certified primary normal human dermal fibroblasts  (Lonza)

Journal: Cell reports

Article Title: TACC3 Regulates Microtubule Plus-End Dynamics and Cargo Transport in Interphase Cells

doi: 10.1016/j.celrep.2019.12.025

Figure Lengend Snippet: (A) NHDFs or SK-N-SHs treated with non-targeting (ctrl), EB1, or CLIP-170 (CLIP) siRNAs were mock-infected or infected with HSV-1 at MOI 10 for 5 h and analyzed by WB. (B) SK-N-SHs treated with the indicated siRNAs were infected, as in (A). (C) SK-N-SHs treated with independent EB2 siRNAs (I or II) were infected, as in (A). (D) SK-N-SHs were treated with 100 nM BafA or DMSO and infected at MOI 10 with VSV for 4 h or HSV-1 for 5 h. (E) NHDFs or SK-N-SHs analyzed by WB using the indicated antibodies. (F) NHDFs or SK-N-SHs stained for tyrosinated (Tyr), detyrosinated (Detyr), and acetylated (Ac) tubulin. Nuclei were stained with Hoechst. (G) NHDFs or SK-N-SHs treated with 500 nM DMSO or 10 μM nocodazole were infected at MOI 20 with HSV-1 for 4 h in the presence of 1 μg/mL actinomycin D. Fixed cells were stained for VP5 and with Hoechst. Assessed for the accumulation of VP5 over 2 biological replicates were ≥ 165 NHDF or ≥ 190 SK-N-SH nuclei; error bars, SEMs; *p < 0.05, **p < 0.01, N.S., not significant; unpaired 2-tailed t test. (H) Cells treated as in (G) were infected at MOI 10 with HSV-1 for 5 h. All of the experiments represent ≥3 replicates unless indicated. See also and .

Article Snippet: Certified Primary Normal Human Dermal Fibroblasts isolated from human male neonatal foreskin (NHDFs, Lonza CC-2509), Phoenix-AMPHO (ATCC), Vero and BSC40 cells (Dr. Ian Mohr, NYU School of Medicine) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% Fetal Bovine Serum (FBS), 1% L-Glutamine, and 1% penicillin-streptomycin.

Techniques: Infection, Staining

Journal: Cell reports

Article Title: TACC3 Regulates Microtubule Plus-End Dynamics and Cargo Transport in Interphase Cells

doi: 10.1016/j.celrep.2019.12.025

Figure Lengend Snippet:

Article Snippet: Certified Primary Normal Human Dermal Fibroblasts isolated from human male neonatal foreskin (NHDFs, Lonza CC-2509), Phoenix-AMPHO (ATCC), Vero and BSC40 cells (Dr. Ian Mohr, NYU School of Medicine) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% Fetal Bovine Serum (FBS), 1% L-Glutamine, and 1% penicillin-streptomycin.

Techniques: Western Blot, Virus, Retroviral, Plasmid Preparation, Recombinant, Transfection, Negative Control, Amplification, Software, Microscopy

Journal: Journal of Cell Science

Article Title: Proteomic and mechanistic dissection of the poxvirus-customized ribosome

