Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 3. The accumulation of cells 24 h after CCL3 injection is hampered in NOD (5–7-week-old) mice (A). Granulocytes (B) and monocytes (C) are not attracted in NOD mice. In response to CCL2, NOD mice show a deficient accumulation of cells into the air pouch after 24 h (D). The accumulation of monocytes to CCL2 is almost absent in NOD (E). Data are cumulative of two individual experiments and represented as mean SEM (n=6/group in A and C; n=4/group in B, C, E). *p<0.05 relative to NOD as determined with Mann-Whitney (A, D) or Student's t-test (B, C, E).

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection, MANN-WHITNEY

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 6. The air pouch of C57BL/6 and BALB/c contains a high number of MU (A) and DC (B) after CCL2 injection, while those of NOD mice do not. MU and DC are defined as shown in Fig. 4. Data are cumulative of two experiments and are represented as mean SEM; each bar represents at least four animals (5–7 weeks old), *p<0.05 as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 7. The air pouch of control mice at 24 h after CCL2 injection shows an increased production of IL-1b; however, this is not seen in NOD (A). NOD mice produce higher amounts of IL-10 in comparison to C57BL/6 and BALB/c mice (B). A representative flow cytometric analysis of intracellular staining for IL-10 (gated for CD11b+ cells) shows that monocytes (CD11bhiF4/80medCD11clow) are producing IL-10, while MU (CD11b+F4/80hiCD11clow) do not (C). Figures shown are representative of five NOD mice 24 h after injection with CCL2. The production of TNF-a in the air pouch of NOD mice is similar to control mice (D). Data are represented as mean SEM (n=5/group; 5–7 weeks old). *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Control, Injection, Comparison, Staining

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 9. Peritoneal exudate MU of NOD mice show a decreased migration towards CCL2 compared to C57BL/6 control mice using a Boyden chamber chemotaxis assay. Data are represent mean SD (n=3), *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Migration, Control, Chemotaxis Assay

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 8. Neutralization of IL-10 does not restore the cell recruitment towards CCL2 to the level of C57BL/6 mice (A). Addition of recombinant IL-10 to the air pouch of C57BL/6 mice decreases the recruitment towards the control level (A). Blocking IL-10 raises the level of IL-1b in the air pouch of NOD to a similar level of C57BL/6 mice (B). Addition of recombinant IL-10 in the air pouch of C57BL/6 does not lower the amount of IL-1b (B). Mean SD (n=6) are shown. Data in (B) are presented relative to the amount of IL-1b that is observed in control-injected animals. Statistical significance was deter- mined with Kruskall-Wallis test and differences between groups using the Mann-Whitney test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Neutralization, Recombinant, Control, Blocking Assay, Injection, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 inhibits CCL2 expression and release by endothelial cells. Confluent HPAECs were serum-restricted for 16 h followed by treatment with BMP9 in 0.1% FBS. (A) HPAECs were treated with 1 ng/ml BMP9 for 2, 4, 8 or 12 h (3 experiments). Data show the fold change relative to 0.1% FBS at each time point. (B) HPAECs were treated with BMP9 (0-10 ng/ml) for 8 h (5 experiments). (C) HPAECs were treated with BMP9 (0-10 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well ( n =4 wells per treatment) and is representative of 3 experiments. (D) HPAECs were treated with BMP10 (0-10 ng/ml) for 8 h (3 experiments). (E) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well. Data ( n =4 wells per treatment) are representative of 3 experiments. (F,G) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 8 h and expression of CCL2 (F) and ID1 and ID2 (G) measured (6 experiments). (H,I) HAECs were treated with BMP9 (0-10 ng/ml) (H) or BMP10 (0-10 ng/ml) (I) for 8 h (3 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to control. Significance was calculated using either one-way repeated measures ANOVA with post-hoc Tukey's HSD test (B,E-G) or Friedman multiple comparison test with post-hoc Dunn's analysis (C,D,H,I). * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS without added BMP9 or BMP10).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Expressing, Control, Comparison

