with a Polymer-HRP reagent for 8 min. Staining was developed for 5 min with DAB as the substrate chromogen.

Techniques: Expressing

Journal: Cells

Article Title: Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research

doi: 10.3390/cells13060507

Figure Lengend Snippet: Development and characterization of two CRISPR/Cas9-labeled FACS-enriched hiPSC populations. ( a ) Schematic representation of the workflow. HiPSCs were nucleofected with CRISPR/Cas9, sgRNA targeting the LMNB1 gene and a plasmid containing the desired modification. HiPSCs that successfully included the knock-in were separated by fluorescence-activated cell sorting (FACS) and further used for cerebral organoid development. ( b ) Box chart depicting the gene-edited and the respective non-edited parental hiPSC lines’ comparable growth rates, represented by the maintenance of a stable cell number 48 h after passage. Significance p < 0.05 according to standard Student’s t -test. n.s: not significant. ( c ) Representative confocal microscopy images of LMNB1 RFP/GFP FACS-enriched cell populations showing GFP+ and RFP+ cells with homogeneous fluorescence intensity levels and perinuclear lamin B1 phenotype. Scale bars: 25 µm. ( d ) Sanger sequencing data obtained after analysis of DNA of LMNB1_RFP hiPSC showing the correct knock-in of the RFP in frame with the LMNB1 sequence.

Article Snippet: The plasmid for tagging the N-terminus of the human LMNB1 gene with RFP (Addgene, Watertown, MA, USA, LMNB1-mTagRFP-T, #114403) and GFP (Addgene, LMNB1-mEGFP, #87422) were purchased from Addgene and their sequence was validated by Sanger sequencing.

Techniques: CRISPR, Labeling, Plasmid Preparation, Modification, Knock-In, Fluorescence, FACS, Stable Transfection, Confocal Microscopy, Sequencing

Journal: Cells

Article Title: Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research

doi: 10.3390/cells13060507

Figure Lengend Snippet: Quality assessment of genomic integrity in hiPSCs after gene editing and long-term culture using optical genome mapping. ( a ) Circos plot of 10-C hiPSC line normalized to the human genome reference GrCh37/h19 presenting multiple SVs and CNVs as detailed in the legend. ( b ) Duo analysis for LMNB_RFP hiPSC normalized to 10 C. The chromosome 22 variant (pink arrow) is detected by CNV pipeline (blue line). The variant on chromosome 20 (purple arrow) is detected by both CNV (blue line) and SV (purple dot). Modification coincident with RFP fluorophore on chromosome 5 (green arrow) is detected by SV pipeline (green dot). ( c ) Duo analysis for PS1-LMNB_GFP hiPSC normalized to PSEN1 +/+. The chromosome 22 copy number gain (pink arrow) is detected CNV pipeline (blue line). The chromosome 1 variant (dark blue arrow) is detected by CNV pipeline (blue line). Copy number losses on in chromosome 6 and 8 (orange arrows) are detected by CNV (red lines) pipeline. Modification coincident with GFP fluorophore on chromosome 5 (green arrow) is detected as SV (green dot). Predicted off-target sites by CasOFFinder tool are indicated in ( b ) and ( c ) (green dashed line). ( d ) Detail view of insertion on chromosome 5 in LMNB1 gene.

Article Snippet: The plasmid for tagging the N-terminus of the human LMNB1 gene with RFP (Addgene, Watertown, MA, USA, LMNB1-mTagRFP-T, #114403) and GFP (Addgene, LMNB1-mEGFP, #87422) were purchased from Addgene and their sequence was validated by Sanger sequencing.

