Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunohistochemical (IHC) staining of the expression of TLR4 and phosphorylated STAT3 (p-STAT3; Tyr705) in a human melanoma tissue microarray. Scale bar: 5 mm. b Quantification of TLR4 and p-STAT3 staining intensity in the melanoma tissue microarray. * p < 0.05, ** p < 0.01. c IHC staining of TLR4 and p-STAT3 in representative melanoma specimens and normal skin tissues. Scale bar: black, 200 μm; red, 50 μm. d Scatter plots of TLR4 and p-STAT3 immunostaining intensity in human melanoma tissues ( n = 208). e Scatter plots of TLR4 and p-STAT3 immunostaining intensity in early-stage melanoma tissues ( n = 7).

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Microarray, Staining, Immunostaining

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunoblot analyses of total STAT3 and p-STAT3 proteins in LPS- and MPLAs-stimulated melanoma cells. Cells were treated with 1 μg/mL of LPS or MPLAs for 48 h. Representative results (upper panels) and relative protein levels (lower panels) are shown. b Immunofluorescence staining of nuclear STAT3 (green) in A375 cells stimulated with LPS (1 μg/mL) for 24 h. DAPI (blue) was used for nuclear staining. Representative images of three independent experiments are shown. Each white arrow denotes the nuclear STAT3. Scale bar: 50 μm. c–e Immunoblot analyses of total STAT3 and p-STAT3 proteins in the indicated siRNA-transfected A375 cells. After 48 h of siRNA transfection, cells were treated with 1 μg/mL of LPS or MPLAs for 48 h. Representative results ( c ) and relative protein levels of p-STAT3 ( d ) and total STAT3 ( e ) are shown. Protein levels of p-STAT3 and STAT3 in LPS- and MPLAs-stimulated cells relative to that in cells without TLR4 ligand simulation are regarded as 1. Data are presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 vs. the group without TLR4 ligand stimulation; ## P < 0.01 vs. the individual TLR4 ligand stimulation group of NC siRNA-transfected cells.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Immunofluorescence, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a RT-qPCR analyses of MCL-1 , BCL-XL , MMP2 , MMP9 and VEGF in MPLAs (1 μg/mL, 24 h)- and LPS (1 μg/mL, 24 h)-stimulated A375 cells. b TLR4 ligands promote A375 cell proliferation. Cells were treated with 2 μg/mL of LPS or MPLAs for 7 days and stained with crystal violet. c TLR4 ligands promote A375 cell invasion. Cells were treated with 1 μg/mL of LPS or MPLAs for 24 h and then subjected to transwell assays. Scale bar: 200 μm. d TLR4 ligands promote HUVECs tube formation. HUVECs were incubated with conditioned media from MPLAs (1 μg/mL, 24 h)- or LPS (1 μg/mL, 24 h)-stimulated A375 cells. Scale bar: 500 μm. In b – d , representative results are shown in the upper panels, and quantitative results of three independent experiments are shown in the lower panels. e Human cytokine array analyses of A375 cells with or without MPLAs (1 μg/mL, 24 h) stimulation. The 1- and 5-min exposure results are shown. f Quantification of levels of altered cytokines detected in e . g ELISA analyses of ICAM-1 in conditioned media from MPLAs (1 μg/mL, 24 h)- and LPS (1 μg/mL, 24 h)-stimulated A375 cells. In a – d , g , * P < 0.05, ** P < 0.01 vs. control group. h Representative immunoblots of STAT3, Mcl-1, Bcl-xL, VEGF and MMP2 in stable B16 NC and B16 STAT3β cell lines. i Photographs of crystal violet-stained B16 NC and B16 STAT3β cells. Cells were treated as in b . j Images of invasive cells in the transwell assay. Cells were treated as in c . Scale bar: 200 μm. In i , j , representative results are shown in the left panels; quantification results of three independent experiments are shown in the right panels. * P < 0.05, ** P < 0.01 vs. control group of B16 NC cells. ## P < 0.01 vs. individual TLR4 ligand stimulation group.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Quantitative RT-PCR, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Transwell Assay

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Representative immunoblots of total and phosphorylated STAT3 in A375 NC and A375 CA-TLR4 cells. b Transcriptional activity of STAT3 in A375 NC and A375 CA-TLR4 cells. c Morphological images of A375 NC and A375 CA-TLR4 cells. Cells expressing green fluorescent protein (GFP) displayed green fluorescence. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. d Representative images (left panel) and weights (right panel) of A375 NC and A375 CA-TLR4 tumors ( n = 7, two independent experiments). e Immunoblot analyses of total STAT3 and phosphorylated STAT3 (p-STAT3) in A375 NC and A375 CA-TLR4 tumors. Representative immunoblotting results are shown in the left panel. Protein levels of p-STAT3 and STAT3 in A375 CA-TLR4 tumors relative to that in A375 NC tumors (regarded as 1) are shown in the right panel. GAPDH was used as a loading control. In a , c – e , data are shown as the mean ± SD of three independent experiments. * P < 0.05 vs. A375 NC group. f Representative IHC staining results of Ki-67, CD31, E-cadherin, N-cadherin and vimentin in A375 NC and A375 CA-TLR4 tumors. Scale bar: 200 μm.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Activity Assay, Expressing, Fluorescence, Staining, Control, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunoblot analyses of TLR4, total STAT3 and p-STAT3 proteins in parthenolide (PTL)- and TAK-242-treated melanoma cells. Cells were treated with parthenolide or TAK-242 for 24 h. Representative results of three independent experiments are shown. GAPDH was used as a loading control. b Pharmacological inhibition of the TLR4/STAT3 pathway inhibit proliferation of melanoma cells. Cells were treated with parthenolide or TAK-242 for 7 days and stained with crystal violet. Photographs of stained cells are shown in the left panel. Quantitative results are shown in the right panel. The area of cell colonies in the control group was regarded as 100%. * P < 0.05, ** P < 0.01, vs. the individual control group. c Parthenolide suppresses tumor growth in melanoma-bearing mice. Representative images of B16 tumors are shown in the left panel. Weights of tumors are shown in the right panel. * P < 0.05 vs. vehicle (PBS)-treated control group. d B16 tumor volumes. * P < 0.05 vs. vehicle-treated control group at each time point. e Body weights of B16 tumor-bearing mice. Data are shown as the mean ± SD, n = 5. f Parthenolide inhibits the TLR4/STAT3 pathway in tumor tissues from melanoma-bearing mice. Immunoblot analyses of TLR4, STAT3 and phosphorylated STAT3 (p-STAT3) in B16 tumors. Representative immunoblotting results are shown in the left panel. Relative protein levels of p-STAT3, STAT3 and TLR4 are shown in the right panel. Data are presented as mean ± SD of three independent experiments. * P < 0.05 vs. vehicle-treated control group.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Control, Inhibition, Staining