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: Reduction of ALK1 attenuates the induction of CCL2 by BMP9, and both ACTR-II and BMPR-II mediate the repression by BMP9 and BMP10. (A-C) HPAECs were transfected with siRNA for ALK1 (siA1), BMPR2 (siB2) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (A) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h. Expression of CCL2 was normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (4 experiments). (B) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. Conditioned media were collected, assayed for CCL2 by ELISA and normalized to cell number for each well. Data ( n =4 wells per treatment) are from a representative of 4 experiments. (C) Specific reduction of ALK1 and BMPR-II by their respective siRNAs was confirmed by western blotting, the numbers below the blots representing band density ratios relative to α-tubulin normalised to the DH1 control. Arrows indicate the positions of the molecular mass markers (kDa). (D-F) HPAECs were transfected with a non-targeting control siRNA pool (siCP) or siRNAs for ACVR2A (siA2A), BMPR2 (siB2) or both in combination (siA2AB2) using DharmaFECT1 (DH1). HPAECs were treated with 1 ng/ml BMP9 or BMP10 in 0.1% FBS for 8 h. Expression of ACTR-IIA ( ACVR2A ; D), BMPR-II ( BMPR2 ; E) and CCL2 (F) were normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (6 experiments). The key for D-F is provided in F. (G-I) Confluent serum-restricted HPAECs were pretreated with 250 nM LDN-193189 or 2µM SD208 for 1 h followed by 1 ng/ml BMP9 in 0.1% FBS for 8 h for mRNA extraction. Expression of CCL2 (G), CXCL8 (H) and ID1 (I) were determined by qPCR and normalized to ACTB . qPCR data are presented as the fold change relative to the DMSO control (1:2500 in 0.1% FBS) (3 experiments). All data are expressed as mean±s.e.m. Significance was calculated using either a paired Students t -test (A,B,G-I), comparing with 0.1% FBS control, or one-way repeated measures ANOVA with post-hoc Sidak test (D-F), comparing with siCP of same treatment. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Extraction

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is dependent on Smad4, but not Smad2 or Smad3. (A) Confluent serum-restricted HPAECs were treated with 0.01-10 ng/ml BMP9 in 0.1% FBS for 1 h. Protein lysates were immunoblotted for phospho-Smad1/5, Smad1, phospho-Smad2 or Smad2. Blots are representative of 4 experiments. (B) Quantification of the blots in A, calculated as the ratio of the density of the phospho-Smad band to the Smad band for each sample and normalised to the 0.1% FBS control. (C-E) HPAECs were transfected with SMAD4 siRNA (siS4) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (C) Reduced Smad4 protein was confirmed by western blotting. (D) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h (3 experiments). (E) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. CCL2 release was measured by ELISA and normalised to cell number. Data are from a representative of 3 experiments ( n =4 wells per treatment). (F,G) HPAECs were transfected with SMAD2 siRNA (siS2) or siCP using DH1 and treated with BMP9 for 8 h as described above. (F) CCL2 (top panel) and CXCL8 (bottom panel) expression (3 experiments). (G) Smad2 protein knockdown was confirmed by western blotting. (H,I) HPAECs were transfected with SMAD3 siRNA (siS3) or siCP using DH1 and treated with BMP9 for 8 h. (H) Smad3 protein knockdown was confirmed by western blotting. (I) CCL2 expression (3 experiments). For western blots, the migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DHI/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey’s HSD test. * P <0.05.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Control, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Migration

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is not dependent on Smad1, Smad5 or Smad9. HPAECs were transfected with siRNAs for SMAD1 (siS1), SMAD5 (siS5) or SMAD9 (siS9) alone or in combination using DharmaFECT1 (DH1). In parallel, cells were transfected with a non-targeting control pool (siCP). (A,B) Knockdown of Smad1 and Smad5 were confirmed by western blotting of cells transfected with siRNAs targeting individual (A) and combinations (B) of Smads. The migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. (C-E) Transfected HPAECs were serum-restricted, followed by treatment with 1 ng/ml BMP9 in 0.1% FBS for 8 h. CCL2 expression (C) and ID2 and CXCL8 expression (D) were determined in cells in which individual Smads were knocked down (5 experiments). CCL2 and ID2 expression (E) were determined in HPAECs in which combinations of Smads were knocked down (4 experiments). (F) Transfected HPAECs were serum-restricted, followed by treatment with 0.3 ng/ml BMP9 or BMP10 in 0.1% FBS for 4h (5 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DH1/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey's HSD (C-E) or Sidak (F) test. * P <0.05, ** P <0.01, *** P <0.001. For CCL2 , data were compared with siCP/0.1% FBS. For ID2 and CXCL8 , data were compared with siCP/BMP9.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Knockdown, Western Blot, Migration, Expressing

