goat hsp27 dy1580 dy1580 goat dy1580 rabbit note  (R&D Systems)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet:

Article Snippet: Macrophages (with or without siRNA transfection) were pretreated with citrulline or recombinant human HSP27 (1580-HS-050, R&D Systems) for 30 minutes and incubated with toxin B for 6 hours.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet: Citrulline promoted IL-10 expression with HSP27 phosphorylation in toxin B-treated macrophages. ( A–B ) Phospho-kinase array. Primary human macrophages were treated with or without citrulline for 30 minutes, followed by either PBS or toxin B for 2 hours. The cells were lysed, and the lysates were assayed with the protein Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems). The images were captured by the Bio-Rad ChemiDoc Imaging system and analyzed by Bio-Rad Image Lab software. The cell lysates were collected for the protein arrays. Citrulline increased phosphorylated HSP27 levels in toxin B-treated macrophages. Results were pooled from 4 experiments (mean ± SD). One-way ANOVA was used. ( C ) Phosphorylated HSP27 levels. The fresh human colonic explants were pretreated with 10 μM citrulline for 30 minutes, followed by incubation with 0.1 mg/mL toxin B for 2 hours. The phosphorylated HSP27 levels were determined by ELISA. Results were pooled from 4 tissue donors (mean ± SD). One-way ANOVA was used. ( D ) Total HSP27 levels. The fresh human colonic explants were pretreated with 10 μM citrulline for 30 minutes, followed by 6 hours of incubation with 0.1 μg/mL toxin A or toxin B. Total HSP27 levels in conditioned media were measured by ELISA. Results were pooled from 4 tissue donors (mean ± SD). One-way ANOVA was used, but no statistically significant difference was found. ( E–F ) Macrophages were transiently transfected with control siRNA (sc-37007) or HSP27 siRNA (sc-29350) from Santa Cruz Biotechnology overnight. ( E ) Total HSP27 ELISA. After overnight transfection, the macrophages were incubated with serum-free RPMI1640 media for 24 hours. Secreted total HSP27 levels of transfected macrophages were measured by ELISA. Results were pooled from 4 experiments (mean ± SD). One-way ANOVA was used. ( F ) IL-10 ELISA. Macrophages (with or without siRNA transfection) were pretreated with citrulline or recombinant human HSP27 (1580-HS-050, R&D Systems) for 30 minutes and incubated with toxin B for 6 hours. IL-10 levels in conditioned media were detected by ELISA. Results were pooled from four experiments (mean ± SD). One-way ANOVA was used.

Article Snippet: Macrophages (with or without siRNA transfection) were pretreated with citrulline or recombinant human HSP27 (1580-HS-050, R&D Systems) for 30 minutes and incubated with toxin B for 6 hours.

Techniques: Expressing, Phospho-proteomics, Imaging, Software, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Control, Recombinant

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Citrulline Inhibits Clostridioides difficile Infection With Anti-inflammatory Effects

doi: 10.1016/j.jcmgh.2025.101474

Figure Lengend Snippet: Information on Fresh Human Colonic Tissues, Primary Macrophages, PBMCs, and Reagents

Article Snippet: Macrophages (with or without siRNA transfection) were pretreated with citrulline or recombinant human HSP27 (1580-HS-050, R&D Systems) for 30 minutes and incubated with toxin B for 6 hours.

Techniques:

Journal: bioRxiv

Article Title: Cellular mechanisms of early brain overgrowth in autistic children: elevated levels of GPX4 and resistance to ferroptosis

doi: 10.1101/2025.01.30.635706

Figure Lengend Snippet: (A) Representative flow cytometric histograms of ferrous iron (Fe 2+ ) accumulation measured using a Far-red Labile Fe 2+ Dye in TD-N, ASD-N, and ASD-DM NPCs. (B) Quantification of the mean fluorescence intensity of Fe 2+ accumulation in NPCs from TD-N, ASD-N, and ASD-DM NPCs. (C) Representative flow cytometric histograms of cellular reactive oxygen species (cROS) measured using a CELLROX Deep Red reagent in TD-N, ASD-N, and ASD-DM NPCs. (D) Quantification of the mean fluorescence intensity of cROS levels in TD-N, ASD-N, and ASD-DM NPCs. (E) Western blot analysis of p-HSP27 and GPX4 proteins in TD-N, ASD-N, and ASD-DM NPCs. GAPDH serves as a loading control. (F) Quantification of protein expression levels of p-HSP27 and GPX4 in TD-N, ASD-N, and ASD-DM NPCs, normalized to GAPDH levels. (G) Quantification of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) using a Luminescent-Based Assay in TD-N, ASD-N, and ASD-DM NPCs. (H) Quantification of the percentage of viable NPCs from TD-N, ASD-N, and ASD-DM conditions after 48h treatment with FIN56 at increasing doses (0, 0.1, 0.5, 1, 2.5, 5 μM), measured using the MTS assay. Data are expressed relative to the corresponding untreated cell lines. (I) Representative electron microscopic images of TD-N, ASD-N, and ASD-DM NPCs. TD-N NPCs treated with 5 μM FIN56 for 20h were used as a positive control for ferroptosis, and the TD-N NPCs treated with 1 μM staurosporine for 3h were used as a positive control for apoptosis. Top row scale bars, 2 μm. Bottom row scale bars, 600 nm. White arrows: shrunken mitochondria. Red arrows: chromatin condensation and stress granules. (J) Quantification of mitochondrial area of 5 mitochondria per cell for a total of 10 cells from each sample (total of 50 mitochondria per cell line; 200 mitochondria per condition). (K) Quantification of mitochondrial cristae area of 5 mitochondria per cell for a total of 10 cells from each sample (total of 50 mitochondria per cell line; 200 mitochondria per condition). Individual crista areas were summed up for the total cristae surface area of each mitochondrion. (L) All data are presented as mean ± standard deviation (*p ≤ 0.05; **p < 0.01; ***p < 0.001; **** p <0.0001). See also Figure S5, S6, and S7.

Article Snippet: The primary antibodies assessed include: Rabbit-anti-PAX6 (1:2000; BioLegend); Apoptosis Western Blot Cocktail (1:250; Abcam); Rabbit-anti-phospho-HSP27 (1:1000; R&D Systems); Rabbit-anti-GPX4 (1:1000; Cell Signaling); Rabbit-anti-gamma H2A.X (1:100000; Abcam); Necroptosis antibody sampler kit (1:1000; Cell Signaling); Rabbit-anti-GAPDH (1:1000; Cell Signaling) and Rabbit-anti-β-actin (1:1000; Cell Signaling).

Techniques: Fluorescence, Western Blot, Control, Expressing, Luminescence Assay, MTS Assay, Positive Control, Standard Deviation

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC chemoresistance through TAMs. A Schematic presentation of the analyzed tumor models, Vehicle or HSP27-shRNA OSCC cells were orthotopically injected into BALB/c nude mice. B - C Tumor pictures ( B ) and growth analysis ( C ) over 21 days show the mean tumor volume at the indicated time points following implantation, statistical significance was determined by 2-way ANOVA. Volume of individual tumors at day 21 is shown in the box plot; statistical significance was determined by Welch t-test. D - E HSP27 mRNA ( D ), protein ( E ) levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR and Western blotting in the whole-cell lysates. F Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR. G Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by RT-qPCR. H Relative cell apoptosis level in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Flow Cytometry. I Relative cell apoptosis level in CAL27 cells without or with rhHSP27 (2μg/mL) induced TAMs-CM, detected by Flow Cytometry. J Analysis of ( H ), determined with relative ratio of apoptosis in total per 10 4 cells. K Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Western blotting in the whole-cell lysates. L Analysis of ( I ), determined with relative ratio of apoptosis in total per 10 . 4 cells. M Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by Western blotting in the whole-cell lysates. N – O Analysis of ( K ) and ( M ). T-tests and two-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: shRNA, Injection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC invasion, migration, and EMT through TAMs. A GSEA of transcriptome data cells shows enrichment of a gene signature associated with increased metastasis in HNSCC. B The correlation between the expression of HSP27 and the metastasis pathway enrichment score. C The EMT pathway enrichment score of HSP27 and TLR4. D Representative invasive (upper) and migratory (lower) images of the transwell assays using CAL27 cells (upper chamber) cocultured with TAMs (lower chamber) and preincubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). E Representative images of the wound-healing assays using CAL27 cells under the stimulation of TAMs-CM from TAMs preincubated with rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). F – H statistical analysis of cell invasion ( F ), migration ( G ), and stretch ( H ). I Representative immunofluorescence staining images showing the colocation of HSP27 and TLR4 on the cell membranes of the RAW 264.7 cells incubated with rhHSP27 (2 µg/mL) in the TCM of the CAL27 cells. Nuclei were counterstained with DAPI. J - L N-cadherin, Vimentin mRNA and N-cadherin protein levels in the CAL27 cells under stimulation with the TAMs-CM from the TAMs incubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM), detected by RT-qPCR and Western blotting. One-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: Migration, Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: HSP27/IL-6 establishes a positive feedback loop between OSCC cells and M2 TAMs. A - B iNOS, IL-12, TNF-α, Arg-1, IL-10, and IL-6 mRNA levels in RAW264.7cells under stimulation with the TCM of the CAL27 cells pretreated without or with HSP27–siRNA, detected by RT-qPCR. C - D iNOS, IL-12, Arg-1, CD163, and IL-6 mRNA levels in RAW264.7cells pretreated without or with rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR. E – F IL-12, Arg-1, and IL-6 mRNA and IL-6 protein levels in RAW264.7cells pretreated without or with TAK-242 (1 µM) under the stimulation of rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR ( E ) and ELISA ( F ). G - H IL-6 mRNA, protein levels in THP-1 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( G ) and ELISA ( H ). I - J IL-6 mRNA, protein levels in THP-1 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( I ) and ELISA ( J ). K - L IL-6 mRNA, protein levels in RAW264.7 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( K ) and ELISA ( L ). M – N ), IL-6 mRNA, protein levels in Raw264.7 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( M ) and ELISA ( N ). O - P HSP27 mRNA levels in CAL27 cells without or with IL-6 (100 ng/mL) treated ( O ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( P ), detected by RT-qPCR. ( Q - S ), HSP27protein levels in CAL27 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( Q ) and ELISA ( R - S ). T - U HSP27 mRNA levels in SCC9 cells without or with IL-6 (100 ng/mL) treated ( T ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( U ), detected by RT-qPCR. V-X HSP27 protein levels in SCC9 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( V ) and ELISA ( W - X ). T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot