Journal: Infection and Immunity

Article Title: STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway

doi: 10.1128/iai.00309-20

Figure Lengend Snippet: Figure 3 JAK2/STAT3 signal pathway was activated in the liver of mice with S. 623

Article Snippet: After dissolution in 10% or 12% SDS-PAGE, 433 the total protein samples were transferred to nitrocellulose membranes for 2 h with 434 transfer buffer and blocked with 5% nonfat dried milk, which were dissolved in TBST 435 (20 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl and 0.02% Tween 20) for 1 h. 436 Membranes were incubated with primary antibodies for GAPDH (Cell Signaling 437 Technology-2118, 1:1500 dilution), α-SMA (abcam-ab5694,1:800), IL-6 438 (abcam-ab6672), p-JAK2 (Cell Signaling Technology-3776), JAK2 (Cell Signaling 439 Technology-3230), p-STAT3 (Cell Signaling Technology-9145), STAT3 (Cell 440 on D ecem ber 17, 2020 at 43709427 http://iai.asm .org/ D ow nloaded from 21 Signaling Technology-9139/4904), Bax (Santa Cruz Biotechnology-SC493), Bcl-2 441 (Santa Cruz Biotechnology-SC492), Sirt3 (Cell Signaling Technology-5490s), 442 Acetylated SOD2 (K68) (abcam-ab137037), SOD2 (abcam-ab13534) overnight at 443 4°C.

Techniques:

Journal: Infection and Immunity

Article Title: STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway

doi: 10.1128/iai.00309-20

Figure Lengend Snippet: Figure 4 Liver p-STAT3 deficiency attenuates hepatic injury caused by S. 635

Article Snippet: After dissolution in 10% or 12% SDS-PAGE, 433 the total protein samples were transferred to nitrocellulose membranes for 2 h with 434 transfer buffer and blocked with 5% nonfat dried milk, which were dissolved in TBST 435 (20 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl and 0.02% Tween 20) for 1 h. 436 Membranes were incubated with primary antibodies for GAPDH (Cell Signaling 437 Technology-2118, 1:1500 dilution), α-SMA (abcam-ab5694,1:800), IL-6 438 (abcam-ab6672), p-JAK2 (Cell Signaling Technology-3776), JAK2 (Cell Signaling 439 Technology-3230), p-STAT3 (Cell Signaling Technology-9145), STAT3 (Cell 440 on D ecem ber 17, 2020 at 43709427 http://iai.asm .org/ D ow nloaded from 21 Signaling Technology-9139/4904), Bax (Santa Cruz Biotechnology-SC493), Bcl-2 441 (Santa Cruz Biotechnology-SC492), Sirt3 (Cell Signaling Technology-5490s), 442 Acetylated SOD2 (K68) (abcam-ab137037), SOD2 (abcam-ab13534) overnight at 443 4°C.

Techniques:

Journal: Infection and Immunity

Article Title: STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway

doi: 10.1128/iai.00309-20

Figure Lengend Snippet: Figure 6 Schematic overview of JAK2/STAT3-mediated effects on S. japonicum 668

Article Snippet: After dissolution in 10% or 12% SDS-PAGE, 433 the total protein samples were transferred to nitrocellulose membranes for 2 h with 434 transfer buffer and blocked with 5% nonfat dried milk, which were dissolved in TBST 435 (20 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl and 0.02% Tween 20) for 1 h. 436 Membranes were incubated with primary antibodies for GAPDH (Cell Signaling 437 Technology-2118, 1:1500 dilution), α-SMA (abcam-ab5694,1:800), IL-6 438 (abcam-ab6672), p-JAK2 (Cell Signaling Technology-3776), JAK2 (Cell Signaling 439 Technology-3230), p-STAT3 (Cell Signaling Technology-9145), STAT3 (Cell 440 on D ecem ber 17, 2020 at 43709427 http://iai.asm .org/ D ow nloaded from 21 Signaling Technology-9139/4904), Bax (Santa Cruz Biotechnology-SC493), Bcl-2 441 (Santa Cruz Biotechnology-SC492), Sirt3 (Cell Signaling Technology-5490s), 442 Acetylated SOD2 (K68) (abcam-ab137037), SOD2 (abcam-ab13534) overnight at 443 4°C.