Techniques: Variant Assay, Modification

Journal: Cells

Article Title: Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research

doi: 10.3390/cells13060507

Figure Lengend Snippet: Cerebral organoids generated from gene-edited cell lines display the expected development of neural cells. ( a ) Representative images of PS1-LMNB1_GFP organoids ( upper panel ) compared with PSEN1 +/+ isogenic non-edited organoids ( bottom panel ) at day 35 immunofluorescence-stained for neural progenitors (PAX6) and immature neurons (β-tubulin III). Both organoids showed primarily neural differentiation. ( b ) Representative organoid sections from LMNB1_RFP ( upper panel ) and ( lower panel ) stained for markers of neural progenitors (PAX6) and immature neurons (β-tubulin III). The gene edited cell line showed a reduction in the number of neural progenitor areas as compared with COs generated from the non-edited isogenic cell line. ( c ) Representative immunofluorescence images of MIX RFP/GFP organoids displaying correct development of PAX6+ and β-tubulin III+ ( upper row ) and fluorescent microscopy pictures for lamin B1-tag analysis showing that progenitor areas were conformed primarily by PS1-LMNB1 hiPSCs ( lower row ). Scale bars: 200 μm (overview) and 25 μm (magnification).

Article Snippet: The plasmid for tagging the N-terminus of the human LMNB1 gene with RFP (Addgene, Watertown, MA, USA, LMNB1-mTagRFP-T, #114403) and GFP (Addgene, LMNB1-mEGFP, #87422) were purchased from Addgene and their sequence was validated by Sanger sequencing.

Techniques: Generated, Immunofluorescence, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: Lamin B1 fluctuations have differential effects on cellular proliferation and senescence

doi: 10.1083/jcb.201206121

Figure Lengend Snippet: Loss of LMNB1 in senescent human keratinocytes. (a and b) Immunofluorescence staining of primary keratinocytes (N 50; a) and hTERT-positive keratinocytes (N/TERT-1; b) with LMNA/C (LA/C), LMNB1 (LB1), and LAP2 antibodies. Merged images are shown for N/TERT-1 cells only. (right) Phase contrast (PH) images are shown for N 50. Arrowheads show large flattened cells. Bars, 10 µm. (c) SA-β-gal staining of hTERT-positive (N/TERT-1) and primary (N 50) keratinocytes at PD 28 and 21, respectively. Same fields shown in phase contrast (top) and bright field (bottom). The percentages of SA-β-gal–positive cells are indicated. Bars, 20 µm. (d) LMNB1, LMNA/C, and LAP2-α levels during a 28 PD time course of primary N 50 cells (left) and one independently passaged N/TERT-1 line (right) by Western blotting. Quantified signal intensities of LMNB1, LMNA/C, and LAP2-α and normalized to GAPDH control are plotted below ( n = 3). The results are presented as the means ± SD of three independent experiments.

Article Snippet: For qRT-PCR, the TaqMan Fast Universal PCR master mix was used with TaqMan primers obtained from Applied Biosystems: LMNA/C (Hs00153462_m1), LMNB1 (Hs01059210_m1), and GAPDH (4326317E). qRT-PCR was performed on a real-time PCR system (7500 Fast; Applied Biosystems).

Techniques: Immunofluorescence, Staining, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Lamin B1 fluctuations have differential effects on cellular proliferation and senescence

doi: 10.1083/jcb.201206121

Figure Lengend Snippet: Loss of LMNB1 and LAP2 during normal skin aging and terminal keratinocyte differentiation in vivo. (a and b) Immunohistochemistry of paraffin sections of normal human skin from young (1 yr; a) versus old (>60 yr; b) individuals. Antibodies are LMNB1 (LB1), LAP2, LAP2-α, LMNA/C (LA/C), Ki-67, keratin-10, and negative control (−ctrl). Different skin layers are indicated: stratum corneum (sc), basal layer (bl), and dermis (d).

Article Snippet: For qRT-PCR, the TaqMan Fast Universal PCR master mix was used with TaqMan primers obtained from Applied Biosystems: LMNA/C (Hs00153462_m1), LMNB1 (Hs01059210_m1), and GAPDH (4326317E). qRT-PCR was performed on a real-time PCR system (7500 Fast; Applied Biosystems).

