Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: A–D Representative whole‐cell currents in HEK 293 cells expressing TRPV1 (A), ASIC3 (B), P2X2 (C), or P2X4 (D) in response to capsaicin (A), acidic pH (B), or ATP (C, D) in the absence of presence of borneol ( n = 5 for each channel). E The effect of different concentrations of borneol on the enzymatic activity of human COX‐2. The number of independent measurements is marked on top of each bar. F Averaged intracellular Ca 2+ increases in cultured mouse DRG neurons in response to consecutive applications of 200 μM borneol, 200 μM menthol, and 67 mM KCl. A total of 81 in 1,689 neurons from four mice were found to be borneol‐sensitive and were included in the analysis. G Representative intracellular Ca 2+ signals in HEK 293 cells expressing human TRPM8 (hTRPM8) in response to different concentrations of borneol. After each application of borneol, Ca 2+ ionophore ionomycin was applied to calibrate Ca 2+ response. RFU: relative fluorescence unit. H Dose–response curves of the borneol‐ or menthol‐induced increase in intracellular Ca 2+ in hTRPM8‐expressing HEK 293 cells. Smooth curves are fit to the Hill equation with an EC 50 of 65 μM and a Hill coefficient of 2.0 for borneol ( n = 15) and an EC 50 of 13 μM and a Hill coefficient of 2.0 for menthol ( n = 6 at concentrations of 0.1, 0.3, and 1 μM; n = 9 at concentration of 3 μM; n = 13 at concentrations of 10, 30, 100, and 300 μM). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. I Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in hTRPM8‐expressing HEK 293 cells ( n = 6). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing, Activity Assay, Cell Culture, Fluorescence, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Representative intracellular Ca 2+ signals in HEK 293 cells transfected with empty vector in response to borneol and subsequent applied Ca 2+ ionophore ionomycin ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing hTRPM8 in response to different concentrations of menthol and the subsequently applied Ca 2+ ionophore ionomycin.

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Quantification of consecutively applied 100 μM menthol‐ and 600 μM borneol‐induced hTRPM8 currents. Currents were normalized to 100 μM menthol‐induced currents at +80 mV ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing mouse TRPM8 in response to different concentrations of borneol. Dose–response curves of borneol‐induced increase in intracellular Ca 2+ in mouse TRPM8‐expressing HEK 293 cells. The smooth curve is a fit to the Hill equation with an EC 50 of 116 μM ( n = 12). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in mouse TRPM8‐expressing HEK 293 cells ( n = 5). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: The expression of ZFAS1 in DN blood samples and HG-induced HGMC ( A and B ) ZFAS1 is highly upregulated DN samples and HG-induced HGMC compared to the normal healthy or control group

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Control

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: The lncRNA ZFAS1 regulates HG-induced proliferation, oxidative stress, fibrosis, and inflammation in mesangial cells A ZFAS1 expression was significantly inhibited in the HG-induced cell, after silencing ZFAS1. Silencing ZFAS1 significantly inhibited the proliferation of the HG-induced cell compared to the negative control group ( B ). Oxidative stress induced after ZFAS1 knockdown in HG-treated HGMC. The level of ROS and MDA were significantly reduced in the HG-treated cell relative to the HG-si-NC group while the SOD activity significantly increased ( C ). The western blot analysis result showed that the protein expression level of fibronectin and collagen type IV was significantly inhibited in the HG si-ZFAS relative to the control HG si-NC group ( D ). The level of inflammatory cytokines IL-6, IL-1β, and TNF-α, produced by HG-induced cells after ZFAS1 knockdown. The inflammatory cytokines were significantly downregulated after ZFAS1 knockdown ( E ). All the experimental data are shown as mean ± SD of three independent experiments and the significance level was defined as p < 0.05 (** p < 0.01 and *** p < 0.001 vs. normal group; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. HG-si-NC group)

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Negative Control, Knockdown, Activity Assay, Western Blot, Control, Produced

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: ZFAS1 endogenously targets the miR-588 sequence. Bioinformatics prediction of target miRNA. Lncbase predicted that the ZFAS1 targets miR-588 seed region sequences. Dual-luciferase reporter assay experiment. MiR-588 mimics significantly reduced the luciferase activity of HGMC co-transfected with the ZFAS1 wild type (WT) reporter plasmid but had no inhibitory effect on that of ZFAS1 mutant type (Mut) plasmid ( A ). Biotinylated RNA pull-down assay. Biotin-coupled probes with wild type (WT) miR-588 sequence pull-down more ZFAS1 than those with the mutated miR-588 sequence (MUT) ( B ). RIP-qRT-PCR analysis. More ZFAS1 and miR-588 were significantly enriched in the immunoprecipitated Ago2-containing complexes compared to the IgG group ( C ). Experiments were carried out in triplicates and p < 0.05 was chosen as the significance level (*** p < 0.001 and ### p < 0.001)

