Journal: Redox Biology

Article Title: Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

doi: 10.1016/j.redox.2024.103102

Figure Lengend Snippet: PXDN and laminin expression are modulated in human lung fibroblasts (HLF) and bone-marrow-derived macrophages (BMDM) upon TGF-β1 stimulation. (A – C) HLFs were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for LAMA1 (A) or PXDN (B) mRNA or PXDN protein expression (C). (D – G) BMDM from C57BL6/NJ mice were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for Pxdn (D) , Lama1 (E) , or Lamb1 (F) mRNA, or Laminin α/β1 protein expression by immunofluorescence imaging (G). Scale bar: 50 μm. Values are the mean of at least three independent biological replicates ± SEM. Differences among groups were evaluated by Student's T-test.

Article Snippet: HLF (ATCC PCS-201-013) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast growth kit-low serum (ATCC PCS-201-041) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Enhancing Drug Delivery with Supramolecular Amphiphilic Macrocycle Nanoparticles: Selective Targeting of CDK4/6 Inhibitor Palbociclib to Melanoma

doi: 10.1101/2023.11.21.567974

Figure Lengend Snippet: Confocal microscopy imaging of NR-MC1 and NR-MC2 NPs internalization in both M14 melanoma and HDFn fibroblast cell lines. The scale bar is 20 µm. Images in each row (left to right): CellMask (green) for staining cell membrane, NR-loaded MC NPs (red), overlapping (green-red), and bright field image (green-red).

Article Snippet: We also obtained RAW 264.7 mouse macrophages, U-87 MG, and adipose stromal cells, human dermal fibroblasts, neonatal (HDFn), human melanoma (Mel29.1) cells from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Imaging, Staining, Membrane

Journal: Cell Reports

Article Title: Notch Signaling Mediates Secondary Senescence

doi: 10.1016/j.celrep.2019.03.104

Figure Lengend Snippet:

Article Snippet: IMR90 normal human diploid fibroblasts , ATTC , ATCC Cat# CRL-7931, RRID:CVCL_0347.

Techniques: Plasmid Preparation, Recombinant, cDNA Synthesis, Imaging, Multiplex Assay, Software

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The association of p62 with cytosolic Salmonella increases shortly after initiation of intracellular replication. (A) GFP-p62- or GFP-expressing HeLa cells were infected with wild-type (WT) or ∆ sifA Salmonella . Cells at 1 hpi and 5 hpi were stained with LAMP-1 (red) and DAPI (cyan). “Zoom” represents a magnified picture of the boxed area. Scale bar, 10 µm. (B to D) Quantification of the percentage of p62 + WT or p62 + ∆ sifA bacteria (B), the percentage of p62 + WT or p62 + ∆ sifA bacteria that are not associated with LAMP-1 (C), and the percentage of LAMP-1 − WT or LAMP-1 − ∆ sifA bacteria (D) in GFP-p62-expressing cells at 1, 5, and 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Staining, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Dynamic association of p62 and/or LC3 correlates with Salmonella replication in the cytosol of HeLa cells. (A) GFP-LC3-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 min), cells were imaged at 30-s intervals with a spinning-disc confocal microscope. A series of live-cell imaging data is shown, and the elapsed time (minutes) is shown in the corner of each picture. Arrowheads 1, 2, 3, and 4 indicate replicating bacteria, whereas arrowheads 5 and 6 indicate nonreplicating bacteria. Scale bar, 10 µm. (B) GFP-p62-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 h), the cells were imaged at 20-s intervals with a spinning-disc confocal microscope. Boxed areas indicate where p62 associates with cytosolic Salmonella . Arrows 1 to 5 indicate bright bacteria that are not replicating; arrowheads 6 and 7 in column 1 (0 h) indicate light-red bacteria that start to replicate. Scale bar, 5 µm.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Microscopy, Live Cell Imaging, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: p62 and autophagy favors cytosolic replication of Salmonella . (A) Western blot analyses of HeLa cells treated with control siRNA (si-NT) or siRNA targeting p62 (si-p62), LC3 (si-LC3), Atg5 (si-Atg5), and p62 and LC3 (si-p62+LC3). These cells were infected with WT Salmonella . Calnexin was used as a loading control. (B) Microscopic analyses of infected cells containing 1 to 5, 6 to 10, 11 to 20, or greater than 20 bacteria per cell at 2 hpi (top) and 6 hpi (bottom). The number of bacteria in at least 50 infected cells was enumerated, and the percentage of cells containing the indicated number of bacteria was presented (mean ± SD, n = 3). (C) Replication of Salmonella was compared in HeLa cells used in panel A. The fold replication was calculated as fold increases of the number of bacteria at late time points compared to the number of bacteria at 2 hpi (mean ± SD, n = 3). (D) HeLa cells were treated with control siRNA (si-NT) or siRNA targeting TBK1 (TANK-binding kinase-1) (si-TBK1), TBK1 and LC3 (si-TBK1+LC3), and TBK1 and ATG5 (si-TBK1+ATG5) and infected with Salmonella for 2 h (top) or 6 h (bottom). The percentage of cells containing the indicated number of bacteria was quantified as in panel B (mean ± SD; n = 3). (E) GFP-LC3-expressing HeLa cells were treated with si-NT, si-TBK1, si-TBK1+LC3, or si-TBK1+Atg5. The percentage of LC3 + bacteria or LAMP-1 − bacteria at 5 hpi was quantified (mean ± SD, n = 3). (F) Western blot analyses of HeLa cells used in panel D.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Western Blot, Control, Infection, Bacteria, Binding Assay, Expressing

