Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet:

Article Snippet: For in vitro study, the siRNA targeting TLR2 (siTLR2, 50 nM, KeyGen Biotech, Jiangsu, China) or nontargeting scrambled controls (scRNA, 50 nM, KeyGen Biotech, Jiangsu, China) was applied by Lipocat3000 transfection reagent (Aoqing Biotechnology, Beijing, China).

Techniques: Recombinant, Protein Extraction, Endotoxin Assay, Western Blot, Virus, Software, Real-time Polymerase Chain Reaction, Imaging

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: TLR4 mRNA and protein levels were examined via real-time PCR and Western blotting after low gucose or high glucose treatment. (A and B) TLR4 mRNA upregulation in the high glucose medium. (C) TLR4 protein levels increased after the high glucose treatment. (D) Experiments were analyzed via densitometry and normalized to the control (0 h). (E) MCP-1 mRNA upregulation in high glucose medium for 24 h. (F) Immunofluorescence analysis of TLR4 expression. PRTC cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium.The cells were fixed for immunofluorescent staining of TLR4 (red). Data are expressed as the mean ± S.D. (n≥3). *, p<0.05 versus the low glucose group; **, p<0.05 versus the control (0 h).

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Expressing, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were treated for 24 hours with low glucose (5.5 mM), high glucose (30 mM), or the PKC inhibitor staurosporine (10 nM, added 1 hour before high glucose treatment), and the cell lysates were collected, TLR4 mRNA, phospho-PKC (pan), p-p38, p38 levels were examined via real-time PCR and Western blotting. (A) PKC phosphorylation and p-p38 upregulation after high glucose treatment. The PKC inhibitor staurosporine decreased phospho-PKC and p-p38 levels but not total PKC levels. (B) Staurosporine reduced TLR4 mRNA levels under the high glucose condition. (C) RPTC were pretreated with staurosporine (10 nM), then treated with high glucose (30mM) for 6, 12, 24 h, and cell lysates were collected at different time points. phospho-PKC (pan), p-p38 levels were examined via Western blotting. (D) Effect of staurosporine on scratch wound healing in low glucose and high glucose. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without staurosporine treatment.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for following experiments. (A) Titration of TAK-242 concentration. RPTC cells were cultured for 12 h in low glucose or high glucose medium with or without TAK-242. (B) MyD88 upregulation was also inhibited by TAK-242 under high glucose conditions by Western blotting. (C) TLR4 mRNA upregulation was also inhibited by TAK-242 under high glucose conditions by real-time PCR analysis. (D) Enhanced wound healing in the high glucose condition with the TLR4 inhibitor. RPTC cells were scratch-wounded and incubated in low glucose or high glucose medium with or without 100 nM TAK-242 for 18 h and then the healing distance was measured. (E) Enhanced cell migration under the high glucose condition with the TLR4 inhibitor. A total of 3x10 5 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of medium with or without 100 nM TAK-242 in low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface of the insert were stained with PI and counted. In C, D and E, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without TAK-242 treatment.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Cell Culture, Titration, Concentration Assay, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were infected with lentiviruses containing a scrambled control sequence (Scr) or the TLR4 shRNA sequence (shRNA) and then cultured for 2 days in low glucose or high glucose medium for the following experiments. (A) TLR4 knockdown caused by the TLR4 shRNA lentivirus. After infection and a high glucose treatment, whole-cell lysates were collected for an immunoblot analysis of TLR4 and MyD88. (B) Scratch-wound healing. After lentiviral infection, RPTC were scratch-wounded and incubated in low glucose or high glucose medium for 18 h to measure the distance over which healing occurred. (C) Transwell cell migration. After lentiviral infection, a total of 3x10 5 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. In B and C, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus high glucose group infected with the scrambled sequence.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Infection, Sequencing, shRNA, Cell Culture, Western Blot, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were infected with lentiviruses containing TLR4 or control lentivirus and then subjected to low glucose and high glucose treatment for 2 days, followed by a scratch-wound healing assay. (A) Increased TLR4 levels were detected in the low-glucose- and high-glucose-treated RPTC. (B) MCP-1 mRNA level in low-glucose-treated and high-glucose-treated RPTC cells after TLR4 overexpression. (C) Overexpression of TLR4 in low-glucose-treated cells also suppressed wound healing, mimicking the effect of high glucose levels. (D) RPTC cells were infected with lentivirus containing TLR4 or control lentivirus, then cultured for 18 h in low glucose or high glucose medium with or without TAK-242, followed by a scratch-wound healing assay. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; #, p<0.05 versus the group without TAK-242.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Infection, Wound Healing Assay, Over Expression, Cell Culture

Journal: Journal of Animal Science and Biotechnology

Article Title: Inhibiting nuclear factor erythroid 2 related factor 2-mediated autophagy in bovine mammary epithelial cells induces oxidative stress in response to exogenous fatty acids

doi: 10.1186/s40104-022-00695-2

Figure Lengend Snippet: Linear and quadratic contrasts in MAC-T cells incubated with increasing concentrations of FFA

Article Snippet: In Exp.3, to explore the effect of NFE2L2 knockdown on autophagy in MAC-T cells, scrambled non-target negative control (Si-Control) or siRNA targeting NFE2L2 (Si-NFE2L2) were used to transfect MAC-T cells for 48 h. The Si-NFE2L2 and Si-Control were designed and synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Incubation

Journal: Cell Proliferation

Article Title: lnc‐RHL, a novel long non‐coding RNA required for the differentiation of hepatocytes from human bipotent progenitor cells

doi: 10.1111/cpr.12978

Figure Lengend Snippet: Inducible shRNA knockdown of lnc‐RHL in HepaRG cells. A, Experimental design. Normal HepaRG cells and HepaRG cells transduced with lentiviral constructs encoding Dox‐inducible shRNAs encoding scrambled shRNA or lnc‐RHL shRNAs sh‐B and sh‐C were used to initiate cultures. These were then grown under 3 experimental conditions: (1) no‐Dox control group (14 days of expansion followed by 19 days of differentiation); (2) terminal Dox induction (14 days of expansion, followed by 19 days of differentiation, with Dox induction during the final 3 days of culture); (3) concurrent Dox induction (14 days of expansion followed by 19 days of differentiation in the presence of Dox. Periods of Dox induction are indicated with red bars. B, Representative images of control HepaRG cells (untransduced or transduced with scrambled shRNA) then imaged at 40X magnification (phase contrast and GFP). HepaRG cells without virus or expressing scrambled shRNA contained numerous hepatocyte colonies on a monolayer background of cholangiocytes. Induction of the lentiviral construct with Dox yielded GFP expression cells. C, lnc‐RHL knockdown in differentiated HepaRG cells transduced with anti‐lnc‐RHL shRNAs sh‐B and sh‐C. Induction of both shRNAs yielded cultures with reduced content of hepatocyte colonies

Article Snippet: Packaged lentivirus containing 3 shRNAs designed to target lnc‐RHL (based on our complete lnc‐RHL sequence) was procured from transOMIC Inc, and expressed from a pZIP‐inducible lentiviral vector as well as a non‐targeting scrambled control shRNA.

Techniques: shRNA, Transduction, Construct, Expressing