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Sequencing, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Pull Down Assay, Quantitative RT-PCR, Immunoprecipitation

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: MiR-588 regulates HG-induced proliferation, oxidative stress, fibrosis, and inflammation in mesangial cells. The expression level of miR-588 in DN and HG-induced HGMC was significantly downregulated compared to the healthy or control group and was inversely related to ZFAS1 expression ( A – C ). CCK-assay and western blot analysis revealed the overexpression of miR-588 in HG-induced HGMC significantly reduced the cell viability, and the fibronectin and collagen type IV protein expression level, compared to the negative control group ( D and E ). overexpression of miR-588 markedly reversed the HG-induced upregulation of ROS and MDA, and downregulation of SOD, in the HG miR-NC treatment group ( F ). The overexpression of miR-588 significantly reduced the level of the HG-induced inflammatory cytokines secreted ( G ). Significance level was defined as p < 0.05 (** p < 0.01 and *** p < 0.001 vs. normal group; ## p < 0.01, and ### p < 0.001 vs. HG-si-NC group)

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Control, Western Blot, Over Expression, Negative Control

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: ROCK1 mRNA was a direct target of miR-588. Targetscan prediction of miR-588 target mRNA. Dual-luciferase reporter experiments. MiR-588 could significantly inhibit the luciferase activity of the HGMC co-transfected with the wild type (WT) ROCK reporter plasmid but had no inhibiting effect on that of the mutant type (MUT) ROCK group ( A ). QRT-PCR and Western blot analysis. Inhibiting miR-588 markedly upregulated the mRNA and protein expression level of ROCK in the HGMC compared to the negative control group while the overexpression of miR-588 reversed this ( B ). Inhibiting miR-588 restored si-ZFAS1-downregulated expression of ROCK1 mRNA and protein in the HGMC cell ( C ). (*** p < 0.001 ### p < 0.001)

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Western Blot, Expressing, Negative Control, Over Expression

Journal: Diabetology & Metabolic Syndrome

Article Title: LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

doi: 10.1186/s13098-022-00791-3

Figure Lengend Snippet: ZFAS1 inhibits HG-induced proliferation, oxidative stress, fibrosis, and inflammation in mesangial cells via the miR-588/ROCK1 axis. Transfecting pcDNA-ROCK1 overexpression plasmid into HGMC significantly upregulated ROCK1 expression ( A ). The overexpression of ROCK1 restored HG-induced cell viability that was reduced by the depleted ZFAS1 ( B ). MiR-588 inhibition or overexpression of ROCK1 partly restored the depleted level of ROS and MDA and repressed the SOD activity that was increased by ZFAS1 knockdown ( C ). MiR-588 inhibitor or overexpressed ROCK1 also recovered the downregulated fibronectin and collagen type IV protein expression that was induced by ZFAS1 knockdown in the HGMC ( D ). The depleted level of IL-6, IL-1β, and TNF-α after ZFAS1 knockdown in the HG-induced cell was also recovered by miR-588 inhibition or ROCK1 overexpression ( E ). Significance level was set at p < 0.05 (** p < 0.01, *** p < 0.001; ### p < 0.001)

Article Snippet: Human glomerular mesangial cells (HGMC) were bought from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Inhibition, Activity Assay, Knockdown

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: Expression of DbpA in human kidney disease and cell lines. (A) Immunohistochemistry shows that DbpA expression is not detected in glomerular cells in minimal change (MC) GN, however DbpA expression is interspersed in tubulointerstitial cells (a–c). In contrast, DbpA protein was detected within the mesangial compartment of the glomeruli from patients with IgA nephritis and in cell infiltrates of the interstitium (d–f). A similar pattern of glomerular expression was observed in patients with lupus ISN/RPS class 4 G(A) GN with upregulated DbpA expression in tubular cells (g–i). The most profound tubular cell and interstitial upregulation of DbpA was seen with interstitial nephritis, where glomerular cells were immune negative (g–l). Images were made with a Leica DM6000 B Microscope (Leica Microsystems) using either a 100× (a, d, g, and j) or a 400× objective (b, c, e, f, h, i, k, and l). Scale bars, 200 μm in a, d, g, and j; 50 μm in b, c, e, f, h, i, k, and l. (B) Immunohistochemistry for DbpA and DbpB/YB-1 in sequential tissue slices from a patient with IgA nephritis reveals concordant upregulation of both proteins in mesangial compartment; at the same time, the staining pattern in the tubular cells and tubulointerstitial infiltrate markedly differs. (C) Western blot analysis of DbpA protein expression in cell lysates of human proximal tubular epithelial cells HK-2 and HKC-8, rMCs, human primary mesangial cells (hMCs), human embryonic kidney cells (HEK-293s), human podocytes, and human umbilical vein endothelial cells (HUVECs). Three major bands are shown: *55 kD; **50 kD; ***44 kD.