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The effector SopB is required for Salmonella to associate with autophagosomes. (A) GFP-LC3-expressing cells were infected with the indicated strains. The ∆ sopB /pACDE mutant expresses the wild-type SopB in a ∆ sopB background. Unlike other strains that are individually used to infect cells, ∆ invA and ∆ sipB bacteria were used in a coinfection model. The percentage of LC3 + bacteria at 5 hpi was quantified for each strain (mean ± SD, n = 3). (B) Western blot analysis of HeLa cells used in panel A. (C) HeLa cells treated with si-NT or si-LC3 were infected with the indicated strains. The fold replication of these strains at 6 hpi was determined (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Mutagenesis, Bacteria, Western Blot

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Cells containing hyperreplicating bacteria and showing decreased autophagy are positive for caspase-1 and caspase-3/7 activation and undergo cell death. (A) GFP-p62-expressing HeLa cells were infected with Salmonella for 8 h and stained with DAPI (blue). A cell containing hyperreplicating bacteria (more than 50 bacteria per cell, i.e., HRBC cells) and showing abnormal nuclear morphology is shown. Scale bar, 10 µm. (B) At least 100 HRBC cells were divided into two groups. The first group exhibited a low level of p62 signals colocalized with Salmonella (<1% of intracellular bacteria), and the second group exhibited a relatively high level of p62 signals (>2% of intracellular bacteria). The number of cells showing abnormal nuclear morphology (such as chromatin condensation and DNA fragmentation) was further quantified and expressed as the percentage of cells with abnormal nuclei in each group of cells (mean ± SD, n = 3). (C) A GFP-p62-expressing HRBC cell shows a relatively enhanced autophagy and normal nuclear morphology. Scale bar, 10 µm. (D) Cells stably expressing monomeric red fluorescent protein (mRFP)-LC3 were infected with WT Salmonella for 7 h and stained with DAPI (blue) and active caspase-1 probe FAM-YVAD-FMK (green) or caspase-3/7 probe FAM-DEVD-FMK (similar to caspase-1 staining [data not shown]). (E) Quantification of the percentage of casapase-1 (left)- or caspase-3/7 (right)-positive cells in uninfected cells, HRBC cells showing decreased autophagy, and HRBC cells showing normal autophagy at 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Activation Assay, Expressing, Infection, Staining, Stable Transfection

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: A proposed model for p62-dependent autophagy favoring Salmonella replication in the cytosol of HeLa cells. In this proposed model, Salmonella likely acquires nutrients supplied by autophagy for its replication. At least three populations of bacteria are present in infected HeLa cells. The first population of bacteria remains in intact Salmonella -containing vacuoles (SCVs). These bacteria replicate slowly, as they do not associate with autophagosomes and are difficult to acquire nutrients from the cytosol. The second population of bacteria is partially exposed to the cytosol through the gaps between damaged SCVs . These bacteria are ineffectively coated with ubiquitin and associated with autophagosomes to a low level. They acquire a limited amount of nutrients for replication and replicate faster than the first population of bacteria. The third population of bacteria is completely exposed to the cytosol. These bacteria are effectively coated with ubiquitin and associated with autophagosomes to a high level. They acquire sufficient nutrients from the cytosol and replicate the fastest. Notably, some cells containing the third population of bacteria will undergo cell death and detach from the epithelial layer, resulting in the release of hyperreplicating bacteria that are capable of initiating secondary infections in neighboring cells. The whereabouts of the first and second populations of bacteria at 8 hpi were not determined (ND).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Infection, Ubiquitin Proteomics