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 does not affect CCL2 induction by TNF-α in HPAECs and HAECs. (A) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Co-treatments were added without BMP9 pre-incubation or after 1 h or 16 h pre-incubation with 5 ng/ml BMP9 (3 experiments). (B) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) and TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Conditioned media were assayed for CCL2 by ELISA. Data are from a representative of 3 experiments ( n =4 wells per treatment). (C) Confluent serum-restricted HPAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-2 ng/ml) in 0.1% FBS for 6 h (4 experiments). (D,E) Confluent serum-restricted HAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h and assayed for CCL2 expression (D) (4 experiments) and CCL2 release (E). ELISA data are from a representative of 3 experiments ( n =4 wells per treatment). (F) Confluent serum-restricted HAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-5 ng/ml) in 0.1% FBS for 6 h. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to 0.1% FBS (no additions). Significance was calculated using Friedman multiple comparisons test with post-hoc Dunn’s analysis. * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 pretreatment augments capsaicin-evoked inward cationic currents in small-diameter dorsal root ganglion neurons. ( A ) Compared with capsaicin (0.3 μM)-induced inward cationic current in a control small dorsal root ganglion (DRG) sensory neuron, the amplitude of capsaicin-evoked inward current was greatly increased in a small-diameter DRG neuron preincubated with 5 nM chemokine CC chemokine ligand 2 (CCL2) for 24 hours. Holding potential (V H ) = −60 mV. ( B ) CCL2 pretreatment increased the density of capsaicin (0.3 μM)-induced inward currents with a concentration-dependent manner. Each point shows the mean ± standard error value of 10 neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 increases density of capsaicin-evoked inward currents by upregulating TRPV1 mRNA expression in sensory neurons. ( A ) The concentration–response curve for the density of capsaicin-induced inward currents was obtained from small-diameter dorsal root ganglion (DRG) neurons in the absence and presence of chemokine CC chemokine ligand 2 (CCL2; 5 nM) pretreatment. Note that CCL2 preincubation increased the density of capsaicin currents without significantly affecting the EC 50 value. Holding potential (V H ) = −60 mV. Each point represents the mean ± standard error (SE) value of 12 neurons. ( B ) Quantitative RT-PCR assays showed that pretreating cultured DRG neurons with 5 nM CCL2 for 24 to 36 hours greatly increased the mRNA level of transient receptor potential vanilloid receptor 1 (TRPV1). Each bar shows the mean ± SE value of five experiments. * P <0.01.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 augments capsaicin-evoked inward currents and increases TRPV1 mRNA in neurons via activating phosphatidylinositol-3 kinase. ( A ) Following co-treating cultured dorsal root ganglion (DRG) neurons with chemokine CC chemokine ligand 2 (CCL2) (5 nM) and phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 (10 μM) for 24 to 36 hours, CCL2 failed to significantly enhance the amplitude of capsaicin (0.3 μM)-evoked inward current in a small-diameter DRG neuron. In the presence of ERK 1/2 inhibitor U0126 (20 μM), CCL2 still greatly augmented the magnitude of capsaicin currents in a small DRG sensory neuron. Holding potential (V H ) = −60 mV. ( B ) Pretreating small-diameter DRG neurons with 5 nM CCL2 significantly increased the density of capsaicin-evoked inward currents. LY294002 (10 μM) almost completely blocked CCL2 enhancement of capsaicin currents, and U0126 (20 μM) failed to affect CCL2 potentiation of capsaicin currents. Each bar shows the mean ± standard error (SE) value of 10 to 13 neurons. ( C ) RT-PCR assays showed that CCL2 (5 nM) pretreatment significantly increased the transient receptor potential vanilloid receptor 1 (TRPV1) mRNA level of cultured DRG neurons. In the presence of 10 μM LY294002, CCL2 pretreatment of DRG sensory neurons for 24 to 36 hours failed to upregulate TRPV1 mRNA expression. Each bar represents the mean ± SE value of five experiments. * P <0.01 compared with control neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 enhances capsaicin currents and upregulates TRPV1 mRNA expression in neurons via activating Akt. ( A ) In the presence of Akt inhibitor IV (1 μM), chemokine CC chemokine ligand 2 (CCL2) (5 nM) pretreatment did not augment the magnitude of capsaicin (0.3 μM)-evoked inward current. Holding potential (V H ) = −60 mV. ( B ) Akt inhibitor IV significantly blocked CCL2 upregulation of density of capsaicin currents. Each bar shows the mean ± standard error (SE) value of 10 neurons. ( C ) CCL2 pretreatment of dorsal root ganglion (DRG) neurons for 24 to 36 hours failed to increase transient receptor potential vanilloid receptor 1 (TRPV1) mRNA level in the presence of 1 μM Akt inhibitor IV. Each bar represents the mean ± SE value of five experiments. * P <0.01 compared with control neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 increases the density of tetrodotoxin-resistant sodium currents in small dorsal root ganglion sensory neurons. ( A ) In the presence of 0.5 μM tetrodotoxin (TTX), the holding potential (V H ) of small dorsal root ganglion (DRG) sensory neurons was held at −80 mV, and depolarizing steps (50 milliseconds) were applied from −50 mV to 50 mV with an increment of 10 mV. The I–V (current–voltage) curve of TTX-insensitive Na + currents was then obtained from control or chemokine CC chemokine ligand 2 (CCL2; 5 nM)-pretreated small-diameter DRG neurons. Each point represents the mean ± standard error (SE) value of 10 neurons. ( B ) Traces of TTX-resistant sodium currents were evoked from a V H of −80 mV to step potentials ranging from −20 mV to 20 mV. Compared with a control small DRG sensory neuron, the magnitude of TTX-insensitive Na + currents was greatly increased in a CCL2-pretreated small-diameter DRG neuron. ( C ) CCL2 pretreatment increased the density of TTX-resistant sodium currents recorded at −10 mV in a dose-dependent manner. Each point shows the mean ± SE value of eight neurons. ( D ) Pretreating cultured DRG neurons with 5 nM CCL2 for 24 to 36 hours significantly upregulated mRNA expression of Na v 1.8 without affecting the Na v 1.9 mRNA level. Each bar represents the mean ± SE value of five experiments. * P <0.01 compared with control neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, Cell Culture, Expressing