Techniques:

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: a Levels of Tyr705-phosphorylated STAT3 (p-STAT3) and total STAT3 in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 as shown by western blotting. Cells were pre-treated with IL-6 (0.1, 1 or 10 ng/mL) for 16 h ( N = 5). b Levels of Tyr705-phosphorylated STAT3 (p-STAT3) in 22Rv1 cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 as shown by western blotting. Cells were pre-treated with 10 ng/mL IL-6 for 16 h ( N = 4). STAT3 phosphorylation at Tyr705 indicates STAT3 activation upon IL-6 treatment. The blots in a , b were cut into two pieces and probed with antibodies against p-STAT3 and GAPDH separately. The p-STAT3 blots were then stripped and blotted with an antibody against STAT3. c Survival of LNCaP cells as assessed by the WST assay after treatment with 1 μM doxorubicin for 48 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with IL-6 (0.1, 1 or 10 ng/mL) for 16 h. Cell survival is shown as fold change relative to cell survival in the absence of JAK/STAT3 inhibitor (S.E.M. is indicated by bars; N = 6). d Left panel: p53 levels in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with IL-6 (10 ng/mL) for 16 h. The blot was cut into two pieces and probed with anti-p53 DO-1 and anti-GAPDH antibody separately. Samples to be compared were loaded on the same gel and transferred onto the same membrane. Right panel: quantification of the western blot data shown as ratio of p53 to GAPDH (S.E.M. is indicated by bars; N = 2). e STAT3 protein phosphorylation and total STAT3 protein levels in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with 10 ng/mL IL-6 for 16 h ( N = 2). The blot was cut into two pieces and probed with antibodies against p-STAT3 and GAPDH separately. The p-STAT3 blot was then stripped and blotted with anti-STAT3 antibody.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, WST Assay, Membrane

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: a Oncoprint from cBioportal of two selected prostate cancer studies showing genetic alteration profiles of IL-6R, STAT3, MDM2, and TP53. Each row represents the genetic alteration according to the figure legend with individual patient in the column. b mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, STAT3 or MDM2 from prostate adenocarcinoma (prad) of the TCGA PanCancer Atlas study based on patients that have no TP53 alterations or putative driver TP53 mutations (missense or truncating). N = 414 no alterations, and N = 56 putative driver mutations. Mann–Whitney test, for IL-6R ** p = 0.003, STAT3 p = 0.47 and MDM2 *** p < 0.0001. c Scatter plots of mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, JAK1, JAK2, STAT3 vs MDM2 in prostate adenocarcinoma patients of the TCGA PanCancer Atlas study that have no alterations in TP53. Indicated r and p values are analysis by Spearman correlation. N = 414 in all plots. d Scatter plots of mRNA expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, JAK1, JAK2, STAT3 vs MDM2 in prostate adenocarcinoma patients of the TCGA PanCancer Atlas study that have putative driver alterations (missense and truncating) in TP53. Indicated r and p values are analysis by Spearman correlation. N = 56 in all plots, except the correlation with JAK2 N = 55. e Survival of patients with metastatic prostate adenocarcinoma from the study by Abida et al. . N is indicated in the figure. Black dot indicates censored patient. Left panel; patients with no TP53 alterations were grouped as having IL-6R alterations (amplification ( N = 12), mRNA high ( N = 1) missense mutation ( N = 1)) or having no IL-6R alterations. Log-rank (Mantel–Cox) test p = 0.09 or Gehan–Breslow–Wilcoxon test ** p = 0.01. Right panel; patients with putative driver TP53 mutations (missense, truncating, inframe) were grouped as having IL-6R alterations (amplification ( N = 4), mRNA high ( N = 3)) or having no IL-6R alterations. Log-rank (Mantel–Cox) test p = 0.5 or Gehan–Breslow–Wilcoxon test p = 0.9.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Expressing, MANN-WHITNEY, Amplification, Mutagenesis

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: CAFs secrete IL-6, which binds to IL-6 receptors (IL-6R) expressed on cancer cells. IL-6 binding to its receptor leads to activation of JAK kinase and STAT3, respectively. STAT3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding. This stimulates Mdm2-mediated p53 ubiquitination and p53 degradation in the proteasome, thereby inhibiting upregulation of the p53 target Bax upon treatment with doxorubicin. As a result, prostate cancer cell survival is enhanced.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Translocation Assay, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: WP1066 Sensitizes Oral Squamous Cell Carcinoma Cells to Cisplatin by Targeting STAT3/miR-21 axis

doi: 10.1038/srep07461

Figure Lengend Snippet: (a) In DDP resistant OSCC tumor tissues, STAT3 was highly expressed in the cytoplasm and nucleus of tumor cells than in DDP sensitive OSCC tissues ( P < 0.05). (B) In DDP resistant OSCC tissues, ISH staining reveals that miR-21 (red) has a relatively high expression than in DDP sensitive OSCC tissues ( P < 0.05) (Bar = 100 μm).