Journal: Neuro-Oncology Advances

Article Title: The brain-penetrant cell-cycle inhibitor p28 sensitizes brain metastases to DNA-damaging agents

doi: 10.1093/noajnl/vdad042

Figure Lengend Snippet: p28 crosses the BBB and preferentially localizes to Brain metastases (BMs). (A) Confocal images of the penetration of normal and cancer cells by p28. Human cancer cell lines (MDA-231BR, BCA-1, Mel-7, and A549) and normal cells (MCF-10A and fibroblasts) were cultured with Alexa Fluor 568-labeled p28 at 37°C for 2 hours, and images were obtained by confocal microscopy. Red, p28; blue, DAPI (nucleus). (B) MDA-231BR brain-specific metastatic triple-negative breast cancer, BCA-1 breast cancer, Mel-7 melanoma, or A549 lung cancer cells were injected into the left cardiac ventricle of athymic mice. ICG-labeled p28 was intravenously injected into the mice. Near-infrared fluorescence imaging of the ICG-p28 signal (gray) in coronal brain sections (yellow dotted line on the anterior-dorsal view) of mice injected with MDA-231BR, BCA-1 or Mel-7 cells or in the anterior-dorsal view of the brain of mice injected with A549 cells. H&E staining of brain sections confirmed the presence of BMs (Tu).

Article Snippet: A549 human lung cancer cells, MCF10A, and fibroblasts were purchased from American Type Culture Collection.

Techniques: Cell Culture, Labeling, Confocal Microscopy, Injection, Fluorescence, Imaging, Staining

Journal: Scientific Reports

Article Title: Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

doi: 10.1038/s41598-018-21073-x

Figure Lengend Snippet: Hypoxia induces pulmonary fibroblast proliferation. Normal human pulmonary fibroblasts (HPFs) and LL29 IPF fibroblasts were exposed to normoxia (21% O 2 ) or hypoxia (5% or 1% O 2 ) for 2–6 days. ( A ) Bright field imaging. Scale bar: 100 µm. ( B ) Cell count. ( C , D ) Cell proliferation by BrdU assay. HPF and LL29 cells were incubated with BrdU for 3 hrs. Data were normalized to normoxia. The absorbance for BrdU at normoxia was 0.08 ± 0.002 (day 3) and 0.09 ± 0.009 (day 6) compared to 0.09 ± 0.004 (day 3) and 0.10 ± 0.006 (day 6) for HPF and LL29 cells, respectively. ( E , F ) Cell viability by MTT assay. The absorbance for MTT at normoxia was 0.23 ± 0.0796 (day 3) and 0.30 ± 0.091 (day 6) compared to 0.07 ± 0.016 (day 3) and 0.28 ± 0.097 (day 6) for HPF and LL29 cells, respectively. ( G ) Additional normal human pulmonary fibroblasts [HPF (with F12K medium), CCD-13Lu, CCD-19Lu, HPF153 and HLF154] and IPF fibroblasts (LL97A, IPF12 and IPF14) were subjected to normoxia and hypoxia (1% O 2 ) for 3 days, and cell proliferation assessed by BrdU assay. Cells were incubated with BrdU for 12 hrs. The absorbance values for BrdU at normoxia were 0.15 ± 0.006 (HPF with F12K medium), 0.09 ± 0.004 (CCD-13Lu), 0.18 ± 0.008 (CCD-19Lu), 0.28 ± 0.003 (HLF153), 0.14 ± 0.008 (HLF154), 0.11 ± 0.004 (LL97A), 0.05 ± 0.003 (IPF12) and 0.10 ± 0.01 (IPF14); values represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normoxia. n = 3 independent experiments.

Article Snippet: IPF fibroblasts LL29 and LL97A were isolated from a 26-year-old female Caucasian patient and a 48-year-old male Caucasian patient, respectively, and were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Imaging, Cell Counting, BrdU Staining, Incubation, MTT Assay