Journal: Journal of Neuroinflammation

Article Title: CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Na v 1.8 sodium channels in dorsal root ganglion neurons

doi: 10.1186/1742-2094-9-189

Figure Lengend Snippet: CCL2 increases tetrodotoxin-resistant Na + current amplitude and Na v 1.8 mRNA through activating the phosphatidylinositol-3 kinase/Akt pathway. ( A ) Tetrodotoxin (TTX)-insensitive sodium currents were evoked from a holding potential (V H ) of −80 mV to step potentials ranging from −20 mV to 20 mV. In the presence of ERK 1/2 inhibitor U0126 (20 μM), chemokine CC chemokine ligand 2 (CCL2) preincubation greatly increased the magnitude of TTX-resistant Na + currents in a small dorsal root ganglion (DRG) sensory neuron. Pretreating small-diameter DRG neurons with 5 nM CCL2 did not augment the amplitude of TTX-resistant sodium currents in the presence of phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 (10 μM) or Akt inhibitor IV (1 μM). ( B ) Pretreating small-diameter DRG neurons with 5 nM CCL2 increased the density of TTX-insensitive sodium Na + currents recorded at −20 mV. PI3K inhibitor LY294002 or Akt inhibitor IV almost completely inhibited CCL2 enhancement of TTX-resistant Na + currents. Each bar shows the mean ± standard error (SE) value of 10 neurons. ( C ) Pretreating DRG neurons with 5 nM CCL2 for 24 to 36 hours significantly increased the mRNA level of Na v 1.8. PI3K inhibitor LY294002 or Akt inhibitor IV significantly inhibited CCL2 upregulation of Na v 1.8 mRNA expression. Each bar represents the mean ± SE value of five experiments. * P <0.01 compared with control neurons.

