Journal: Scientific Reports

Article Title: Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory

doi: 10.1038/s41598-020-77380-9

Figure Lengend Snippet: SAGs regulate pro-inflammatory signaling networks in mouse and human FLS. ( A ) Venn diagram showing the overlap between genes assigned to ATAC-seq and DNase-seq peaks in short-term (16 h) and chronic-TNF conditions with that of mSAGs identified from Fig. A. Overlapping SAGs are shown in blue. ( B ) Venn-diagram showing the overlap between gene upregulated by > twofold in human RA-FLS treated with 10 ng/mL recombinant TNF for 8 h and 24 h with that of 141 mSAGs identified in Fig. 3A. 52 overlapping human SAGs are shown in blue. ( C ) Heatmap of hSAGs. Each box represents one gene and is color-coded based on normalized expression levels. ( D ) Functional protein–protein interaction between hSAGs by STRING analysis. Proteins interacted with RELB are highlighted. ( E ) Top 10 transcriptional factors predicted to regulate hSAGs expression identified by ChEA3 online tool.

Article Snippet: Mice fibroblast cells (FLS) treated with 5 ng/mL recombinant human TNF (R&D Systems) for 16 h were harvested at 95% confluence.

Techniques: Recombinant, Expressing, Functional Assay

Journal: Scientific Reports

Article Title: Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory

doi: 10.1038/s41598-020-77380-9

Figure Lengend Snippet: SRGs regulate BMP signaling in mouse and human FLS. ( A ) Venn diagram showing the overlap between genes assigned to ATAC-seq and DNase-seq peaks in short-term (16 h) and chronic-TNF conditions with that of mSRGs identified from Fig. B. Overlapping SRGs are shown in red. ( B ) Venn-diagram showing the overlap between genes downregulated by > twofold in human RA-FLS treated with 10 ng/mL recombinant TNF for 8 h and 24 h with that of 34 mSAGs identified in Fig. 5A. 7 overlapping human SRGs are shown in red. ( C ) Heatmap of hSRGs. Each box represents one gene and is color-coded based on normalized expression levels. ( D ) Functional protein–protein interaction between hSRGs by STRING analysis. ( E ) Top 5 transcriptional factors predicted to regulate hSAGs expression identified by ChEA3 online tool.

Article Snippet: Mice fibroblast cells (FLS) treated with 5 ng/mL recombinant human TNF (R&D Systems) for 16 h were harvested at 95% confluence.

Techniques: Recombinant, Expressing, Functional Assay

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence

Journal:

Article Title: The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-09-4380

Figure Lengend Snippet: CD200R1+ stromal cells are CD11b+/Gr-1+ MDSCs. A, CD200R1 immunofluorescence in WD SCC, LN Met and Lung Met (white arrows). DAPI counterstain was conducted to visualize nuclei. Dashed line demarcates SCC keratinocytes in LN Met. B, For CD200R1 FACS analysis of CD200R1, CD11b, and Gr-1 or MHC II expression. The total population of CD200R1+ cells were gated and subsequently analyzed for expression of CD11b and Gr-1 or MHC class II. The percentage of the total CD200R1 pool for a single experiment is shown. Far right panel: murine SCC were stained with antibodies against CD200R1 (red) and CD11b (green) and counterstained with DAPI (blue). Arrows point to CD200R1+/CD11b+ (yellow) stromal MDSCs. C, Left: Bar graph showing the densitometric units representing GM-CSF and G-CSF levels in pRS-NS Lung Met/CD200R1+ co-cultures versus pRS-CD200 Lung Met/CD200R1+ co-cultures. *- statistically significant difference (GM-CSF, p = 0.049; G-CSF, p = 0.002). Right: H&E staining (top left) and G-CSF (red) and CD200 (green) immunofluorescence in murine PD SCC. Nuclei were delineated with DAPI (blue). Scale bars mark 50µm.