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 1. Growth curve of WI38 and VA13 cells. On day 0, 8.8 103 cells/cm2 were seeded in a 35-mm dish in 3 ml of medium and kept at 37 °C in 5% CO2. On subsequent days (1–8) the cells were trypsinized and counted in a Neubauer chamber. The growth curve for WI38 (f) in the upper panel was expressed with a different ordinate scale than that for VA13 (G) and WI38 (f) in the lower panel. Data from two independ- ent experiments with similar results are shown.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 2. GEM components from WI38 and VA13 cells and their distribution pattern. A-1, fractions 1–3, 4–6, 7–9, and 10–12 were separated by sucrose density gradient centrifugation from the postnuclear fraction (PNF) prepared in 1% Brij98 from WI38 and VA13 cells as described under ‘‘Materials and Methods.’’ The above fractions, having the same protein concentration, were passed through a C18 column and washed with water, and the GSL fraction was eluted with C/M (2:1, v/v). This eluate was evaporated to dryness under a stream of N2 and dissolved in the appropriate volume of C/M. Aliquots equivalent to 20 g of protein were applied in each lane, and HPTLC was developed in solvent C/M/ 0.2% CaCl2 (50:40:10, v/v/v) and stained with orcinol. GM3 was not detectable in Fr. 1–3 or 7–9 from either WI38 or VA13 (data not shown). The exclusive presence of GM3 in GEM prepared in the 1% Triton X-100 postnuclear fraction was reported previously (see ‘‘Discussion’’). A-2, distribution pattern of membrane components in low density GEM (Fr. 4–6) versus the high density soluble fraction (Fr. 10–12). Cells were grown to 90% confluence, and the GEM fraction and high density soluble fraction were prepared in 1% Brij98 or 1% Triton X-100 lysis buffer as described under ‘‘GEM Preparation and Determination of GEM Components’’ of ‘‘Materials and Methods.’’ An equal protein quantity (1 g) of each fraction was analyzed. B, major components showing a clear difference in WI38 versus VA13 GEM. Data from Fr. 5 and Fr. 12 prepared in 1% Brij98 lysis buffer are shown. S, sparse growing cells; C, confluent cells. An equal protein quantity (1 g) was analyzed. C, quantitative densitometry of immunoblotted bands for FGFR, cSrc, CD81, and CD9 in Fr. 5 of WI38 versus VA13. Note that the FGFR level is 4–5 times higher in VA13 than in WI38. cSrc was 2–3 times higher in VA13 than in WI38, whereas CD9 and CD81 were 3–4 times higher in WI38 than in VA13. Results similar to those shown in A, B, and C were obtained in three separate experiments.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gradient Centrifugation, Protein Concentration, High Performance Thin Layer Chromatography, Solvent, Staining, Membrane, Lysis

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 3. Effect of -CD and saponin on distribution pattern of GEM components in WI38 and VA13 cells. Nearly confluent cells were harvested and pretreated with -CD or saponin as described under ‘‘Materials and Methods.’’ The effects of these treatments on distribution patterns of major components are shown by Western blot- ting pattern. Note that FGFR and cSrc were barely reduced in GEM from VA13 but were significantly reduced in GEM from WI38. Csk, CD9, and CD81 were slightly reduced in WI38, but caveolin was com- pletely resistant to -CD. All components in both WI38 and VA13 were resistant to saponin. P, postnuclear fraction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 6. Presence of Csk in WI38 GEM fraction. GEM fractions from sparse and confluent WI38 and VA13 cultures were analyzed for the presence of the Src family inhibitor Csk. Cells were processed as described under ‘‘Materials and Methods’’ using 1% Brij98 lysis buffer. After protein quantification, 2.3 g of protein was loaded on an 8% gel and analyzed by Western blot using anti-Csk Ab (Santa Cruz Biotech- nology, Inc.). Note that Csk was present in WI38 GEM (Fr. 5 and 6) but absent in VA13 GEM. P, postnuclear fraction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Lysis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 4. HPTLC immunostaining of total GSLs extracted from WI38 and VA13 cells. A, cells were grown, and GSLs were extracted and stained with mAb DH2 as described under ‘‘GM3 Composition of Various Fractions’’ of ‘‘Materials and Methods.’’ B, cells were pretreated with () or without () P4, and GSLs were extracted and stained as in A. For both panels, GSLs extracted from 20 g of cell protein were loaded per lane. Note that the level of GM3 is essentially the same in WI38 as in VA13. GM3 was completely depleted by P4 treatment in both WI38 and VA13 regardless of sparse (S) versus confluent (C) condition. Similar results to those shown in A and B were obtained in three separate experiments.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: High Performance Thin Layer Chromatography, Immunostaining, Staining

