rhhsp27  (R&D Systems)

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

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. The fluorescence in one unbleached cell (Ref) was used to correct the artefact loss of fluorescence. Note the fluorescence recovery follows an exponential time course when the bleached cells (circles) are interconnected by open gap-junction channels to unbleached cells (black squares are Ref). The relative permeability of gaps is given by the time constant k . B. Recombinant human HSP27 (rhHSP27) effect on GJIC. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP27 (5 μg/ml). C. Histogram shows k values measured after the rhHSP27 addition for 0, 30 and 60 min (mean ± SD, n = 8; * P < 0.05 vs control [ t = 0 min]). D. Both rhHSP27 and SW480-conditioned media (-CM; collected after 6 h) increase the GJIC in a TLR3-dependent manner. Cells exposure for 30 min, in the absence or the presence of neutralizing anti-TLR3 antibody (20 μg/ml) (mean ± SD, n = 4; * P < 0.01 vs control). E. LPS (1 μM) blocks GJIC within 60 min. This inhibitory effect was prevented by OxPAPC (30 μg/ml), a TLR4/TLR2 inhibitor (mean ± SD, n = 4; * P < 0.01 vs control).

Article Snippet: Low endotoxin rhHSP27 was purchased from Enzo Life Sciences (Villeurbanne, Fr) and rabbit anti-HSP27 from ABR (AffinityBioReagent, ThermoFisher, Fr).

Techniques: Fluorescence, Permeability, Recombinant

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. Immunofluorescence detection of Cx43 in SW480 cells and SW620 cells (Bar 20 μm). The dotted areas are enlarged in the inserts below. Arrows indicated the Cx43 plaques at the plasma membrane in cells. Representative of 5 experiments. B. Western blot of Cx43 in whole cell lysates from SW480 and SW620 cells, exposed or not the HMEC-conditioned media (-CM, collected after 6 h). P0, P1 and P2 denote the three major Cx43 migration bands (Hsc70 as loading control). C. Time-dependent increase in Cx43 phosphorylation induced by the SW480-CM in confluent HMEC. Whole cell lysates in HMEC exposed to SW480-CM (collected after 6 h) for time periods as indicated (Hsc70 as loading control; representative of 3 experiments). D. No change in the phosphorylation state of the endothelial Cx43 was induced by the SW620-CM ( n = 3). E. Serine phosphorylation and Cx43 immuno-precipitate in HMEC exposed to SW480-CM. Note that the Cx43 interaction with the protein 14–3-3 precedes its phosphorylation in serine sites. Data are representative of 3 independent experiments. IP, immunoprecipitation; IB, immunoblot; Input material, total amount of proteins per lane. IgG heavy chain at 55 kDa. F. Neither rhHSP27 nor SW480-CM affect the low amount of CIP75 interacting with Cx43 (representative of 3 experiments).

Article Snippet: Low endotoxin rhHSP27 was purchased from Enzo Life Sciences (Villeurbanne, Fr) and rabbit anti-HSP27 from ABR (AffinityBioReagent, ThermoFisher, Fr).

Techniques: Immunofluorescence, Western Blot, Migration, Immunoprecipitation

Journal: Cancer Research

Article Title: Heat Shock Protein 27 Differentiates Tolerogenic Macrophages That May Support Human Breast Cancer Progression

doi: 10.1158/0008-5472.can-10-1778

Figure Lengend Snippet: Figure 1. Elevated serum Hsp27 levels in breast cancer patients and increased expression and release of Hsp27 in human primary breast tumor cells and cell lines. A, serum samples (n ¼ 32 patients and 26 controls) were tested for Hsp27 by ELISA. B, healthy volunteers’ (normal) primary breast epithelial cells, patients’ breast tumor cells, and normal (HMEC) and breast tumor (MCF-7 and ZR-75-1) cell lines were tested for intracellular expression of Hsp27 by flow cytometry. CD326 (Ep-CAM, an epithelial cell marker) is used for the gating of epithelial cells. Representative data are shown. C, normal primary breast epithelial cells, primary breast tumor cells, and normal and breast cancer cell lines were cultured for 24 hours (5 105 cells/mL), and culture supernates were tested for Hsp27; **, P < 0.01; ***, P < 0.0005; ****, P < 0.0001. D, culture supernates of primary breast tumor cells and cell lines were concentrated and subjected to 4–15% gradient Tris-glycine gel electrophoresis [each containing approximately 10 ng Hsp27 (determined by ELISA) in 30 mL loading volume]. rhHsp27 (20 ng) was loaded as the positive control. Blot was probed with anti-Hsp27 antibody followed by reprobing with anti-phospho-Hsp27 (Ser82) antibody.

Article Snippet: rhHsp27 and phospho-Hsp27 (Ser82) antibody were purchased from Stressgen Laboratories/Enzo Life Science.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Marker, Cell Culture, Nucleic Acid Electrophoresis, Positive Control

Journal: Cancer Research

Article Title: Heat Shock Protein 27 Differentiates Tolerogenic Macrophages That May Support Human Breast Cancer Progression

doi: 10.1158/0008-5472.can-10-1778

Figure Lengend Snippet: Figure 4. Hsp27-differentiated MACs induce anergy in T cells. A, C, and D, monocyte macrophages were cocultured (1:5 ratio) with autologous T cells in the presence of iCD3 for 6 days. T cells were re-isolated, rested for 24 hours, and tested for anergy by restimulation of the T cells (1 105/well) with iCD3þsCD28 in the presence or absence of IL-2 (100 U/mL) for 4 days for assessing proliferation (n ¼ 14; A) and cytokine production (n ¼ 6; B, C; *, P < 0.05; ***, P < 0.0005, compared with adherence alone group; #, P < 0.05; ###, P < 0.0005, compared with M-CSF group; $, P < 0.05, compared with corresponding IL-2–treated group). B, monocytes were cultured in the presence of M-CSF alone or in the presence of ZR-75-1 culture supernate [added to monocyte culture at 20% (v/v) at a final concentration of 100 ng Hsp27/mL, as determined by ELISA]. The tumor culture supernate was also incubated for 1 hour with Hsp27 antibody or control IgG (each 5 mg/mL) before their addition to the monocyte culture. rhHsp27 (250 ng/mL) was also added to the M-CSF group to serve as positive control. Differentiated MACs were then cocultured with autologous T cells, and the proliferation of re-isolated T cells was assessed as described above. Cul Sup, culture supernate.

Article Snippet: rhHsp27 and phospho-Hsp27 (Ser82) antibody were purchased from Stressgen Laboratories/Enzo Life Science.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Positive Control