Article Snippet: Paraffin-embedded tumor tissue samples were selected for ISH with Cy3-labeled miR-21 probe (Boster, China) and IHC for STAT3 (Cell signaling, USA) expression.

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: WP1066 Sensitizes Oral Squamous Cell Carcinoma Cells to Cisplatin by Targeting STAT3/miR-21 axis

doi: 10.1038/srep07461

Figure Lengend Snippet: (a) MTT assay indicated that Tca8113/DDP and Tca8113 cells showed different sensitivity to DDP treatment. The cells were incubated in different doses of DDP (0.1, 0.2, 0.5, 1, 2, 4, 8, 12 and 16 μg/ml) for 48 h, and survival rate was determined by MTT assay. (b) QPCR showed that miR-21 was 3.7 folds higher in Tca8113/DDP cells than in Tca8113 cells ( P < 0.05). (c) Western blot assay indicated that STAT3 protein expression was 3 folds higher in Tca8113/DDP cells than in Tca8113 cells ( P < 0.05). Anti-GAPDH antibody was used as a loading control.

Article Snippet: Paraffin-embedded tumor tissue samples were selected for ISH with Cy3-labeled miR-21 probe (Boster, China) and IHC for STAT3 (Cell signaling, USA) expression.

Techniques: MTT Assay, Incubation, Western Blot, Expressing

Journal: Scientific Reports

Article Title: WP1066 Sensitizes Oral Squamous Cell Carcinoma Cells to Cisplatin by Targeting STAT3/miR-21 axis

doi: 10.1038/srep07461

Figure Lengend Snippet: ISH was used to determine the expression of miR-21 in treated OSCC cells; and Western blot was used to detect the expression of its target genes. (a) In Tca8113 and Tca8113/DDP cells, WP1066 combined with DDP treatment could inhibit MMP2/9, Bcl-2, mTOR, Ki67 expression, while in DDP + WP1066 treated group, Caspase-3 expression was upregulated. (b) In Tca8113 and Tca8113/DDP cells, WP1066 combined with DDP treatment could induce the expression of miR-21 (Red signal labeled by Cy3, cell nuclei was labeled by DAPI). IL-6 treatment could inhibit the expression of miR-21. (Bar = 20 μm) (c) In Tca8113 and Tca8113/DDP cells, DDP + WP1066 treatment could induce the expression of PTEN, TIMP3 and PDCD4. While IL-6 treatment could inhibit their expression in both cell lines. The gels were cropped, but those gels were run under the same experimental condition.

Article Snippet: Paraffin-embedded tumor tissue samples were selected for ISH with Cy3-labeled miR-21 probe (Boster, China) and IHC for STAT3 (Cell signaling, USA) expression.

Techniques: Expressing, Western Blot, Labeling

Journal: Scientific Reports

Article Title: WP1066 Sensitizes Oral Squamous Cell Carcinoma Cells to Cisplatin by Targeting STAT3/miR-21 axis

doi: 10.1038/srep07461

Figure Lengend Snippet: (a) The growth curves of blank control, DMSO, WP1066-treated, DDP, and DDP + WP1066 treated Tca8113/DDP xenograft tumors. The growth rate in WP1066, DDP and DDP + WP1066 group were inhibited compared to control and DMSO groups. (b) ISH showed that, in WP1006, WP1066 and DDP treated Tca8113/DDP tumors, miR-21 expression was inhibited (Red signal labeled by Cy3, cell nuclei was labeled by DAPI). (c) TUNEL assay showed an induced apoptotic nucleus (green) in WP1066 combined DDP-treated Tca8113/DDP tumors. (d) IHC staining showed the upregulation of PTEN, TIMP-3, and PDCD4 in WP1066-treated and DDP + WP1066 treated group.(e) IHC assay was used to determine the expression of tumor growth related proteins in Tca8113/DDP xenograft tumors. Downregulation of mTOR, Ki67, Bcl-2, and MMP-2 was observed in WP1066 and WP1066 combined DDP-treated group. In combined treated group Caspase-3 expression was upregulated. (Bar = 100 μm).