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 5. Src and MAPK status in response to FGF and depletion of GM3 in WI38 versus VA13 cells. A, sparse growing (S) and con- fluent (C) cultures of WI38 (W) and VA13 (V) cells were prepared as described under ‘‘Materials and Methods.’’ The stimulatory effect of 10 pg/ml acidic FGF and the inhibitory effect of GM3 (probed by P4 treatment) were determined as described under ‘‘Materials and Meth- ods.’’ Three major points should be noted. (i) FGF-induced MAPK acti- vation in sparse growing WI38 was inhibited by GM3 as probed by P4 treatment (W, S, row 6). This inhibitory effect was stronger in confluent cells (W, C, row 6). (ii) Inactive Src with Tyr-527 was higher in confluent (W, C, row 4) than in sparse growing (W, S, row 4) WI38. The inactive Src expression was inhibited by GM3 (in the absence of P4) (W, S, row 4, two right lanes). (iii) Both MAPK and Src activation in VA13 were observed regardless of cell population density or P4 treatment (V, S, rows 3, 4, and 6; V, C, rows 3, 4, and 6). B, [3H]thymidine incorporation of WI38 after P4 treatment and FGF stimulation. Cells were seeded and processed as described under ‘‘Materials and Methods.’’ The statistical difference for each of the comparisons indicated by a bracket (*, between P4-treated versus P4-nontreated cells; **, between FGF-treated versus FGF-non-treated cells) was significant at the p 0.001 level.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 8. Interaction of FGFR in WI38 GEM with GM3 or other GSLs probed by polystyrene beads coated with GSLs. This interaction was determined by incubation of GSL-coated polystyrene beads with GEM and determination of the relative amount of FGFR adsorbed on beads by SDS-PAGE with Western blotting as described under ‘‘Materials and Methods.’’ Upper panel, level of FGFR adsorbed on GSL-coated beads determined by Western blot. ‘‘GEM,’’ FGFR in 1 g of protein in GEM for reference. ‘‘No GSL,’’ beads without GSL coating. GM3, GM2, GM1, SPG, Gb4, and LacCer: beads coated with 10 g of the respective GSLs. ‘‘GM3 sialidase,’’ beads coated with 10 g of GM3 treated with C. perfringens sialidase; beads were incubated with a defined amount of GEM under defined conditions (see ‘‘Materials and Methods’’) and subjected to Western blot. Lower panel, densitometric analysis (by Scion imaging) of intensity of FGFR in upper panel bands. Note that the FGFR interaction with GM3-coated beads was much higher than that with beads coated with GM2, GM1, or SPG. Neutral GSLs (Gb4 and LacCer) showed no interaction.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Incubation, SDS Page, Western Blot, Imaging

Journal: Journal of Biological Chemistry

Article Title: Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

doi: 10.1074/jbc.m403857200

Figure Lengend Snippet: FIG. 10. Components and their possible role in GEM of WI38 versus VA13 cells. Possible growth control mechanisms of WI38 versus VA13 cells are illustrated based on differences in components and signal transducer activity. A, in WI38 cells (i) FGF causes FGFR acti- vation and minimal signaling (2) through its association with FGFR and cSrc, and consequently minimal MAPK activation (5). This lack of response is due to the fact that the FGFR level in WI38 is low (compared with VA13), and GM3 inhibits FGFR activity (4). This inhibitory effect of GM3 may be enhanced by formation of a complex with CD9/CD81. (ii) GM3 clustering, or GM3 interaction with FGFR, may occur upon cell contact, which in turn activates cSrc (1, 2). However, activation of cSrc is minimal because Csk, the physiological cSrc inhibitor (3), is associ- ated in the same microdomain. Which molecule interacts with GM3 upon cell contact (i.e. trans interaction between interfacing GEM) to induce contact inhibition is unknown. FGFR was shown to interact preferentially with GM3 based on direct binding of FGFR to GM3- coated beads and data from confocal microscopy. Such trans interaction is stronger in WI38 than in VA13. B, in VA13 cells, because the CD9/ CD81 level is minimal and Csk is absent, signaling through FGFR to cSrc (1) and that through clustered GM3 to cSrc (2) is relayed to MAPK without interruption (3). The inhibitory effect of GM3 on FGFR is nearly absent since the CD9/CD81 level is low. Thus, FGF-dependent activation of FGFR through cSrc is relayed to MAPK (3), and both activated cSrc and MAPK are continuously present regardless of cell contact. trans interaction between FGFR and GM3, as indicated by dashed arrows, is weaker than in WI38. Levels of FGF and cSrc in VA13 GEM are much higher than in WI38 as reflected by the size of the shapes and symbols.

