Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Transformation Assay, Western Blot, Inhibition, Activation Assay

Journal: BMC Microbiology

Article Title: First human cell-based cultivation system for the syphilis spirochete Treponema pallidum

doi: 10.1186/s12866-026-04856-5

Figure Lengend Snippet: Human foreskin fibroblast cell lines support the growth of T. pallidum in vitro . A Human foreskin fibroblast cell lines (MoNa, HFF1, and HFFC) support the growth of T. pallidum in long-term, continuous, in vitro cultivation. While cultivation of strain SS14 was discontinued after 10 weeks, in vitro cultures of the DAL-1 strain have been ongoing for over one year. During this period, scheme of subcultures was optimized (up to 1:20). A booster passage (*), extending the standard 7-day subculture to a 14-day period with a cultivation medium exchange on day 7, was employed to enrich the treponemal culture during critical declines. In vitro cultivation was performed in triplicate (i.e., in three cultivation wells) and the mean values for each subculture are presented in graph. Source data from individual wells are shown in Supplementary Table . B Parallel cultivation for seven days ( n = 15) with defined DAL-1 inoculum (10 6 treponemes) validated the differences in growth support of individual foreskin cell lines. Treponemal growth on human foreskin fibroblast cells was slower compared to growth on rabbit epithelial Sf1Ep cells. Red bar, mean. C Human foreskin-based cultivation system supports the growth of various T. pallidum strains ( n = 8), from the Nichols-like as well as the SS14-like cluster. Note that human foreskin fibroblast cells were prepared as an equal mixture of three tested cell lines. Each T. pallidum strain was cultivated in a single in vitro well, representing a sole experimental replicate used for data acquisition

Article Snippet: Human foreskin fibroblasts HFF1 (SCRC-1041; ATCC) and HFFC (300715; Cytion) were purchased, while the third cell line (MoNa) was kindly provided by Dr. Vladimir Rotrekl (Masaryk University), and was originally obtained from the National Tissue Centre (Czech Republic).

Techniques: In Vitro

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Spatial ratiometric images and histograms of all pixels within each field of view are shown for dimeric amine/cys Fn-DA in 0 and 1 M GdnHCl and monomeric amine/cys Fn-DA in 1 and 4 M GdnHCl (A). Amine/cys Fn-DA was added to the culture medium of fibroblasts for 24 h, and excess Fn-u was added to suppress intermolecular energy transfer. Confocal microscopic images of acceptor and donor peak intensities taken 1 μm above the glass–cell interface were background subtracted, averaged, and thresholded, and the I A / I D ratiometric image of acceptor to donor was color-coded within the range of 0.05 to 1.0. A histogram (B) for all pixels of amine/cys Fn-DA–containing ECM (C) and an overlay of I A / I D on the DIC image (D) are shown in a region in which the matrix showed a transition from low to intermediate I A / I D within a single Fn fiber. Histograms are overlaid in (B) for regions of extended (E; purple) and unfolded Fn (F; pink). Histograms were generated with 0.01-ratio-unit bin widths. Scale bars = 25 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Generated

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Amine/cys Fn-DA and excess Fn-u were added to the culture medium of fibroblasts for 24 h. Color-coded I A / I D ratiometric images are shown for control cells (A), extracted cell-free matrix (B), and fibroblast cells after 60 min exposure to the ROCK inhibitor Y-27632 (C). Histograms with 0.01-ratio-unit bin widths for all pixels of control (black), cell-free (purple), and ROCK-inhibited matrix (pink) were derived from three random fields of view each from three separate experiments in each group (D). Solution denaturation values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Control, Derivative Assay

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: A schematic of the strain device is shown in the relaxed configuration with length L before (A) and length L + DL after (B) application of strain. PDMS sheets were covalently modified with Fn-u as described in Materials and Methods, and fibroblast cells were cultured for 24 h in the presence of amine/cys Fn-DA and excess Fn-u. Cells were extracted in mild detergent. Color-coded I A / I D ratiometric images are shown for a field of view without application of stretch (C) and after application of 70% elongation strain with 28% transverse compression (D). Region of interest analysis on individual fibrils was used to determine the impact of elongation on I A / I D on a per fibril basis (circles, mean ± standard deviation), and binned averages were calculated for fibrils between −37% and −20%, −20% and −10%, −10% and 10%, 10% and 40%, and 40% and 73% strain (red squares, mean ± standard deviations) (E). Abscissa is also plotted as relative length change. Solution values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as horizontal red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Modification, Cell Culture, Standard Deviation

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Cys/cys Fn-DA (A–C) or amine/cys Fn-DA (D–G) was incorporated into fibroblast matrix on Fn-u that was adsorbed to plasma cleaned PDMS, and after cell extraction the substrate was relaxed to 4/5 (A and B; 3.7% transverse stretch) or 3/5 the starting length (D–F; 10% transverse stretch). I A / I D ratiometric images of cys/cys Fn-DA–containing matrix are shown at the PDMS–ECM interface (A), where a portion of the cell-free fibers are still attached to the substrate, and from the same field of view but acquired 3 μm above the PDMS surface (B), where the strain-free Fn mat randomly diffused around its points of attachment to the underlying ECM. Histograms are shown for all pixels within the field of view at the substrate (C; black) and from the upper, strain-free confocal slice (C; pink). An I A / I D ratiometric image of amine/cys Fn-DA is shown with both detached (E) and still-attached (F) regions of matrix within the same confocal slice. Region of interest analysis was used to generate histograms (G0 for all pixels within the detached (E and G; purple) and attached (F and G; pink) regions of matrix, which were overlaid on a histogram of all pixels in the field of view (black). Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Clinical Proteomics, Extraction