Article Snippet: Antibodies Antibodies were used against human CD200, mouse CD200, CD3ε, CD86, α6 integrin-FITC (BD Biosciences); NK1.1-488, c-kit, CD123, MHC II, CD11b-FITC, CD11b-PE, Gr-1-FITC, Gr-1-PE, CD11c (BioLegend); Langerin, Foxp3 (eBioscience); Keratin 14 (Covance), CD200R1 (R&D Systems), CD4-APC (Abcam), G-CSF (Santa Cruz), anti-rabbit HRP (Jackson Immunoresearch); species-specific Alexa Fluor-488, -594, -647, and -680 (Invitrogen); and human α6 integrin (kindly provided by Fiona Watt).

Techniques: Immunofluorescence, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: A, B, Prevention and reversal of paclitaxel-induced hyperalgesia by intrathecal injection of a TRPV1 antagonist (AMG9810). The baseline (BL) behavioral test in A and B were collected before paclitaxel (Pac) or vehicle (Veh) treatments. In A, the gray shading indicates the time of treatment with 15 μg of AMG9810 (intrathecal) or vehicle solution. In B, paclitaxel-induced mechanical hypersensitivity was confirmed as significant from Veh–Veh-treated rats (open squares, n = 5) at 14 d after treatment (P) in two groups (open and filled circles, n = 5 each); rats were then treated with 15 μg of the TRPV1 antagonist AMG9810 intrathecally (filled circles) or vehicle solution (open circles) as indicated by the arrow. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test. The representative image in C shows the baseline staining of TRPV1 (red) in the DRG in vehicle-treated rats that was not different from naive rats (data not shown); TRPV1 staining becomes elevated by day 7 after paclitaxel treatment (D). Cotreatment of rats with PBS (intrathecally) with paclitaxel did not affect the increased staining of TRPV1 (E), whereas cotreatment with LPS-RS (intrathecally) completely prevented the increase in TRPV1 (F). As indicated by the bar graphs in G, the increase in TRPV1+ neurons by paclitaxel was statistically significant (**p < 0.01), whereas in H, this was significantly less in the LPS-RS-treated rats versus the PBS-treated rats. Scale bar, 100 μm. **p < 0.01.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Injection, Staining

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 is colocalized with TLR4 in DRG neurons and afferent terminals in the spinal cord. TRPV1 alone is shown in red in the left column for DRG neurons in A and in spinal terminals in D. TLR4 staining in subsets of DRG neurons is shown in green in B and spinal terminals in E (center column). Colocalization of the two are shown in the merged image by yellow for DRG in the right column (C), as well as in fiber profiles in the superficial spinal dorsal horn (F). Scale bar, 100 μm. The representative recording in G shows that acute application of paclitaxel (12.5 μm) evoked spontaneous action potentials in subsets of DRG neurons from animals treated with paclitaxel. In H and I, representative action potential waveforms for the neuron in G evoked by direct current injection after 5 min of vehicle treatment and then after 5 min of acute perfusion with 12.5 μm paclitaxel are shown. The bar graphs in J show the group data for the effects of paclitaxel on several action potential properties. AP, Action potential; RMP, resting membrane potential; AHP, after-hyperpolarization; AHPxx%, interval to each percentage of maximal amplitude. *p < 0.05; **p < 0.01; ***p < 0.001 paclitaxel versus vehicle; paired t test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Staining, Injection

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 sensitization by paclitaxel and LPS in DRG neurons shown using calcium imaging. Representative calcium imaging results of change in 340/380 ratio in dissociated DRG neurons after perfusions of capsaicin (CAP, 200 nm) alone and in combination with paclitaxel (12.5 μm) or paclitaxel plus LPS-RS (2 μg/ml) are shown in A–D, each colored line is a single neuron and the time of each application is indicated by the bars over the traces. The bar graphs show the grouped results for experiments testing the interactions between paclitaxel on the responses to capsaicin (E–G) on the effects of LPS on DRG neurons alone (H) and the effects of LPS on DRG neurons from vehicle- and paclitaxel-treated rats (I). **p < 0.01; ***p < 0.001, vehicle rats versus paclitaxel rats; ###p < 0.001, CAP+Pac versus CAP+Pac+LPS-RS.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Imaging