Article Snippet: Human embryonic lung fibroblast cell line WI38 (10, 11) and SV40transformed WI38, termed VA13 (12), were from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Activity Assay, Activation Assay, Inhibition, Binding Assay, Confocal Microscopy

Journal: Stem Cells

Article Title: Three‐dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1

doi: 10.1002/stem.3441

Figure Lengend Snippet: Human amniotic fluid stem cell (hAFSC) characterization for cell morphology and cell size. A,B, Phase contrast imaging for the evaluation of cell morphology in c‐Kit‐selected hAFSC lines (A) and low passage human amniotic fluid cells obtained via amniocentesis performed for routine prenatal diagnosis (B). The bottom panels represent a twofold magnification of the corresponding image section marked with a rectangle. C, Volume‐based cell size distribution of logarithmically growing hAFSCs ( AFS ). For comparison, human amniotic fluid cells ( AF ) and primary as well as immortalized/transformed cells of mesenchymal origin were co‐analysed. The latter include fetal/neonatal primary human lung and foreskin fibroblasts (IMR‐90 and CCD‐1079Sk, HFF‐1, respectively), adult primary cardiac fibroblasts and chondrocytes (HCF and HCH, respectively), mouse embryonic fibroblasts (NIH/3T3) and human fibrosarcoma cells (HT1080). “AFS‐all” represents the average of all values obtained for the hAFSC lines shown

Article Snippet: Amniotic fluid was provided by Gabor Joo (Semmelweis University Medical School, Budapest, Hungary); IMR‐90, primary human lung fibroblasts (ATCC, US, #CCL‐186); CCD‐1079Sk, primary human foreskin fibroblasts (ATCC, #CRL‐2097); HFF‐1, primary human foreskin fibroblasts (ATCC, #SCRC‐1041); HCF, primary human cardiac fibroblasts isolated from the ventricle of an adult heart (Promocell, Germany, #C‐12375); HCH, primary human chondrocytes isolated from normal human articular cartilage from the femoral head (Promocell, #C‐12710); NIH/3T3, immortalized mouse embryonic fibroblasts (ATCC, #CRL‐1658); HT1080, human fibrosarcoma cells (ATCC, #CCL‐121); MCF7, human mammary carcinoma cells (ATCC, #HTB‐22); MDA‐MB‐231, human mammary carcinoma cells (ATCC, #HTB‐26); HCC‐1.2 and HCC‐1.1, human hepatocellular carcinoma cells ; HUVEC, primary human umbilical vein endothelial cells (Promocell, #C‐12200); Hep G2, human hepatocellular carcinom cells (ATCC, #HB‐8065); and ARPE‐19, human retinal pigmented epithelium cells (ATCC, #CRL‐2302).

Techniques: Imaging, Biomarker Discovery, Comparison, Transformation Assay

Journal: Stem Cells

Article Title: Three‐dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1

doi: 10.1002/stem.3441

Figure Lengend Snippet: The epithelial‐mesenchymal transition (EMT)‐related marker profile of human amniotic fluid stem cells (hAFSCs). A, Immunoblotting for the detection of EMT‐related marker proteins in epithelial ( ep ) and mesenchymal ( ms ) cells of fibroblast (CCD‐1079Sk, HT1080), mammary (MCF7, MDA‐MB‐231), and hepatic (HCC‐1.2, HCC‐1.1) origins. l.e ., long exposure; pro , pro‐form; act , active form. B, Immunoblotting for the analysis of EMT‐related marker proteins in hAFSCs. Epithelial and mesenchymal cells were co‐analysed as a reference. For data presented in the left panel, the same lysates were separated on two different gels ( Blot#1 and Blot#2 ). l.e ., long exposure; pro , pro‐form; act , active form; FL , full‐length; CL , cleaved. C, Gelatin zymography for the detection of secreted MMPs in hAFSCs. Recombinant human MMP2 and MMP9, and cells of epithelial and mesenchymal origin were co‐analyzed as controls. For better visualization of results, a color‐inverted version of the long exposure was included ( l.e. INV ). D, Summary of results obtained in B and C including the hAFSC passage numbers and the quantification results for inactive, cleaved vimentin

Article Snippet: Amniotic fluid was provided by Gabor Joo (Semmelweis University Medical School, Budapest, Hungary); IMR‐90, primary human lung fibroblasts (ATCC, US, #CCL‐186); CCD‐1079Sk, primary human foreskin fibroblasts (ATCC, #CRL‐2097); HFF‐1, primary human foreskin fibroblasts (ATCC, #SCRC‐1041); HCF, primary human cardiac fibroblasts isolated from the ventricle of an adult heart (Promocell, Germany, #C‐12375); HCH, primary human chondrocytes isolated from normal human articular cartilage from the femoral head (Promocell, #C‐12710); NIH/3T3, immortalized mouse embryonic fibroblasts (ATCC, #CRL‐1658); HT1080, human fibrosarcoma cells (ATCC, #CCL‐121); MCF7, human mammary carcinoma cells (ATCC, #HTB‐22); MDA‐MB‐231, human mammary carcinoma cells (ATCC, #HTB‐26); HCC‐1.2 and HCC‐1.1, human hepatocellular carcinoma cells ; HUVEC, primary human umbilical vein endothelial cells (Promocell, #C‐12200); Hep G2, human hepatocellular carcinom cells (ATCC, #HB‐8065); and ARPE‐19, human retinal pigmented epithelium cells (ATCC, #CRL‐2302).