Article Snippet: Paraffin-embedded tumor tissue samples were selected for ISH with Cy3-labeled miR-21 probe (Boster, China) and IHC for STAT3 (Cell signaling, USA) expression.

Techniques: Expressing, Labeling, TUNEL Assay, Immunohistochemistry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: Activin-A–induced STAT3 activation enhances CD73 expression in Th17 cells. (A) Representative FACS plots of Act-A–Th17 or Th17 cells showing pSTAT3 expression. Shaded histogram represents isotype control. Cumulative data are shown as mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (B) ChIP analyses demonstrating the binding of STAT3 on the Nt5e locus (site 2, +1,700 bp, Left) and on the Entpd1 promoter, at the SRE1 locus (−3,740 bp) (Right). Results are mean ± SEM; each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (C) Act-A–Th17 cells or Th17 cells were cultured in the presence of STA-21. Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (D) Cumulative data showing the percentages of CD39+, CD73+, and CD39+CD73+ among CD4+ T cells. Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (E) IL-10 in culture supernatants. Each symbol represents the mean ± SEM of triplicate wells and corresponds to one of four independent experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In some experiments, the AhR antagonist (CH-223191, 5 μM; Sigma-Aldrich), the CD73 antagonist (AMP-CP, 100 μM; Sigma-Aldrich), the Smad3 inhibitor (SIS3, 20 μm; Sigma-Aldrich), the neutralizing anti-ALK4 antibody (10 μg/mL; R&D), the STAT3 inhibitor (STA-21, 10 μM; Cayman Chemical) or vector (PBS) were administered in T cell cultures, as indicated.

Techniques: Activation Assay, Expressing, In Vitro, Binding Assay, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: AhR drives activin-A–mediated up-regulation of CD73 and antiinflammatory genes in Th17 cells. (A) Representative immunoblots showing AhR and c-Maf protein levels in act-A–Th17 or Th17 cells. (B) Quantification of relative AhR and c-Maf expression is shown; TATA binding protein (TBP). Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (C) ChIP analyses demonstrating the binding of AhR and c-Maf on the Nt5e locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (D) ChIP analyses demonstrating the binding of AhR and c-Maf on the Entpd1 locus and (E) on the Il10 locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (F) Sequential ChIP analysis demonstrating STAT3 and AhR cobinding on the Il10 conserved noncoding sequence-9 (−9.0 kb) locus. Data are mean ± SEM; each symbol corresponds to one of three independent in vitro experiments. (G) Act-A–Th17 or Th17 cells were differentiated, in the presence of the AhR antagonist, CH-223191 or control (DMSO). Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of three independent in vitro experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In some experiments, the AhR antagonist (CH-223191, 5 μM; Sigma-Aldrich), the CD73 antagonist (AMP-CP, 100 μM; Sigma-Aldrich), the Smad3 inhibitor (SIS3, 20 μm; Sigma-Aldrich), the neutralizing anti-ALK4 antibody (10 μg/mL; R&D), the STAT3 inhibitor (STA-21, 10 μM; Cayman Chemical) or vector (PBS) were administered in T cell cultures, as indicated.

Techniques: Western Blot, Expressing, Binding Assay, In Vitro, Sequencing

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Western blot analysis of MDA-MB-231 cell line stably transduced with a lentiviral vector encoding for control sh-RNA (CTR) or for sh-RNAs directed against human STAT3 (sh), serum starved and then stimulated for the indicated times with 5% wound fluids (WF). Vinculin expression was used as loading control. (B) qRT-PCR analysis of Bcl-2 and Survivin expression in MDA-MB-231 control cells (sh-no Target) or in STAT3 silenced clones (sh-STAT3). Cells were serum starved and then stimulated for the indicated times with wound fluids (WF). Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (C) Growth curve analysis of MDA-MB-231 control cells (sh-no Target) or STAT3 silenced clones (sh-STAT3). Cells (50×10 3 /well) have been seeded in complete medium (CM) on day 0, and then counted by Trypan Blue exclusion test, every day for 5 days. Two independent cell clones have been evaluated. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (D) Growth curve analysis of MDA-MB-231 cell line in the presence of the indicated inhibitors. Cells (50×10 3 /well) have been seeded in complete medium (CM) on day 0, in the presence of S3I-201 (50 μM) or STA-21 (30 μM) or vehicle (CTR) and then counted by Trypan blue exclusion test, every day for 5 days. Fresh medium containing the inhibitor was replaced on day 3. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (E) Same as in (C), but seeding cells in serum free medium supplemented with 3% wound fluids (WF). Three independent cell clones have been evaluated. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (F) Same as in (D), but seeding cells in serum free supplemented with 3% wound fluids (WF). Data represents the mean (± S.D.) of two independent experiments performed in triplicate.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Western Blot, Stable Transfection, Transduction, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Clone Assay

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Western blot analysis of MDA-MB-231 and MDA-MB-468 cell lines, serum starved and then stimulated for the indicated times with EGF (20 ng/mL) or 5% wound fluids (WF), as indicated. (B) Images show primary mammospheres formed by MDA-MB-231 cells. Control or STAT3-silenced (sh2 and sh3) cells were plated on poly-HEMA coated dishes in mammosphere growing medium supplemented with 5% wound fluids, in the presence of IL-6 blocking antibody (0.2μg/ml) or STAT3 inhibitors (S3I-201, 50 μM; Stattic, 10 μM; STA-21, 30 μM; and Galiellalactone, 12 μM), as indicated, and grown for ten days. (C) Same as in (B) but using MDA-MB-468 cells. STAT3 inhibitors were used as follows: S3I-201, 100 μM; Stattic, 10 μM; STA-21, 30 μM; Galiellalactone, 25 μM. (D) Graphs report the percent of mammosphere forming efficiency (MFE%) in MDA-MB-231 (left) and MDA-MB-468 (right) cells of the experiment described in (B) and (C). MFE was calculated as the ratio between the numbers of mammospheres counted/number of cells seeded, per well. (E) Graphs report the self-renewal in MDA-MB-231 (left) and MDA-MB-468 (right) cells treated with the inhibitors only during the second generation. Self-renewal was calculated as the ratio between number of secondary mammospheres/number of primary mammospheres. (F) Flow cytometry analysis of CD44 high CD24 low/neg stem cell-like subpopulation in MDA-MB-231 cells. Percent of CD44 high CD24 low/neg (Q4) and of CD44 high CD24 high (Q2) is reported in the plots.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Western Blot, Control, Blocking Assay, Flow Cytometry

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Graph reports the volume (mm 3 ) of primary tumors derived from injection of 2×10 6 MDA-MB-231 control (CTR) or STAT3 silenced (sh-STAT3) cells in thoracic mammary fat pads of nude mice (2 MFP/mouse) in 50 μl Matrigel/PBS (1:1). (B) Graph reports the time dependent appearance of primary tumors derived from injection of 2×10 6 MDA-MB-231 control (CTR) or STAT3 silenced (sh-STAT3) cells in the nude mouse thoracic mammary fat pads (2 MFP/mouse) in 50 μl Matrigel/PBS (1:1). (C) Graph reports the rate of tumor growth, independently from the time of appearance, in mice described in (A). Values are expressed as ratio of the tumor volume over the value of 20 mm 3 , considered as cut off. The red and the green lines represent the trend of growth of the MDA-MB-231 CTR and sh-STAT3, respectively. (D) Same as in (A), but injecting 2×10 5 MDA-MB-231 (CTR) or STAT3 silenced (sh-STAT3) cells, in place of 2×10 6 cells. (E) Same as in (B), but injecting 2×10 5 cells. (F) Same as in (C), but injecting 2×10 5 cells. In all graphs, statistical significance was calculated using the Student's t-test. One asterisk (*) indicates a p value ≤ 0.05, two asterisks (**) a p value ≤ 0.01 and three asterisk (***) a p value ≤ 0.005.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Derivative Assay, Injection, Control

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Table reports the percentage of tumor take-rate, following injection of the indicated numbers of MDA-MB-231 CTR or sh-STAT3 clones, in the presence of Matrigel. The media between 2 to 3 different sh-STAT3 clones is reported. (B) Graph reports the disease free survival in the indicated cohorts of mice, after injection of decreasing number of MDA-MB-231 CTR or two different sh-STAT3 clones. Data are reported as percentage of mice that developed primary tumors during 12 weeks of follow-up. (C) The picture shows an example of primary masses excised from nude mice after surgery. It is evidenced the similar dimension of the tumors in the different groups. (D) Table reports the percentage of local recurrences formed by mice injected with MDA-MB-231 CTR cells or two different clones of sh-STAT3 after 8 weeks of follow up. (E) Graph reports the disease free survival in the indicated cohorts of mice, after removal of the primary tumor. Data are reported as percentage of mice that developed recurrent disease during 8 weeks of follow-up.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Injection, Clone Assay