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, Immunohistochemistry, Microscopy, Staining, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: Overexpression of DbpA in rMCs leads to increased cell proliferation. (A) Cellular morphology of rMCs after overexpression of DbpA isoforms DbpA_a and DbpA_b for 3 days. Both interventions result in increased cells proliferation. (B) Western blot analysis reveals the overexpression of both isoforms of DbpA (40 and 55 kD; asterisks in left panel) by pCDH lentiviral transduction, which is accompanied by upregulation of cell proliferation markers (e.g., PCNA and cyclin D1). *P<0.05 (n=4). (C) Immunofluorescence staining shows both cytoplasmic and nuclear DbpA expression, whereas overexpressed DbpA protein mainly localizes in a punctuate manner within the cytoplasm. Scale bar, 50 μm. (D) BrdU cell proliferation assay shows increased cell proliferation in DbpA_a and DbpA_b overexpressing mesangial cells compared with transfection of control vector. **P<0.01 (n=3). (E) Overexpression of DbpA in rMCs with stable knockdown of DbpA leads to much stronger induction of BrdU incorporation. **P<0.01 (n=3).

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Over Expression, Western Blot, Transduction, Immunofluorescence, Staining, Expressing, BrdU Cell Proliferation Assay, Transfection, Control, Plasmid Preparation, Knockdown, BrdU Incorporation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: DbpA protein expression is upregulated by PDGF-BB stimulation in mesangial cells in vitro. (A, upper panel) Western blot analysis of DbpA protein expression in PDGF-BB–challenged primary human mesangial cells (hMCs). *55 kD; **50 kD; ***44 kD. (A, lower panel) Quantification of band intensities reveals 2.5-fold induction of DbpA protein expression (44 and 55 kD) after 24 hour incubation with human PDGF-BB (50 ng/ml). *P<0.05 (n=3). (B, upper panel) Western blot analysis of DbpA protein expression in rMCs stimulated with rat PDGF-BB for 24 hours at increasing doses. A dose-dependent upregulation of DbpA is shown from 5 to 10 ng/ml PDGF-BB stimulation. No significant change of DbpA expression was observed after stimulation with 50–200 ng/ml PDGF-BB. *55 kD; **50 kD; ***44 kD. (B, lower panel) Two bands at 50 and 44 kD are seen, with the predominant band at 44 kD. ctrl, Control. **P<0.01 (n=3). (C) Real–time PCR analysis for DbpA transcripts in rMCs reveals a profound upregulation after 24 and 48 hours of PDGF-BB incubation. *P<0.05 (n=3). (D) Immunofluorescence staining for DbpA in rMCs. Compared with vehicle, DbpA expression increases after 24 hours of incubation with rPDGF-BB (50 ng/ml). DbpA protein accumulates mainly within the cytoplasm. Scale bar, 50 μm. (E) Changes of BrdU incorporation rates are determined for rMCs undergoing overexpression of DbpA_a and DbpA_b or knockdown of DbpA. Stimulation with rPDGF-BB (50 ng/ml) was performed for 24 hours. *P<0.05 (n=3).

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, In Vitro, Western Blot, Incubation, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, BrdU Incorporation Assay, Over Expression, Knockdown

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: DbpA protein expression is induced in experimental models of anti-Thy1.1 and mice MsPGN. (A) DbpA protein was identified by immunohistochemistry in kidney tissue from healthy rats (a and b) and the time course of anti-Thy1.1 nephritis (c and d, 4 hours; e and f, day 4; g and h, day 7; and i and j, day 21). Whereas no DbpA was detected in the glomeruli of healthy rats, significant DbpA expression was observed within the cytoplasm of mesangial cells after the induction of anti-Thy1.1 nephritis on day 4, peaking at day 7 and returning to background after 3 weeks. Magnification, ×100 in a, c, e, g, and i; ×400 in b, d, f, h, and j. Scale bars, 100 μm in a, c, e, g, and i; 25 μm in b, d, f, h and j. (B) Western blot analysis reveals profound upregulation of DbpA protein (44 kD) expression that peaks at day 7 and subsequently, returns to basal levels. DbpA protein levels normalized to β-actin are shown. **P<0.01; ***44 kD. (C) DbpA protein expression by immunohistochemistry staining in kidney tissue from healthy mice (a and b) and after induction of MsPGN for 3 (c and d) and 6 (e and f) days. Quantification of the DbpA staining is shown for 50 visual fields. **P<0.01.

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, Immunohistochemistry, Western Blot, Staining