Techniques: Marker, Western Blot, Zymography, Recombinant

Journal: Biomaterials advances

Article Title: Electrospun fiber-based micro- and nano-system for delivery of high concentrated quercetin to cancer cells.

doi: 10.1016/j.bioadv.2023.213582

Figure Lengend Snippet: Fig. 2. PLA/Q and PLA/PEO/Q release (A), uptake (B) and mediated effects on metabolic activity (C), apoptosis induction (D) and cell number (E) in three oste osarcoma cell lines, namely MG-63, U-2 OS, and SaOS-2 cells. (A) The concentration of released quercetin into an aqueous solution was monitored using UV/VIS spectroscopy between 2 and 72 h incubation in cell-free in vitro system. The concentration of quercetin (μg/ml) was calculated using the calibration curve (Fig. S8A). The quercetin release is presented as ΔC (μg/ml). Bars indicate SD, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05 compared to quercetin release at time 0 (ANOVA and Dunnett's a posteriori test), ###p < 0.001, ##p < 0.01, #p < 0.05 compared to quercetin release from PLA/PEO/Q at appropriate time point (Student's t-test). Cells were treated with the fibers for 48 h (B–D). (B) Quercetin uptake was documented using imaging flow cytometry and dedicated software. Two parameters were applied, namely Normalized Frequency and Intensity_MC_Ch03. Representative microphotographs and histograms are presented. Ch01 and Ch09, bright field (BF); Ch03, Cyanine-3 (CY3)/orange; Ch06, side scatter (SSC). (C) Metabolic activity was analyzed using MTT test. Metabolic activity at untreated conditions is considered as 100 %. BJ human fibroblasts were used to also assess the effects of Q-loaded fibers on normal cells. (D) Apoptosis was evaluated using flow cytometry and dual staining approach, namely Annexin V and 7-AAD staining. Four subpopulations were distinguished, namely live cells (dual staining-negative), early apoptotic cells (Annexin V-positive), late apoptotic cells (dual staining-positive) and necrotic cells (7-AAD-positive). (E) Cell number was assessed using an automated cell counter. Data were normalized to control conditions. Bars indicate SD, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05 compared to CTR untreated control (ANOVA and Dunnett's a posteriori test). CTR, control conditions; Q, treatment with a free quercetin; PLA, treatment with polylactide-based fibers; PLA/Q, treatment with polylactide-based fibers containing quercetin; PLA/PEO, treatment with polylactide and polyethylene oxide-based fibers; PLA/PEO/Q, treatment with polylactide and polyethylene oxide-based fibers containing quercetin.

Article Snippet: The following human normal and cancer cell lines were used, namely normal foreskin fibroblasts (BJ cells, CRL-2522TM, ATCC, Manassas, VA, USA), osteosarcoma cell lines MG-63 (86051601), U-2 OS (92022711) and SaOS-2 (89050205) (ECACC, Public Health England, Porton Down, Salisbury, UK) and breast cancer cells SK-BR-3 (HTB-30TM), MCF-7 (HTB-22TM), MDA-MB-231 (HTB-26TM), MDA-MB-468 (HTB-132TM), Hs 578T (HTB-126TM), and BT-20 (HTB-19TM) (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Concentration Assay, UV-Vis Spectroscopy, Incubation, In Vitro, Imaging, Flow Cytometry, Software, Staining, Control

Journal: Journal of medicinal chemistry

Article Title: Discovery of 6-Phenylhexanamide Derivatives as Potent Stereoselective Mitofusin Activators for the Treatment of Mitochondrial Diseases

doi: 10.1021/acs.jmedchem.0c00366

Figure Lengend Snippet: Diastereomer-selective correction of mitochondrial fragmentation and depolarization in Mfn knockout cells for 13. A and B. Dose-response curves for 13 stereoisomers increasing mitochondrial aspect ratio in cells expressing only Mfn2 (A) or only Mfn1 (B). Prototype mitofusin activator 2 data are shown for comparison. C. Effects of compounds on mitochondrial inner membrane polarization in Mfn1 knockout (KO; top) and Mfn2 KO (bottom) cells. D. Representative confocal imaging of mitochondrial morphology and polarization status in Mfn2 null MEFs treated with different compounds (1 μM, 24 h). Green mitochondria are depolarized and have damaged respiratory function. Scale bars are 10 μm. MT Green (mitotracker green), TMRE (tetramethylrhodamine ethyl ester, red) and nuclear Hoescht (blue) were used for staining. Data are means ± SEM of three independent experiments.

