Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 2. Ex vivo analysis of hPRL gene promoter activation. A, qPCR of hPRL promoter activity of PE , BM cells, and PBMC in T24 and S24 rats. A minimum of four samples from each treatment group was assayed in duplicate. Data are presented as mean SEM; *, P 0.05. B, Luciferase activity of PE and BM cells isolated from S24 and T24 rats. ***, P 0.001. C, Ex vivo bioluminescent imaging of FITC-labeled, CD11b adherent cells from the PE, BM, and PBMC of S24 transgenic rats. Scale bar, 50 m. D, Comparison of bioluminescence between PE cells from a S24 rat and a T24 rat. S24, saline 24 h; T24, TG 24 h; RLU, Relative luminescence units. Scale bar, 100 m.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Activation Assay, Activity Assay, Luciferase, Isolation, Imaging, Labeling, Transgenic Assay, Comparison, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 3. Identification of subpopulations of CD11b cells expressing luciferase in the PE. A, FACS analysis of cells isolated from PE of S24 rats: two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11B/ssclow. Diff-Quick staining of the corresponding sorted cell populations (right panels). Small black arrow, Mast cell, dark purple granules; small white arrow, eosinophils, red granules; large white arrow, B-cell, small cell with little cytoplasm; large black arrow, macrophage. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 CD11b/ssclow population (n 5). C, FACS analysis of cells isolated from PE of T24 rats. Two-dimensional dot plot (ssc vs. fsc), representing three populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11b/Gra; pink, CD11b/Gra. Diff-Quick staining of the corresponding sorted cell populations (right panels). Scale bar, 50 m. D, qPCR quantification of luciferase transcript in the T24 CD11b/Gra (n 3) and CD11b/Gra (n 5) populations. E, RT-PCR for detection of extrapituitary luciferase and endogenous rPRL gene expression. In addition to the expected 122-bp product, a larger band (260 bp) was produced by alternative splicing as described previously (31). M, marker; 1, Luciferase from T24 CD11b/Gra; 2, rPRL from T24 CD11b/Gra; 3, Luciferase from T24 CD11b/Gra; 4, rPRL from T24 CD11b/Gra; 5, rPRL on pituitary (control); S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Produced, Alternative Splicing, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 4. Subpopulations of CD11b cells expressing luciferase in BM and PBMC. A, FACS analysis of cells isolated from BM of T24 rats. Two- dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh (Gra); green, CD11b/ssclow (Gra). Diff-Quick staining of corresponding sorted cell populations (right panels). Black arrow, Monocytes; white arrow, neutrophils. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 6) CD11b/ssclow population in BM. C, RT-PCR for the detection of extrapituitary luciferase and endogenous rPRL gene expression. M, Marker; 1 and 3, Luciferase on T24 CD11b/sschigh; 2 and 4, rPRL on T24 CD11b/sschigh; 5, rPRL on pituitary (control). D, FACS analysis of cells isolated from PB of T24 rats. Two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/Gra and green, CD11b/Gra. E, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 5) CD11b/Gra and T24 CD11b/Gra (n 5 in each group). S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 7. Ex vivo stimulation of PRL expression in human peripheral blood monocytes. A, Peripheral blood monocytes stained with CD11b antibody (left panel). Diff-Quick stain of CD11b cells to confirm monocytes morphology. Scale bar, 50 m. B and C, RT-PCR analysis of two distinct donors, showing the bad pattern of extrapituitary hPRL gene expression. D and E, qPCR of hPRL extrapituitary gene expression in human CD11b PBMC of two donors. Cells were treated with TNF- (TNF), LPS, and TG for 16 h, then analyzed for hPRL gene expression. CycloA, Cyclophilin A; Ctrl, no-stimulation control.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Expressing, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder exhibited excellent security by targeting lung fibroblasts based on pY levels in vivo. A , Schematic showing progression of SH2 superbinder treatment in control mice. B , Percent survival during last 7 days after SH2 superbinder injection (n = 8). C , HE staining after SH2 superbinder challenge. Images showing the panoramic and partial view of lungs. D , Neutrophil accumulation measured by MPO assay in lung tissues after GST, GST-SH2 WT, GST-SH2 TrM or SH2-TrM treatment in saline groups (n = 6). E , Total cell counting in BALF (n = 6). F-G , Changes in different kinds of cells by Giemsa and Diff-quick staining in BALF (n = 6). H , Inflammatory cytokines in BALF measured by ELISA (n = 6). I , The determination of liver function index in serum (n = 6). J , Schematic showing progression of SH2 superbinder treatment in BLM-treated mice. K , Western blot of GST tag of different tissues in BLM group treated with GST-SH2 TrM. L , The process of FACS for mice lungs. M , Western blot of pY levels in different lung cells which were isolated by FACS of BLM treated 14 days mice. N , The fluorescence images of SH2 superbinder entering into activated fibroblasts, epithelial cells (EpCAM + ), leukocytes (CD45 + ) and endothelial cells (CD31 + ). O-R , Representative images of SH2 superbinder in myofibroblast (α-SMA + ) ( O ), epithelial cells (EpCAM + ) ( P ), leukocytes (CD45 + ) ( Q ) and endothelial cells (CD31 + ) ( R ) of BLM-treated mice.