Article Snippet: Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by streptozotocin (STZ) administration. Non-diabetic control mice received vehicle (Veh). A REDD1 protein was evaluated in kidney cortical tissue homogenates by western blotting. Representative blots are shown. Molecular mass in kDa is indicated at right of each blot. B Correlation between fasting blood glucose and urine ACR is shown for REDD1 +/+ mice ( blue ; Pearson r = 0.72; p < 0.0001) and REDD1 −/− mice ( red ; Pearson r = 0.55; p = 0.029). C Ccl2 mRNA expression was quantified in kidney homogenates by qPCR. D CCL2 protein abundance was quantified in kidney homogenates by western blotting. E Il1b mRNA expression was quantified in kidney homogenates by qPCR. F IL-1β protein levels were determined in kidney homogenates by western blotting and quantified by ELISA. Individual data points are plotted with values presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh; # p < 0.05 versus REDD1 +/+ . n.d., not detected.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by administration of streptozotocin (STZ). Non-diabetic control mice received vehicle (Veh). A Nuclear isolates were prepared from kidney homogenates. NF-κB and Lamin B were examined in nuclear isolates by western blotting and NF-κB activity was quantified by DNA-binding ELISA. Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. B REDD1 ( red ) and Nephrin ( green ) were visualized in kidneys by immunofluorescence microscopy. White box indicates area shown at increased magnification. Representative micrographs are shown (scale bar 50 μm). C – I Wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol (OC) for 48 h. NF-κB phosphorylation at S536 and REDD1 protein abundance was determined in cell lysates by western blotting ( C ). Nuclear localization of NF-κB p65 ( white arrowheads ) was evaluated by immunofluorescence ( D ). Nuclei were visualized with DAPI (scale bar 25 μm). NF-κB activity was measured in lysates from cells expressing NF-κB firefly luciferase/ Renilla luciferase reporter plasmids by dual luciferase assay ( E ). Relative expression of IL1B and CCL2 mRNA were determined by qPCR ( F ). IL-1β secreted into culture media was determined by ELISA ( G ). Chromatin immunoprecipitation (ChIP)-PCR analysis was carried out in WT and REDD1 KO podocytes to determine binding of p65 NF-κB to the promoter region of the CCL2 gene ( H ). CCL2 protein levels were determined in cell lysates by western blotting ( I ). J NF-κB p65 phosphorylation and NF-κB luciferase reporter activity was evaluated in REDD1 KO cells expressing either an empty vector control (EV) or hemagglutinin (HA)-tagged REDD1. Individual data points are presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh or NG; # p < 0.05 versus REDD1 +/+ , WT, or EV.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Knock-Out, Phospho-proteomics, Quantitative Proteomics, Expressing, Luciferase, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Differentiated wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol as an osmotic control (OC) for 48 h. Transwell migration assay was used to evaluate chemotaxis in a co-culture model with CIHP-1 and THP-1 macrophages ( A ). Macrophages were stained with crystal violet and cells that migrated across the Transwell were counted ( B ). C Cre-lox recombination was used to achieve conditional podocyte-specific REDD1 knockout (REDD1 PodKO). D – H Diabetes was induced in REDD1 fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic groups were administered a vehicle (Veh) control. All assessments were performed after 16 weeks of diabetes. Urine albumin to creatinine ratio (ACR) was determined (D). Kidney sections from diabetic and non-diabetic mice were immunolabeled for REDD1 ( red ) and the podocyte marker Nephrin ( green ) ( E ). Protein abundance of CCL2 was determined in renal homogenates by western blotting ( F ). Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. Kidney sections were immunolabelled for F4/80 ( red ) and nuclei were counterstained with Hoechst 33342 ( blue ) ( G ). Representative micrographs (scale bar 50 µm) are shown. Immune cell populations of CD11b + F4/80+ macrophages ( H ) and CD86 + M1 macrophages ( I ) were determined by flow cytometry. Individual data points are plotted. Significance was analyzed by two-way ANOVA and pairwise comparisons were made using the Tukey’s test for multiple comparisons. * p < 0.05 versus OC or Veh; #, p < 0.05 versus WT or REDD1 fl/fl .

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Knock-Out, Control, Transwell Migration Assay, Chemotaxis Assay, Co-Culture Assay, Staining, Immunolabeling, Marker, Quantitative Proteomics, Western Blot, Flow Cytometry