Techniques: In Vivo, Immunohistochemistry, Negative Control

Journal: The Journal of Cell Biology

Article Title: Lamin B1 fluctuations have differential effects on cellular proliferation and senescence

doi: 10.1083/jcb.201206121

Figure Lengend Snippet: shRNA-mediated depletion of LMNB1 impairs proliferation but does not cause cellular senescence. (a) Western blot depicting LMNB1, LMNA/C, and GAPDH levels in clones expressing a scrambled control (ctrl) or LMNB1 shRNA (clones A, B, C, and D). (b) Immunofluorescence staining of control and LMNB1 (LB1)-depleted cells using an antibody against LMNB1 (green). DAPI staining is in blue. Bars, 50 µm. (c) Nuclear abnormalities in LMNB1-deficient fibroblasts. Bar, 10 µm. Quantification of nuclear abnormalities is shown below. The results are presented as the means ± SD of three independent experiments. (d) LMNB1-deficient fibroblasts exhibit growth retardation. (top) Growth curve of primary and hTERT-positive normal dermal fibroblasts (NDFs) stably infected with doxycyclin (DOX)-inducible LMNB1 shRNA construct. Doxycyclin induced (LMNB1 deficient: light blue and red) and noninduced (dark blue and dark red) are shown. Dotted lines indicate SEM ( n = 3). (bottom) 6-d mean growth rate of doxycyclin induced (+) versus noninduced (−). Error bars represent ± SEM. **, P < 0.01. (e) Expression of dominant-negative p53 (p53DD) does not rescue LMNB1 loss–induced growth retardation. Doxycyclin-inducible down-regulation of LMNB1 in cells expressing vector control (first and second lanes) or a dominant-negative allele of p53 (p53DD; third and fourth lanes). (f) Growth curve of p53DD-expressing fibroblasts ± doxycyclin-inducible LMNB1 shRNA (dotted lines indicate SEM [ n = 3]). Inset shows 8-d mean growth rate for noninduced (−) and induced (+). Error bars indicate ± SEM. **, P < 0.01. (g) LMNB1 loss–induced proliferation defect is not rescued by growth under low oxygen (hypoxia 1.5% O 2 ) conditions. Western blot of cells constitutively expressing a scrambled control shRNA or an shRNA against LMNB1 . LA, LMNA; LC, LMNC. (h) Proliferation assay of hTERT-positive dermal fibroblasts cells expressing scrambled control (sc) or shRNA against LMNB1 under normal (21% O 2 ) and hypoxic (1.5% O 2 ) conditions. (i) Proliferation assay of WI-38 cells expressing control or LMNB1 shRNA. Assays were performed in triplicates, and means ± SD from three independent experiments are shown. ***, P < 0.001. Cell numbers of scrambled controls were normalized to 1. a.u., arbitrary unit; WT, wild type.

Article Snippet: For qRT-PCR, the TaqMan Fast Universal PCR master mix was used with TaqMan primers obtained from Applied Biosystems: LMNA/C (Hs00153462_m1), LMNB1 (Hs01059210_m1), and GAPDH (4326317E). qRT-PCR was performed on a real-time PCR system (7500 Fast; Applied Biosystems).

Techniques: shRNA, Western Blot, Clone Assay, Expressing, Control, Immunofluorescence, Staining, Stable Transfection, Infection, Construct, Dominant Negative Mutation, Plasmid Preparation, Proliferation Assay

Journal: The Journal of Cell Biology

Article Title: Lamin B1 fluctuations have differential effects on cellular proliferation and senescence

doi: 10.1083/jcb.201206121

Figure Lengend Snippet: Overexpression of LMNB1 causes senescence, which is reversible by telomerase or p53 inactivation. (a) Doxycyclin-inducible overexpression of v5-tagged LMNB1 (LB1). (top) Western blot showing LMNB1 levels upon doxycyclin induction (+) in hTERT-negative and hTERT-positive normal dermal fibroblasts. Samples were run in parallel on two gels and probed with the following antibodies: LMNB1, LMNA/C (LA and LC), and v5 tag. GAPDH loading control is shown for both gels. (bottom) Quantification of LMNB1 in noninduced versus induced cells (normalized to GAPDH/hTERT negative or hTERT positive/no doxycycline [Dox]; n = 5). The means ± SD of five independent experiments are shown. (b, top) Growth curve of hTERT-negative (red and light red) and hTERT-positive (blue and light blue) cells expressing LMNB1 or a vector control. Dotted lines indicate SEM ( n = 6). (bottom) Mean growth rate of cells ± LMNB1. Error bars indicate ± SEM. **, P < 0.01. (c) Quantification of SA-β-gal–positive cells in LMNB1-overexpressing cells versus vector control ( n = 3). Data are presented as means ± SD from three independent experiments. (d) Expression of LMNB1 or vector control, dominant-negative p53 (p53DD), or pBABE-hygro control in hTERT-negative and hTERT-positive fibroblasts. Antibodies are p53, LMNB1, and GAPDH. (e) Growth curve of hTERT-deficient fibroblasts overexpressing LMNB1 (or pBABE-neo) in the presence of dominant-negative p53DD or pBABE-hygro control (ctrl). Dotted lines indicate SEM ( n = 3). a.u., arbitrary unit; NDF, normal dermal fibroblast.

