Journal: Frontiers in Plant Science

Article Title: Improving transient expression in N. benthamiana by suppression of the Nb-SABP2 and Nb-COI1 plant defence response related genes

doi: 10.3389/fpls.2024.1453930

Figure Lengend Snippet: Schematic representation of expression constructs used in this study. (A) The pLH- 35S-COI1-INT-COI1 RNAi construct. The pLH- 35S-COI1-INT-COI1 plasmid contains the bar and Nb-COI1 RNAi expression cassettes. The components of the pLH- 35S-COI1-INT-COI1 are designated as follows: bar , bar gene; Nb-COI1 , the 186 bp fragment of the Nb-COI1 gene in sense and antisense orientation; INT, the second intron ( IV2 ) of the potato ST-LS1 gene. (B) The pLH- 35S-SABP2-INT-SABP2 RNAi construct. The pLH- 35S-SABP2-INT-SABP2 plasmid contains the bar and Nb-SABP2 RNAi expression cassettes. The components of the pLH- 35S-SABP2-INT-SABP2 are designated as follows: bar, bar gene; Nb-SABP2, the 230 bp fragment of the Nb-SABP2 gene in sense and antisense orientation; INT, the second intron ( IV2 ) of the potato ST-LS1 gene. (C) The pLH -TMV-GOI expression vector. Features are as follows: RdRp, RNA-dependent RNA polymerase; MP, movement protein; genes of interest (GOI): βNGF (βNGF) and GFP (GFP); CP, coat protein encoding sequence; (D) The pLH- PVX-βNGF expression vector. Features are as follows: RdRp, RNA-dependent RNA polymerase; 25K, 12K, 8K, triple gene block; βNGF, βNGF gene; CP, coat protein encoding sequence. All expression constructs are based on the pLH7000 vector backbone. The regulatory elements used in expression vectors are pAct2, Act2 promoter from A thaliana ; tNos, nopaline synthase terminator, p35S, CaMV 35S promoter; t35S, CaMV 35S terminator. LB, RB, left and right border of T-DNA, respectively.

Article Snippet: At first, the βNGF gene containing Nhe I restriction site at the 5’ of the start codon and Sal I restriction site at the 3’ end of the stop codon was synthesized by BioCat (Heidelberg, Germany).

Techniques: Expressing, Construct, Plasmid Preparation, Sequencing, Blocking Assay

Journal: Arthritis Research & Therapy

Article Title: High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain

doi: 10.1186/ar4449

Figure Lengend Snippet: Application of the 8 hours/16 hours/8 hours stretch protocol to human intervertebral disc cells. (A) Representative morphological images of static and high mechanical strain (HMS) cultured human annulus fibrosus (AF) and nucleus pulposus (NP) cells. Scale bar: 200 μm. (B) Gene expression analysis of both AF and NP cells immediately after the stretch protocol ended. IVD, Intervertebral disc; NGF, Neuronal growth factor; TLR, Toll-like receptor; TNF, Tumour necrosis factor. Error bars represent SEM. Three independent experiments were performed. * P < 0.05 and ** P < 0.01 by Student’s t -test.

Article Snippet: After cell attachment, control wells were changed to 0.1% serum RPMI 1640 medium supplemented with 50 ng/ml recombinant βNGF (BioShop Canada, Burlington, ON, Canada) or sterile water vehicle (control).

Techniques: Cell Culture, Gene Expression

Journal: Arthritis Research & Therapy

Article Title: High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain

doi: 10.1186/ar4449

Figure Lengend Snippet: Application of 8 hours/16 hours/8 hours/16 hours stretch protocol. (A) Representative morphological images of human static and high mechanical strain (HMS) cultured annulus fibrosus (AF) and nucleus pulposus (NP) cells. Scale bar: 200 μm. (B) Gene expression analysis of both NP and AF cells after the additional 16-hour resting period. IVD, Intervertebral disc; NGF, Neuronal growth factor; TLR, Toll-like receptor; TNF, Tumour necrosis factor. Error bars represent SEM. Three independent experiments were performed. * P < 0.05 and ** P < 0.01 by Student’s t -test.

Article Snippet: After cell attachment, control wells were changed to 0.1% serum RPMI 1640 medium supplemented with 50 ng/ml recombinant βNGF (BioShop Canada, Burlington, ON, Canada) or sterile water vehicle (control).

Techniques: Cell Culture, Gene Expression

Journal: Arthritis Research & Therapy

Article Title: High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain

doi: 10.1186/ar4449

Figure Lengend Snippet: Media from nucleus pulposus and annulus fibrosus cells cultured under high mechanical strain promote neurite outgrowth of PC12 cells. (A) Conditioned media from static and high mechanical strain (HMS) nucleus pulposus (NP) and annulus fibrosus (AF) cells were incubated with PC12 cells, and neurite outgrowth and viability were observed and compared to vehicle (−NGF) and neural growth factor (NGF)-treated (+NGF) controls. Black arrows in phase images indicate neurites. Scale bar: 200 μm. (B) Quantification of total and multiple neurite outgrowth and viability. Error bars represent SEM three independent experiments were performed. * P < 0.05, ** P < 0.01, and # P < 0.05 (all by Student’s t -test) for samples with three or more neurites per cell body. All samples were compared to –NGF controls.

Article Snippet: After cell attachment, control wells were changed to 0.1% serum RPMI 1640 medium supplemented with 50 ng/ml recombinant βNGF (BioShop Canada, Burlington, ON, Canada) or sterile water vehicle (control).

Techniques: Cell Culture, Incubation

Journal: Arthritis Research & Therapy

Article Title: High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain

doi: 10.1186/ar4449

Figure Lengend Snippet: Cytokine analysis of conditioned media. Cytokine array blots were used to compare high mechanical strain (HMS) conditioned media of nucleus pulposus (NP) cells (A) and annulus fibrosus (AF) cells (B) with their respective static controls. Quantitation was carried out by performing enzyme-linked immunosorbent assays with conditioned media for neural growth factor (NGF) (C) and tumour necrosis factor α (TNFα) (D) . GCSF, Granulocyte colony-stimulating factor; GM-CSF, Granulocyte-macrophage colony-stimulating factor; GRO, growth-related oncogene; IFN, Interferon; MCP, monocyte chemoattractant protein; MIG, monokine induced by γ interferon; IL, Interleukin; TGF, Transforming growth factor. Error bars represent SEM. Three independent experiments were performed. * P < 0.05 and # P = 0.0504 by Student’s t -test (the latter statistic approaching significance).

Article Snippet: After cell attachment, control wells were changed to 0.1% serum RPMI 1640 medium supplemented with 50 ng/ml recombinant βNGF (BioShop Canada, Burlington, ON, Canada) or sterile water vehicle (control).

Techniques: Quantitation Assay