Article Snippet: Functional evaluation of mitofusin agonist fusogenicity was performed in MFN1- or MFN2-deficient MEFs purchased from ATCC (Cat #: CRL-2992, CRL-2993) and cultured at 37°C, 5% CO 2 -95% air in Dulbecco’s minimal essential medium (DMEM) containing glucose (4.5 g/l) with 10% (v/v) fetal bovine serum (FBS; Gibco, 26140–079), 1x nonessential amino-acids (Gibco, 11130051), 2 mM L-glutamine (Corning, 34717007), and 1% (v/v) penicillin/streptomycin (Gibco, 15140–122).

Techniques: Knock-Out, Expressing, Imaging, Staining

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: Quantitative Temporal Analysis of VACV Infection (A) Schematic of experimental workflow for each of three biological replicates. Cells were infected at MOI of 5 or mock infected ( Figure S1 A). Additionally, one mock and one infected sample were treated for 6 h with the viral DNA replication inhibitor cytosine arabinoside (AraC). (B) Hierarchical cluster analysis of all proteins quantified. An enlargement of three subclusters is shown (right panel), including multiple proteins that were substantially up- or downregulated. (C) Scatterplot of all proteins quantified at 18 h of infection. For all analyses in this manuscript, a mean fold change at each time point was calculated by averaging fold changes from each of the biological replicates in which the protein was quantified. For the purposes of comparison, the 18-h mock sample from each replicate was used, because the 0-, 6-, and 18-h mock samples behaved extremely similarly ( Figure S1 E). To perform a comprehensive analysis, “sensitive” criteria were employed, examining proteins down- or upregulated >2-fold on average across all replicates in which the protein was quantified ( A). Sensitive criteria were used in each analysis apart from where indicated. Data from “stringent” criteria that examined only proteins quantified in all three replicates with an average fold change > 2 and p < 0.05 are shown in C. For proteins quantified in all three replicates, a Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. Only proteins quantified in all three replicates are shown in this scatterplot. (D) Example of a previously unreported target of VACV infection. Data are represented as mean ± SEM; ∗ p < 0.05 (see ). Immunoblot of HFFF-TERTs infected with VACV (MOI = 5) confirmed rapid HDAC5 downregulation.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Comparison, Two Tailed Test, Western Blot

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: VACV Protein C6 Downregulates HDAC5 (A) HDAC5, but not HDAC1, is proteasomally degraded during VACV infection. Bar charts and statistics were generated as described in Figure 5 . (B) Viral class centroid profiles compared to an inverted profile of HDAC5, which had additionally been scaled from 0 to 1. (C) Profile of HDAC5 scaled as in (B), and Tp2-class VACV proteins with known roles in regulation of IFN or ISGs. (D) C6 targets HDAC5. HFFF-TERTs were infected in biological triplicate with WT VACV or vΔC6 (lacking gene C6L ) ( Unterholzner et al., 2011 ) (MOI = 5 and 12 h). The scatterplot shows all proteins quantified. A Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. (E) Representative immunoblot demonstrating that C6 specifically targets HDAC5. By comparison, HDAC1 was unmodified (MOI = 5). Quantitation of all three replicate immunoblots are shown (right panel). Data are represented as mean ± SEM, ∗ p < 0.05 from a two-tailed t test. (F) Representative immunoblot demonstrating rescue of HDAC5 expression by inhibition of the proteasome (MOI = 5). MG132 was added 2 h after VACV infection. Quantitation of all three replicate immunoblots is shown (right panel). Data are represented as mean ± SEM, ∗∗ p < 0.00005 from a two-tailed t test. (G) Immunoblot demonstrating that inducible expression of C6 is sufficient for HDAC5 degradation. HEK293T cells inducibly expressing C6 with an N-terminal Myc or C-terminal TAP tag were either treated or untreated with 100 ng/ml doxycycline overnight then lysates immunoblotted.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Generated, Two Tailed Test, Western Blot, Comparison, Quantitation Assay, Expressing, Inhibition

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy