Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery

doi: 10.1186/s13046-019-1027-0

Figure Lengend Snippet: N-EV and H-EV treatment promote macrophage M2 polarization by delivering miR-21-5p that targets PTEN. a , western blot analysis of PTEN protein expression level in induced macrophages. H/i-miR-EV, monocytes were induced with the presence of EV secreted by miR-21-5p-inhibited, hypoxia pre-challenged MSCs; H-EV + i-miR, monocytes were transfected with miR-21-5p inhibitor-expressing vector before induction with the presence of H-EV. Macrophages induced without MSC-EV were used as negative control (NC). b, c , flow cytometry determining the percentage of CD163 + CD206 + cells among total CD68 + cells after induction. N-EV + O/E PTEN or H-EV + O/E PTEN, monocytes were transfected with PTEN overexpressing vector before N-EV or H-EV treatment, respectively. d – f , western blot detecting Akt and STAT3 protein expression as well as their activating phosphorylation (p-Ser473 for Akt and p-tyr705 for STAT3) in macrophages after induction. g – i , ELISA evaluating IL-10, TGF-β and VEGF-α in macrophage culture medium after induction. Macrophages induced with the presence of N-EV were used as negative control in b – i . Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Article Snippet: Protein level of N-cadherin, E-cadherin, and Vimentin (NBP1–48309, NBP2–19051 and NBP1–31327, respectively, Novus Biologicals), CD9 and CD81 (NBP2–22187 and NB100–65805, Novus Biologicals) Arginase-1 and iNOS (P05089 and MAB9502, R&D Systems), PTEN (4C11A11, BioLegend, San Diego, USA), PDCD4 (NBP2–26138, Novus Biologicals, Littleton, USA), RECK (MA5–14781, Invitrogen), AKT (ab8805, Abcam, Cambridge, USA), STAT3 (NBP2–22471, Novus Biologicals), GAPDH (NB300–221, Novus Biologicals) as well as Ser473 phosphorylation of Akt protein (649,001, BioLegend) and Tyr705 phosphorylation of STAT3 (AF4607, R&D Systems) in cell lysate was analyzed by western blot.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) The transfection efficiency of the siRNA was analysed in OS cells at different time points after transfection using flow cytometry. (B) Western blot analysis of STAT3 and pSTAT3 expression in OS cells treated with ADSC-conditioned medium. OS cells were pretreated with the STAT3 siRNA (50 nM) for 48 h before treatment with ADSC-conditioned medium for an additional 12 h. STAT3 expression levels were detected by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. (C) OS cell invasion was analysed using transwell assay. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (D) The MMP2 and MMP9 levels in the supernatants were detected using ELISAs. (E) OS cells were treated with the STAT3 siRNA or ADSC-conditioned medium, and MMP2/9 and E-cadherin expression in OS cells was examined by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. The results are expressed as mean±SD. Abbreviation: C Control; L Lipofectamine 2000; S siRNA. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: Transfection, Flow Cytometry, Western Blot, Expressing, Transwell Assay, Control

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A-B) Cell proliferation was evaluated using the CCK8 assay and EdU analysis. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (C-D) OS cells were transfected with the STAT3 siRNA or treated with ADSC-conditioned medium, and apoptosis rates were determined using flow cytometry. OS cells treated with Lipofectamine 2000 alone served as negative controls. The percentages of Annexin V-positive cells are presented in bar charts. *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: CCK-8 Assay, Transfection, Flow Cytometry

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) An in vivo imaging system was used to monitor OS xenograft luminescence activity, which represented tumour growth and metastasis. (B) Living Image Software was used to analyse tumour bioluminescence intensity weekly. The quantitation of the normalized image counts is shown. (C) Lungs of the tumour-bearing mice were excised, and the bioluminescence intensity was analysed to determine the level of tumour metastasis in the lungs. (D) Survival curves of the three groups are shown, and the median survival of the OS group was 43 days, which was significantly longer than the survival of the OS + conditioned-medium group (25 days, P<0.01). (E) The immunohistochemical analysis of Ki67, STAT3, MMP2 and MMP9 expression in the orthotopic tumour xenografts is shown. (F) Quantitation of the intensity of Ki67, STAT3 and MMP2/9 staining in the xenografts. Scale bar: 25 μm. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: In Vivo Imaging, Activity Assay, Software, Quantitation Assay, Immunohistochemical staining, Expressing, Staining

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Article Snippet: Biotinylated-anti-galectin-3 (BAF1154) antibody and antibodies against galectin-3 (MAB1154), STAT1 (MAB1490), Phospho-STAT1(MAB2894), STAT3 (MAB1799), Phospho-STAT3 (MAB4934), PYK2 (AF4589), phosphorPYK2 (MAB6210), GSK3α/β (AF2157), Phospho-GSK3α/β (AF1590); Proteome Profiler Human Protease Array (ARY021B), Proteome Profiler Human Phospho-Kinase Array (ARY003C); Human MMP-13 DuoSet ELISA (DY511), Human Cathepsin-B DuoSet ELISA (DY2176) PYK2 inhibitor PF-431396 (4278) and GSK3α/β inhibitor SB 216763 (1616/1) were all purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Activation Assay, Comparison, Control, Phospho-proteomics, Western Blot

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 4. STAT3 is activated in fibroblasts upon keratinocyte growth factor (KGF)–induced oncostatin M (OSM) secretion. Western blot analyis was performed for STAT3 and phosphorylated STAT3 (pSTAT3) using b-actin as a loading control. (a) STAT3 is constitutively expressed in normal fibroblasts (NFs) but phosphorylation only occurs upon direct OSM treatment or treatment with OSM-containing keratinocyte-conditioned media (KCMs) as confirmed by the OSM ELISA in Figure 3c. (b) Direct KGF treatment had no unspecific effect on STAT3 phosphorylation. NFs showed decreased STAT3 phosphorylation when treated with KCM:kFCM:siKGF compared with KCM:kFCM:siCTRL, but increased phosphorylation after treatment KCM:nFCM:KGF compared with KCM:nFCM:mock. (c) pSTAT3 and STAT3 expression status in NFs, keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (d) pSTAT3 and STAT3 expression in NFs treated with rOSM and KCM in the presence or absence of OSM-neutralizing antibodies ( þ antiOSM).

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Western Blot, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 5. STAT3 mediates fibroblast activation. (a) Collagen type I-a1 (ColIa1) expression and (b) fibroblast migration cannot be induced upon oncostatin M (OSM) stimulation when pretreated with the STAT3 inhibitor S3I-201. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (c) The expression of the STAT3 target gene urokinase-type plasminogen activator (uPA) is induced upon direct OSM treatment and upon incubation with KCM (keratinocyte-conditioned medium):kFCM, KCM:sFCM, and KCM:rKGF. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with analysis of variance (ANOVA)þ Kruskal–Wallis test and Mann–Whitney test, respectively. (d) uPA mRNA expression in normal fibroblasts (NFs), keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (e) The induction of fibroblast migration measured upon OSM treatment and incubation with KCM:kFCM or KCM:sFCM is completely suppressed when the uPA expression is decreased by means of small interfering RNA transfection. The graphs in d and e represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (f) The uPA expression is significantly higher in keloid and scleroderma tissue compared with normal skin. The graphs represent mean values of four independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVAþ Kruskal–Wallis test. (g) OSM-induced STAT3-mediated uPA expression can be suppressed by pretreatment with S3I-201. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Migration, MANN-WHITNEY, Incubation, Recombinant, Small Interfering RNA, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Hedgehog Signalling Modulates Immune Response and Protects against Experimental Autoimmune Encephalomyelitis

doi: 10.3390/ijms23063171

Figure Lengend Snippet: Histological and immuno-histochemical analyses of spinal cord cross-sections from ptch +/− and wt mice. MOG 35–55 -inoculated mice were sacrificed on day 20 after inoculation, and spinal cord sections were subjected to haematoxilin-eosin staining ( A , B ) or immuno-histochemical analysis with antibodies against CD11b ( C ), Iba-1 ( D ) and GFAP ( E ) or double staining for GFAP and Stat3 ( F ). Arrows in D point to microglia with typical activated or resting morphologies in wt or ptch +/− samples, respectively. Thick arrows in F indicate double-positive cells with anti-GFAP (brown cytoplasm) and anti-Stat3 (red nuclei), thin arrows show infiltrating immune cells and the asterisk marks polymorphic leukocytes. Magnitude scales are indicated on each micrograph.

Article Snippet: Primary antibodies used for immunohistochemistry analysis were CD11b rabbit monoclonal (Abcam, Cambridge, United Kingdom, Ab133357), GFAP rabbit monoclonal (MilliporeSigma, Burlington, MA, USA, 04-1062), Phospho Stat3 (Tyr705) rabbit polyclonal (Elabscience E-AB-20983), β-galactosidase rabbit polyclonal (Cappel, MP Biomedicals, Fountain Parkway Solon, Ohio, USA, 55976), NG2 rabbit polyclonal (Abcam ab101807), human/mouse, Olig1 mouse monoclonal (R&D systems, Minneapolis, MN, USA, MAB2417), Olig2 Rabbit monoclonal (EPR2673) and Iba-1 goat polyclonal (Abcam Ab5076).

Techniques: Staining, Double Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thymosin β4 Alleviates Autoimmune Dacryoadenitis via Suppressing Th17 Cell Response

doi: 10.1167/iovs.64.11.3

Figure Lengend Snippet: rTβ4 inhibited Th17 immune response via suppressing STAT3 phosphorylation. ( A ) Administration of 1000 µg/mL rTβ4 suppressed the protein level of phosphorylated STAT3 in vivo. ( B ) PBLs induced by pLGECs were pretreated with the STAT3 activator (Colivelin) for 1 hour and coincubated with rTβ4 for 72 hours. The protein expression of IL-17 and pSTAT3 was measured and quantitatively analyzed by Western blot. Data were representative of three independent experiments ( n = 3), and bar graphs showed mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We used primary antibodies against IL-17 (1:666, cat. MAA063Rb21; Cloud-Clone Corp, Wuhan, China), phospho-stat3 (Y705) (1:500, cat. MAB4607; R&D Systems, Minneapolis, MN, USA), IL-1β (1:250, cat. PAA563Rb51; Cloud-Clone Corp), IL-6 (1:1000, cat. MAA079Rb21; Cloud-Clone Corp), and β-actin antibody (1:2000, cat. ZB-5301; ZSGB-BIO, Beijing, China).

Techniques: Phospho-proteomics, In Vivo, Expressing, Western Blot