Article Snippet: For qRT-PCR, the TaqMan Fast Universal PCR master mix was used with TaqMan primers obtained from Applied Biosystems: LMNA/C (Hs00153462_m1), LMNB1 (Hs01059210_m1), and GAPDH (4326317E). qRT-PCR was performed on a real-time PCR system (7500 Fast; Applied Biosystems).

Techniques: Over Expression, Western Blot, Control, Expressing, Plasmid Preparation, Dominant Negative Mutation

Journal: The Journal of Cell Biology

Article Title: Lamin B1 fluctuations have differential effects on cellular proliferation and senescence

doi: 10.1083/jcb.201206121

Figure Lengend Snippet: Reduced levels of LMNA/C exacerbate the effects of LMMB1 overexpression. (a, top) Western blot showing LMNB1 (LB1) and LMNA/C (LA/C) levels in cells expressing pBABE-neo control or pBABE-neo-LMNB1 in cells previously transduced with control (ctrl) or LMNA/C shRNA. Antibodies are LMNB1, LMNA/C, and actin. (bottom) Quantification of LMNB1 and LMNA/C levels normalized to actin ( n = 3). Data are from three independent experiments. (b) Proliferation assay of cells expressing pBABE-neo or pBABE-neo-LMNB1 in cells expressing control shRNA (first two bars) or in cells expressing shRNA against LMNA/C (last two bars). *, P < 0.05; **, P < 0.01; n = 4. (c) SA-β-gal staining of control versus LMNB1 in shLMNA/C cells (PH, phase contrast; BF, bright field). Bars, 50 µm. (d) Quantification of SA-β-gal–positive cells in cells described in b. ( n = 4). (e) Cell cycle analysis of control versus LMNB1 in shLMNA/C cells. Percentages of cells in different cell cycle stages are indicated ( n = 4). (f) Quantification of DNA damage foci by 53BP1 staining in control versus LMNB1 in shLMNA/C cells. At least 400 cells were counted for each condition. (g) Confocal immunofluorescence microscopy showing telomere dysfunction–induced DNA damage foci (TIFs). Cells were stained with 53BP-1 (red) and TRF1 (green). Shown are cells expressing TRF2ΔBΔM (positive control) or vector control (scrambled shRNA + pBABE-neo) and cells with reduced levels of LMNA/C overexpressing LMNB1 (bottom two rows). Merged images also show DAPI staining. Bar, 20 µm. White boxes highlight TIFs. Thick framed white boxes are enlarged on the right. Bar, 2.5 µm. (h) Quantification of TIFs from control versus shLMNA/C:LMNB1 cells. Percentages of cells showing zero, one, two or more than three TIFs are shown. 100 cells were counted for each condition. Error bars represent ± SD.

Article Snippet: For qRT-PCR, the TaqMan Fast Universal PCR master mix was used with TaqMan primers obtained from Applied Biosystems: LMNA/C (Hs00153462_m1), LMNB1 (Hs01059210_m1), and GAPDH (4326317E). qRT-PCR was performed on a real-time PCR system (7500 Fast; Applied Biosystems).

Techniques: Over Expression, Western Blot, Expressing, Control, Transduction, shRNA, Proliferation Assay, Staining, Cell Cycle Assay, Immunofluorescence, Microscopy, Positive Control, Plasmid Preparation