doi: 10.1242/jcs.246603

Figure Lengend Snippet: Threonine 278 and serine 281 of RPS2 are phosphorylated during VacV infection. (A) Western blot analysis of HAP1 cells infected with VacV, HSV-1 or VSV (MOI=5) at 20 hpi (for VacV and HSV-1) or 6 hpi (for VSV). Arrowheads point to bandshifted species. (B) Western blot analysis of NHDFs infected with VacV, HSV-1, or VSV (MOI=5) at 20 hpi (for VacV and HSV-1) or 6 hpi (for VSV). Arrowheads point to bandshifted species. (C) Western blot analysis of NHDF lysates that were either mock infected (–) or infected with VacV (+), then treated with recombinant shrimp alkaline phosphatase (rSAP) as indicated. Arrows point to the loss of band-shifted RPS2 in SAP-treated samples. (D,E) NHDFs infected with temperature sensitive (Ts) viral mutants of B1 or F10 kinase for 20 h at permissive (32°C) or non-permissive (39.5°C) temperatures. (F) Structural model of RPS2 with labeled amino acid residues identified as being phosphorylated during VacV infection. (G) MS/MS spectra of RPS2 peptides from GFP-TRAP ribosomes isolated from VacV-infected HAP1 cells. The peptide amino acid sequence, phosphorylated residue, b-series ions (blue), y-series ions (red), charge state (+), and ions that support phosphorylation (^) are all indicated. (H) Western blot analysis of NHDFs expressing HA–RPS2 or HA–RPS28 protein. Ex, exogenous RPS2; En, endogenous RPS2. (I) Western blot analysis of NHDFs expressing HA–RPS2 or site-specific alanine substitution mutants (T276A; T278A; S281A) infected with VacV (MOI=5) for 20 h. Exogenous and endogenous forms of RPS2 are indicated. Arrowheads point to unmodified (black) and modified (red) forms of each. (J) Western blot analysis of NHDFs expressing HA–RPS2 or site-specific alanine (T278A; S281A) or glutamic acid (T278E; S281E) substitution mutants infected with VacV (MOI=0.003) for 72 h. (K) Quantification of relative change in viral protein levels presented as mean±s.e.m. (n=4) of NHDFs expressing HA–RPS2 or site-specific alanine mutants (T276A; T278A; S281A) infected with VacV (MOI=0.003) for 72 h. *P<0.05; NS, not statistically significant (unpaired Student's t-test).

Article Snippet: Certified primary normal human dermal fibroblasts (NHDFs) isolated from human male neonatal foreskin were purchased from Lonza (CC-2509) and grown in Dulbecco's Modified Eagle's Medium (DMEM; Fisher Scientific; MT15013CV) supplemented with 5% FBS, 2 mM L-Glutamine, and penicillin-streptomycin and maintained at 37°C in 5% CO 2 .

Techniques: Infection, Western Blot, Recombinant, Labeling, Tandem Mass Spectroscopy, Isolation, Sequencing, Expressing, Modification

Journal: Journal of Cell Science

Article Title: Proteomic and mechanistic dissection of the poxvirus-customized ribosome

doi: 10.1242/jcs.246603

Figure Lengend Snippet: RPS28 is expressed at low levels and its modification does not enhance infection in NHDFs. (A) MS/MS spectra of RPS28 peptides from GFP-TRAP ribosomes isolated from VacV-infected HAP1 cells. The peptide amino acid sequence, phosphorylated residue, b-series ions (blue), y-series ions (red), charge state (+), and ions that support phosphorylation (^) are all indicated. (B) Structural model of RPS28 with labeled amino acid residues identified as being phosphorylated during VacV infection. (C) Western blot analysis of NHDFs expressing HA–RPS28, or site-specific alanine (T38A; S39A) or glutamic acid (T38E; S39E) substitution mutants of HA–RPS28 infected with VacV (MOI=0.003) for 72 h. (D) Western blot analysis of NHDFs expressing HA-RPS28, or double alanine (AA) or glutamic acid (EE) substitution mutants of HA–RPS28 infected with VacV (MOI=0.003) for 72 h. (E) To estimate the relative abundance of RPS2 and RPS28 peptides identified by mass spectroscopy, spectra counts were used as a semi-quantitative measurement and the percentage difference between the total spectra count of identified RPS2 peptides compared to the total spectra count of RPS28 peptides was determined and displayed as the mean±s.e.m. percentage difference relative to the total spectra count of RPS2 (n=5). *P<0.05; NS, not statistically significant (unpaired Student's t-test). (F) Western blot analysis of NHDFs expressing HA–RPS2 or HA–RPS28 protein. Short and long exposures are shown to illustrate the relative difference in HA–RPS2 versus HA–RPS28 expression.

Article Snippet: Certified primary normal human dermal fibroblasts (NHDFs) isolated from human male neonatal foreskin were purchased from Lonza (CC-2509) and grown in Dulbecco's Modified Eagle's Medium (DMEM; Fisher Scientific; MT15013CV) supplemented with 5% FBS, 2 mM L-Glutamine, and penicillin-streptomycin and maintained at 37°C in 5% CO 2 .

Techniques: Modification, Infection, Tandem Mass Spectroscopy, Isolation, Sequencing, Labeling, Western Blot, Expressing, Mass Spectrometry