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Interactions between TLR4 and TRPV1 in HEK 293 cells studied using whole-cell patch clamp. Inward currents were recorded in HEK293 cells transfected with TLR4 only (top line), TRPV1 only (second line), and both TLR4 and TRPV1 (line 3). The bar graphs at the bottom show the summarized responses with statistical differences determined by paired t tests. Capsaicin (200 nm) did not induce inward currents in cells expressing TLR4 alone, whereas the responses to repeated capsaicin showed desensitization in cells expressing TRPV1 alone. LPS (10 ng/ml) and paclitaxel (12.5 μm) sensitized the responses to capsaicin in cells expressing both TLR4 and TRPV1. *p < 0.05, LPS+capsaicin; ***p < 0.001, paclitaxel+capsaicin; *p < 0.05 vehicle solution+capsaicin versus first capsaicin response.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Patch Clamp, Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Representative examples of whole-cell recordings for substantia gelatinosa (SG) neurons before (left) and after (right) administration of the TRPV1 antagonist AMG9810 (5 μm) in the vehicle-treated group (A, B), day 7 paclitaxel-treated group (C, D), and day 14 paclitaxel-treated group (E, F) show increased sEPSCs only in the day 7 paclitaxel-treated group that is suppressed by the AMG9810. The washout segment is not shown. Bar graphs in G and H summarize the mean (±SEM) change in amplitude (G) and frequency (H) of sEPSCs before and after 5 min of AMG9810 application. **p < 0.01, day 7 paclitaxel-treated group versus vehicle and day 14 paclitaxel-treated groups; two-way ANOVA followed by Newman–Keuls post hoc test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Paclitaxel application increased mEPSC frequency in superficial dorsal horn neurons in rat spinal cord slice. A, Native recording of mEPSC activity before and after paclitaxel (50 nm) application. B, The TRPV1 antagonist SB366791 (10 μm) did not change the mEPSC frequency but prevented its increase during coapplication with paclitaxel. C, Averaged responses demonstrate that paclitaxel treatment induced a significant increase in mEPSC frequency compared with the baseline (control, 100%) value (140.7 ± 11.1%; n = 14). This increase was prevented by the TRPV1 antagonist (SB366791+paclitaxel) treatment, whereas the antagonist alone had no effect (SB366791; n = 10). D, Paclitaxel (50 nm) application did not change the frequency or amplitude of the sEPSCs or the amplitude of the dorsal root sEPSCs (E). ***p < 0.001 versus control values; ###p < 0.001 versus paclitaxel; one-way ANOVA followed by Student–Newman–Keuls test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Immunofluorescent double staining shows that TRPV1 (red, A) is colocalized with TLR4 (green, B) in human DRG neurons (yellow in merged image at right, C). The red arrows in the merged image indicate cells only showing TRPV1, the green arrows indicate cells only expressing TLR4, and the yellows arrow points to cells positive for both TRPV1 and TLR4. Scale bar, 200 μm. Three types of responses were observed when human DRG neurons were tested by application of capsaicin (Cap) and paclitaxel (Pac). Type 1 neurons did not respond to either capsaicin or paclitaxel (data not shown). Type 2 neurons (D) responded positively to capsaicin (left column), showed no responses to paclitaxel (center column), and then showed desensitization to a second application of capsaicin (right column). Type 3 neurons (E) showed responses to capsaicin (left column) and to paclitaxel (center column) and then showed a facilitation of response to the repeated application of capsaicin (right column). The bar graphs at the bottom show the summarized response for the type 2 and 3 neurons. The baseline response to capsaicin was not different between groups. The second response to capsaicin was significantly reduced compared with the first in the type 2 neurons. The type 3 neurons showed significantly greater responses to paclitaxel than did type 2 neurons; and type 3 neurons showed a significantly increased response to the second application of capsaicin compared with the first. *p < 0.05; Mann–Whitney U test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Double Staining, Expressing, MANN-WHITNEY