human fibroblast cell lines  (ATCC)

Journal: Langmuir

Article Title: Traction Force Microscopy with DNA FluoroCubes

doi: 10.1021/acs.langmuir.5c04971

Figure Lengend Snippet: Demonstration of TFM with FluoroCubes and fluorescent beads as fiducial markers. (a) (i) and (ii) bright-field image of a murine kidney fibroblast with GFP-labeled kindlin-2 (Kind Ko+K2GFP cell line) and the corresponding fluorescent image; (b) displacement correlation between the bead and FluoroCube channel resulting from the stage shift in the x direction. The black dots represent the shifts measured by the conventional KLT method from the FluoroCube channel as a function of the corresponding shifts in the bead channel. The fitted curve has a slope of 0.9975 with R 2 = 0.9940; (c) (i) and (ii) displacement magnitudes measured in the bead and FluoroCube channels, respectively; (d) correlation of the displacements shown in (c) (i,ii); the micrometer-to-pixel factor is 0.103 μm/pix; (e) (i) traction forces calculated from the displacements in (c) (i) with BFTTC; (e) (ii) traction forces calculated from the displacements in (c) (ii); (e) (iii) traction forces calculated from dual-channel optical flow tracking results (see Figure S7 ). All scale bars represent 12.5 μm.

Article Snippet: Human foreskin fibroblasts (ATCC; SCRC-1041) and Kind Ko+K2GFP cells (Kind Ko immortalized murine kidney fibroblast reconstituted with green fluorescent protein-tagged kindlin-2 expression plasmids, generous gift from Prof. Dr. Reinhard Fässler, PMID: 26821125) were cultured at 37 °C and in 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6546), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, F2442), 4 mM l -glutamine (Sigma-Aldrich, G7513), and 20 μg/mL gentamicin (Sigma-Aldrich, G1397).

Techniques: Labeling

Journal: Neuro-Oncology

Article Title: MBD3 deficiency decommissions the nucleosome remodeling and deacetylase complex and orchestrates the epigenetic regulation of gene expression to suppress neuroblastoma progression

doi: 10.1093/neuonc/noaf297

Figure Lengend Snippet: MBD3 knockdown induces cell growth inhibition and apoptosis in human neuroblastoma cell lines. (A-C) Data used for plotting are from DepMap database ( https://depmap.org ). The mRNA expression levels (A, n = 33) and gene effect scores (B, n = 37) of MBD2 and MBD3 across a panel of neuroblastoma cell lines were plotted. Values are log2(TPM + 1) (A) or gene effect scores (B). For (C), the gene effect scores of MBD3 across a panel of cancer cell lines ( n = 1150) derived from different tissues were plotted. The number of cell lines analyzed for each tissue is noted in parentheses after the tissue name. Boxes indicate the median (horizontal line), 25th percentile and 75th percentile; Whiskers indicate the minimum to the maximum (A, B), or distances from the largest and smallest value to each end of the box that are within 1.5× box length (C). Dashed line indicates mean value of the gene effect scores (C). Values were compared with the 2-tailed unpaired t -test. *** P < .001, **** P < .0001. TPM, transcripts per kilobase of exon per million mapped reads. (D-I) Neuroblastoma cell lines (IMR32, SK-N-DZ, SH-SY5Y, and SK-N-SH) and a foreskin fibroblast cell line HFF-1 engineered with a doxycycline-inducible shRNA knockdown system were untreated or treated with 100 ng/mL doxycycline for 4 days. sh Luc was used as a control. The expression of MBD3 was detected by western blot analysis; β-actin was used as a loading control (D). Relative cell growth (E-I) at days 2, 4, and 6 was evaluated, and the color scheme is shown in (I). Values are means ± SEM of triplicate experiments. Mean values were compared with 2-way ANOVA by Dunnett's test. **** P < .0001. (J) Colony formation assay in neuroblastoma cells with a doxycycline-inducible shRNA. Cells were treated with 100 ng/mL doxycycline for 7 days and stained with 0.05% crystal violet. sh Luc was used as a control. (K, L) Neuroblastoma cells with a doxycycline-inducible shRNA were treated with 100 ng/mL doxycycline for 4 days and subjected to cell cycle and apoptosis analysis by flow cytometry. The percentages of cells in G0/G1, S, and G2/M phase of the cell cycle were analyzed with propidium iodide (PI) staining (K). Apoptotic cells were determined by the percentage of Annexin V-positive cells after staining with Annexin V-FITC and PI (L). Values are means ± SEM of 3 independent experiments. Values were compared with the 2-tailed unpaired t -test. * P < .05, ** P < .01, *** P < .001, **** P < .0001, ns, not significant.

Article Snippet: Neuroblastoma cell lines IMR32, SH-SY5Y, SK-N-SH and fibroblast cell line HFF-1 were purchased from National Collection of Authenticated Cell Cultures (NCACC), and SK-N-DZ and SK-N-AS from American Type Culture Collection (ATCC).

Techniques: Knockdown, Inhibition, Expressing, Derivative Assay, shRNA, Control, Western Blot, Colony Assay, Staining, Flow Cytometry