Article Snippet: The amounts of IL-1β, IL-6, IL-10 and TNF-α in BALF of mice were measured by using mouse ELISA kits from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vivo, Control, Injection, Staining, MPO Assay, Saline, Cell Counting, Diff-Quik, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation, Fluorescence

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: TNFα is correlated with human oral cancer pain scores. ( a ) TNFα protein concentration is higher in cancer tissues compared to anatomically matched contralateral healthy tissues from the same patient (n = 10, * P < 0.05, paired t-test). ( b ) Patients were asked to answer the Oral Cancer Pain Questionnaire before surgery. The mean pain score from patients correlated positively with percentage change in TNFα concentration between cancer and matched contralateral normal tissues ( r = 0.7, P < 0.05).

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Protein Concentration, Concentration Assay

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Blocking TNFα or JNK inhibits nociception in mice with cancer. ( a ) After 16 weeks of 4NQO treatment, mice exhibited significant increase in gnaw-time from its respective baseline (pre-injection). Propylene glycol (PG) treatment did not affect gnaw-time. In 4NQO tongue cancer mice, C-87 (12.5 mg/kg) IP injection significantly reduced percentage of gnaw-time change from baseline (n = 8) 1 h post-injection than the vehicle (10% DMSO) treated cancer mice (n = 5). C-87 (n = 6) or vehicle (n = 4) had no effect in non-cancer mice treated with PG alone. ( b ) Mice with paw SCC developed cancer pain at PID7. C-87 and the JNK inhibitor SP600125 treatment significantly reduced mechanical nociception compared to vehicle at 1, 3, and 6 h after treatment compared to the control group. 24 h after the treatment the analgesic effect of C-87 was gone (n = 5 per group, Two-way ANOVA). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Blocking Assay, Injection, Control

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Blocking TNFα inhibits cancer cell growth, migration, and cytokine release. ( a ) Growth rate, measured with the RTCA, following different doses of C-87 treatment in HSC-3 cell culture. C-87 inhibited oral cancer cell growth in a dose dependent manner. One-way ANOVA with Tukey's post hoc analysis. ( b ) Mice with C-87 treatment (n = 7) exhibited a significant decrease in the paw volume compared to the vehicle control mice (n = 6) at PID14, 18, and 21 (two-way ANOVA). Arrow indicates C-87 injection. ( c ) C-87 treated paw cancer mice (n = 6) had smaller tumor area relative to the total paw area compared to vehicle treated paw cancer mice (n = 4). Tumor areas and total paw areas were quantified using H&E stained paw sections. Mann–Whitney U-test. ( d ) Representative H&E stained pictures showing a normal mouse paw, a cancer mouse paw, and a cancer paw treated with C-87 (10 × inset). Scale bar: 100 μm. Images were taken and quantified using Nikon imaging software NIS-Elements F Ver4.60.00. ( e ) C-87 treatment reduced the concentration of TNFα, NGF, IL1β, IL4, MIP3α, IL28β, and IL33 in the paw tumor. Data were presented as fold change of cytokines/chemokines measured from tumor paws over normal paws. n = 6 per group. Mann–Whitney U test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Blocking Assay, Migration, Cell Culture, Control, Injection, Staining, MANN-WHITNEY, Imaging, Software, Concentration Assay

