Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Coomassie blue stained gel demonstrating the purity of IL-36α preparation. Ten µg IL-36α was loaded on the lane. B) Western immunoblotting of IL-36α protein preparation detects a band around 18 KDa, the predicted molecular weight of mouse IL-36α. C) Cytospin preparations demonstrating neutrophil influx in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. D) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. E) Differential cell count percentages and F) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group. G) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c − CD11b + Ly6G + neutrophils. Depicted flow cytometry plots are representative of 4–5 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 4–5 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Staining, Western Blot, Molecular Weight, Cell Counting, Flow Cytometry, Isolation

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. I–K) Protein expression of TNFα, IL-1α, IL-1β and CXCL1 in the BAL fluid recovered from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Total lung resistance and B) lung compliance were not significantly different in the lungs of IL-36α challenged mice compared to PBS controls. Airway responses in mice were measured using invasive plethysmography 24 h following i.t instillation of IL-36α or PBS. Data presented are percentage changes from baseline (0mg/mL) measurements. Data represent mean±SEM from 5–7 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques:

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. B) Differential cell count percentages and C) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 3–4 mice/group. D) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c − CD11b + Ly6G + neutrophils. Depicted flow cytometry plots are representative of 3–4 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 3–4 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Cell Counting, Flow Cytometry, Staining, Isolation

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–F) Transcript expression of early response cytokines (TNFα and IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. G–H) Protein expression of TNFα and CXCL1 in the BAL fluid recovered from IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 3–4 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R as well as the neutrophil specific chemokines CXCL1 and CXCL2 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P<0.05 ) compared to 0 µg/mL group. Data represent mean±SD of quadruplicate samples from one of two representative experiments. I) Diff-quik stained cells from cytospun BAL cells from the lungs of naïve mice demonstrates that the majority of lung resident immune cells are alveolar macrophages. J) PCR on cDNA from naïve mouse alveolar macrophages demonstrating the constitutive mRNA expression of an endogenous control (β-actin), IL-1R1, IL-36R and IL-1RAcP. Image of a DNA electrophoresis gel has been color-inverted for clarity. bp – base pairs.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Diff-Quik, Staining, Control, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–C) Transcript expression of the co-stimulatory molecules CD80, CD86 and CD40 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P<0.05 ) compared to 0 µg/mL group. Data represent mean±SD from quadruplicate samples from one of two representative experiments. D) Flow cytometric evaluation of splenic CD11c + cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. E) Cell surface expression of co-stimulatory molecules in splenic CD11c + cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. MFI – mean fluorescence intensity. *Indicates significant differences ( P<0.05 ) compared to 0.1 µg/mL group. Data represent mean±SD from triplicate samples from one of two representative experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Fluorescence

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Flow cytometric evaluation of CD4 + T cell proliferation responses induced by IL-36α stimulated splenic CD11c + cells. Splenic CD11c + cells were incubated with increasing doses of IL-36α for 2 h. Following incubation, media containing IL-36α was removed and CFSE-labeled CD4 + T cells were co-cultured with IL-36α stimulated CD11c + cells. CFSE dilution was used to evaluate T cell proliferation responses 96 h following co-culture. CD4 + T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c + cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments. B) Flow cytometric evaluation of antigen-specific CD4 + T cell proliferation responses induced by IL-36α stimulated splenic CD11c + cells. Splenic CD11c + cells were incubated with increasing doses of IL-36α and 100 ng/mL OVA 323-339 for 2 h. Following incubation, media containing IL-36α and the OVA peptide was removed and CFSE-labeled CD4 + T cells from OTII TCR transgenic mice were co-cultured with IL-36α stimulated, OVA peptide pulsed CD11c + cells. CFSE dilution in the culture was used to evaluate T cell proliferation responses 96 h following co-culture. CD4 + T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c + cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Incubation, Labeling, Cell Culture, Co-Culture Assay, Concentration Assay, Flow Cytometry, Transgenic Assay

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Cells from a mouse macrophage NF-κB reporter cell line (RAW-ELAM cells) were stimulated with increasing concentrations of IL-36α. Green fluorescent protein (GFP) expression, indicative of NF-κB activation, was increased in a dose-dependent manner upon incubation with IL-36α. Flow cytometry plot presented is representative of triplicate samples in one out of two independent experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Activation Assay, Incubation, Flow Cytometry

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Identification of a homozygous AMZ2 variant in an individual with teratozoospermia from a consanguineous family. ( a ) Diff-Quik staining of human spermatozoa obtained from one individual with an AMZ2 variant and one healthy control. Most spermatozoa from the individual with the AMZ2 variant showed sperm-head vacuolation. Sperm-head vacuoles are indicated with white triangles. ( b ) Validation of the AMZ2 c.520A>G (p.Thr174Ala) variant in the proband’s pedigree using Sanger sequencing. The mutation (M) was homozygous in the proband (IV-1) and both parents were heterozygous carriers. The double horizontal line indicates the consanguineous union. Squares and circles denote male and female family members, respectively. The proband is indicated with an arrow. I–V represent five generations from the oldest to the youngest. AMZ2 : archaelysin family metallopeptidase 2; M: mutation; WT: wild-type.

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Variant Assay, Diff-Quik, Staining, Control, Biomarker Discovery, Sequencing, Mutagenesis

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Archaelysin family metallopeptidase 2 (AMZ2) variant data for the patient analyzed in this study

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Variant Assay, Mutagenesis

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Effects of the AMZ2 missense mutation on protein level. ( a ) Structural model of wild-type AMZ2 protein (in grey) and the mutant p.Thr (in green) 174Ala (in magenta). Cyan indicates the zinc-binding motif. Dashed lines represent hydrogen bonds. ( b ) Western blot analysis of AMZ2 levels in human spermatozoa from the individual with the AMZ2 variant and a healthy control. AMZ2 was absent in the spermatozoa of the individual with the AMZ2 variant. GAPDH was used as the loading control. ALA: alanine; AMZ2 : archaelysin family metallopeptidase 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ILE: isoleucine; M: mutation; PHE: phenylalanine; THR: threonine; WT: wild-type.

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Mutagenesis, Binding Assay, Western Blot, Variant Assay, 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