Journal: Viruses

Article Title: Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

doi: 10.3390/v12010105

Figure Lengend Snippet: The EBOV NP-R111 residue is unannotated, but could impact NP–NP interaction. ( A ) Schematic of NP. R111 (yellow) lies in an un-annotated region within the N-terminal lobe. Key residues of known NP interactions are highlighted; ( B ) Crystal structure (PDB #4YPI) of NP. Though the precise location of the oligomerization domain has yet to be determined by crystallography (orange dashed line), the R111 residue (yellow) is located on the same face as residues proximal to the oligomerization domain (orange: residues 39, 40), but opposite to the VP35 (magenta: residues 160, 171, 174) and RNA (red: residues 240, 248, 252) interaction interfaces; ( C ) Alignment of ebolavirus sequences. The basic residues at 109, 110, and 111 (blue), and a recently identified electrostatic interaction between K110 and E349 [ , ], are conserved in all known ebolaviruses except Sudan virus (SUDV, red).

Article Snippet: NP-R111, the adjacent basic residues K109 and K110, and the K109-E349 salt bridge identified by cryo-EM [ , ], are highly conserved among ebolaviruses, including in the newly described Bombali virus [ ] ( C).

Techniques:

Journal: Regenerative Therapy

Article Title: Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

doi: 10.1016/j.reth.2018.04.001

Figure Lengend Snippet: Morphology and proliferation in keratinocyte line cells cultured on molecular type I collagen and type I collagen gels. HaCaT cells in DMEM (10) were cultured on molecular type I collagen (10 μg/mL) (A, C) or type I collagen gels (1 mg/mL) (B, D) for 2 h (A, B) or 3 days (C, D). FEPE1L-8 cells in K110 were cultured on molecular type I collagen (10 μg/mL) (E, G) or type I collagen gels (1 mg/mL) (F, H) for 2 h (E, F) or 3 days (G, H). White bars indicate 100 μm. Viable cell numbers of HaCaT (I) and FEPE1L-8 cells (J) were estimated on molecular type I collagen (solid line, filled circle), type I collagen gels (dotted line, open circle), or non-treated dish surfaces (dotted line, grey circle) at 2 h, 1day and 3days. Experiments were performed in triplicates and values are shown as means ± SD. Cell proliferation ratios are estimated by the averages of OD450 values at 1- or 3-day over those at 2 h for each culture conditions (K).

Article Snippet: After conditioning from DMEM (10) to serum-free keratinocyte medium, K110 (Kyokuto Seiyaku Inc., Tokyo, Japan), proliferating HaCaT cells were passaged using 0.05% trypsin at least five times (data not shown).

Techniques: Cell Culture

Journal: Regenerative Therapy

Article Title: Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

doi: 10.1016/j.reth.2018.04.001

Figure Lengend Snippet: FEPE1L-8 cells cultured for 6 days in K110 (Ca: 30 μM). Annexin V and PI positive ratio (%).

Article Snippet: After conditioning from DMEM (10) to serum-free keratinocyte medium, K110 (Kyokuto Seiyaku Inc., Tokyo, Japan), proliferating HaCaT cells were passaged using 0.05% trypsin at least five times (data not shown).

Techniques: Cell Culture

Journal: Regenerative Therapy

Article Title: Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

doi: 10.1016/j.reth.2018.04.001

Figure Lengend Snippet: Morphology and proliferation in HaCaT cells cultured on molecular type I collagen and type I collagen gels in K110. HaCaT cells in K110 were cultured for 2 days on molecular type I collagen (10 μg/mL) (A, C) or type I collagen gels (1 mg/mL) (B, D) with 30 μM (A, B) or 1.8 mM (C, D) calcium concentration. White arrow indicates cell aggregate (D). White bars indicate 100 μm. Viable cell numbers were estimated on molecular type I collagen (E) or type I collagen gels (F) with 30 μM (solid line, filled circle) or 1.8 mM (dotted line, open circle) calcium concentrations. All experiments were performed in triplicates and values are shown as means ± SD. Cell proliferation ratios are estimated by the averages of OD450 values at 2-day over those at 2 h for each culture conditions (G).

Article Snippet: After conditioning from DMEM (10) to serum-free keratinocyte medium, K110 (Kyokuto Seiyaku Inc., Tokyo, Japan), proliferating HaCaT cells were passaged using 0.05% trypsin at least five times (data not shown).

Techniques: Cell Culture, Concentration Assay

Journal: Regenerative Therapy

Article Title: Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

doi: 10.1016/j.reth.2018.04.001

Figure Lengend Snippet: HaCaT cells cultured for 6 days in K110 (Ca: 30 μM). Annexin V and PI positive ratio (%).

Article Snippet: After conditioning from DMEM (10) to serum-free keratinocyte medium, K110 (Kyokuto Seiyaku Inc., Tokyo, Japan), proliferating HaCaT cells were passaged using 0.05% trypsin at least five times (data not shown).

Techniques: Cell Culture

Journal: Regenerative Therapy

Article Title: Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8

doi: 10.1016/j.reth.2018.04.001

Figure Lengend Snippet: Proliferation ratios of keratinocyte line cells cultured on type I collagen gels in K110 with various calcium concentrations. HaCaT (A) and FEPE1L-8 cells (B) in K110 were cultured on type I collagen gels with 30, 60, 90, 120, 150, 180, 450, 900 μM, 1.8 mM and 3.6 mM calcium concentrations for 2 days (dotted line) or 7 days (solid line), estimated living cell numbers using MTT assay. All experiments were performed in triplicates and values are shown as means ± SD. After 2 to 7 day-culture, proliferation ratios are estimated by average OD450 values at 7 days over those at 2 days in HaCaT (solid line) and FEPE1L-8 (dotted line) (C). Schematic views of exogenous calcium range from the beginning of cell proliferation to a plateau between 2 and 7 days were shown in D. Survival signals are activated by adhesion to both forms of type I collagen (solid black arrows) and exogenous calcium stimulus (dotted black arrows). In cells on molecular type I collagen, apoptosis induction did not occur. On type I collagen gels, apoptosis was induced in 20–30% both of HaCaT and FEPE1L-8 cells ( Table 3 , Table 4 ). On type I collagen gels, apoptotic signals are activated as well (grey arrow). Increasing exogenous calcium concentration reinforces activation of survival signals and cell proliferation to some extent (D).

Article Snippet: After conditioning from DMEM (10) to serum-free keratinocyte medium, K110 (Kyokuto Seiyaku Inc., Tokyo, Japan), proliferating HaCaT cells were passaged using 0.05% trypsin at least five times (data not shown).

Techniques: Cell Culture, MTT Assay, Concentration Assay, Activation Assay