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: TNFα mediates Schwann cell proliferation and migration in vitro. ( a ) The presence of either DOK or HSC-3 cells in cell inserts increased Schwann cell proliferation 48 h following co-culture in the MTS assay. Increased Schwann cell proliferation induced by the presence of HSC-3 cells was inhibited by adding C-87 into inserts. Representative images of cells with Hoechst stain were shown under each culture condition. OD: optical density. ( b ) Schwann cells are more migratory in the presence of HSC-3 cells compared to the DMEM control while DOK reduced Schwann cell migration. Adding C-87 into the HSC-3 culture reduced Schwann cell migration. ( c ) Adding TNFα to the media at the bottom chamber increased Schwann cells migration compared to the DMEM control. Neutralizing TNFα with C-87 decreased Schwann cell migration. ( d ) Schwann cells induced increased HSC-3 cell migration compared to the DMEM control; adding C-87 (20 µM) into the Schwann cell culture in the bottom chamber blocked this increase. ( b – d ), images shown are representative diff-quick stained migrated cells. a-d, one-way ANOVA with Tukey's post hoc analysis. SCs: Schwann cells. Scale bar: 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001. Images were taken using Nikon imaging software NIS-Elements F Ver4.60.00.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Migration, In Vitro, Co-Culture Assay, MTS Assay, Staining, Control, Cell Culture, Diff-Quik, Imaging, Software

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: The effect TNFα on the expression of Schwann cell activation markers in vitro. TNFα treatment increased c-Jun ( a , b ), GFAP ( c , d ), and p75 ( e – f ) immunofluorescence intensity and protein expression in cultured Schwann cells compared to the DMEM control. TNFα treatment decreased MBP immunofluorescence intensity and protein expression in cultured Schwann cells compared to the DMEM control ( g – h ). Full-length gel blots were provided in the Supplemental Fig. online. Scale bar: 100 μm. Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Expressing, Activation Assay, In Vitro, Immunofluorescence, Cell Culture, Control

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Activated Schwann cells release increased NGF and TNFα. ( a ) Schwann cells co-cultured with HSC-3 cells overexpressed c-Jun, GFAP, p75 but downregulated MBP compared to control Schwann cells (media alone). Full-length gel blots were provided in Supplemental Fig. online. ( b ) Both DOK and HSC-3 co-cultures increased TNFα mRNA expression in Schwann cells compared to control Schwann cells. ( c ) TNFα protein concentration in Schwann cells co-cultured with either DOK or HSC-3 cells compared to control Schwann cells. ( d ) HSC-3 cell or DRK co-culture increased NGF release in Schwann cells compared with control Schwann cells. ( e ) Adding TNFα in cell culture media stimulated increased NGF release compared with control Schwann cells. One-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Cell Culture, Control, Expressing, Protein Concentration, Co-Culture Assay

Journal: Antioxidants

Article Title: Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma

doi: 10.3390/antiox9070563

Figure Lengend Snippet: Effects of LOL on inflammatory cell number, Th2 cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in OVA-challenged asthma model. ( A ) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. ( B ) The levels of IL-4, IL-5, IL-13, eotaxin, and MUC5AC in the BALF were determined by ELISA. ( C ) The total IgE and OVA-specific IgE in the serum were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD ( n = 7/group). # p < 0.01, significantly different from NC group; †† p < 0.01, significantly different from OVA-challenged group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, IL-5, IL-6, IL-13, and eotaxin (R&D system, Minneapolis, MN, USA), MUC5AC (Cusabio Biotech Co. Wuhan, China) and IgE (BioLegend, CA, USA) were used according to the manufacturer’s instructions.

Techniques: Staining, Diff-Quik, Enzyme-linked Immunosorbent Assay, Control

Journal: Antioxidants

Article Title: Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma

doi: 10.3390/antiox9070563

Figure Lengend Snippet: Effects of LOL on activation of MAPKs/AP-1, p65NF-κB and expression of MUC5AC in OVA-challenged asthma model. The protein levels of ( A – E ) MAPKs/AP-1, ( F ) p65NF-κB and ( G ) MUC5AC in the lung tissues were determined by western blot analysis. β-actin was used to confirm equal protein loading. NC: normal control mice; OVA: OVA-challenged asthma mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged asthma mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged asthma mice. The values are expressed as the means ± SD ( n = 7/group). # p < 0.01, significantly different from NC group; †† p < 0.01, significantly different from OVA-challenged group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, IL-5, IL-6, IL-13, and eotaxin (R&D system, Minneapolis, MN, USA), MUC5AC (Cusabio Biotech Co. Wuhan, China) and IgE (BioLegend, CA, USA) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Western Blot, Control