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: TLR4 signaling activates STAT3 through MYD88 and TRIF, and subsequently upregulates a series of STAT3 target genes to promote melanoma progression.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques:

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunohistochemical (IHC) staining of the expression of TLR4 and phosphorylated STAT3 (p-STAT3; Tyr705) in a human melanoma tissue microarray. Scale bar: 5 mm. b Quantification of TLR4 and p-STAT3 staining intensity in the melanoma tissue microarray. * p < 0.05, ** p < 0.01. c IHC staining of TLR4 and p-STAT3 in representative melanoma specimens and normal skin tissues. Scale bar: black, 200 μm; red, 50 μm. d Scatter plots of TLR4 and p-STAT3 immunostaining intensity in human melanoma tissues ( n = 208). e Scatter plots of TLR4 and p-STAT3 immunostaining intensity in early-stage melanoma tissues ( n = 7).

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Microarray, Staining, Immunostaining

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunoblot analyses of total STAT3 and p-STAT3 proteins in LPS- and MPLAs-stimulated melanoma cells. Cells were treated with 1 μg/mL of LPS or MPLAs for 48 h. Representative results (upper panels) and relative protein levels (lower panels) are shown. b Immunofluorescence staining of nuclear STAT3 (green) in A375 cells stimulated with LPS (1 μg/mL) for 24 h. DAPI (blue) was used for nuclear staining. Representative images of three independent experiments are shown. Each white arrow denotes the nuclear STAT3. Scale bar: 50 μm. c–e Immunoblot analyses of total STAT3 and p-STAT3 proteins in the indicated siRNA-transfected A375 cells. After 48 h of siRNA transfection, cells were treated with 1 μg/mL of LPS or MPLAs for 48 h. Representative results ( c ) and relative protein levels of p-STAT3 ( d ) and total STAT3 ( e ) are shown. Protein levels of p-STAT3 and STAT3 in LPS- and MPLAs-stimulated cells relative to that in cells without TLR4 ligand simulation are regarded as 1. Data are presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 vs. the group without TLR4 ligand stimulation; ## P < 0.01 vs. the individual TLR4 ligand stimulation group of NC siRNA-transfected cells.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Immunofluorescence, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a RT-qPCR analyses of MCL-1 , BCL-XL , MMP2 , MMP9 and VEGF in MPLAs (1 μg/mL, 24 h)- and LPS (1 μg/mL, 24 h)-stimulated A375 cells. b TLR4 ligands promote A375 cell proliferation. Cells were treated with 2 μg/mL of LPS or MPLAs for 7 days and stained with crystal violet. c TLR4 ligands promote A375 cell invasion. Cells were treated with 1 μg/mL of LPS or MPLAs for 24 h and then subjected to transwell assays. Scale bar: 200 μm. d TLR4 ligands promote HUVECs tube formation. HUVECs were incubated with conditioned media from MPLAs (1 μg/mL, 24 h)- or LPS (1 μg/mL, 24 h)-stimulated A375 cells. Scale bar: 500 μm. In b – d , representative results are shown in the upper panels, and quantitative results of three independent experiments are shown in the lower panels. e Human cytokine array analyses of A375 cells with or without MPLAs (1 μg/mL, 24 h) stimulation. The 1- and 5-min exposure results are shown. f Quantification of levels of altered cytokines detected in e . g ELISA analyses of ICAM-1 in conditioned media from MPLAs (1 μg/mL, 24 h)- and LPS (1 μg/mL, 24 h)-stimulated A375 cells. In a – d , g , * P < 0.05, ** P < 0.01 vs. control group. h Representative immunoblots of STAT3, Mcl-1, Bcl-xL, VEGF and MMP2 in stable B16 NC and B16 STAT3β cell lines. i Photographs of crystal violet-stained B16 NC and B16 STAT3β cells. Cells were treated as in b . j Images of invasive cells in the transwell assay. Cells were treated as in c . Scale bar: 200 μm. In i , j , representative results are shown in the left panels; quantification results of three independent experiments are shown in the right panels. * P < 0.05, ** P < 0.01 vs. control group of B16 NC cells. ## P < 0.01 vs. individual TLR4 ligand stimulation group.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Quantitative RT-PCR, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Transwell Assay

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Representative immunoblots of total and phosphorylated STAT3 in A375 NC and A375 CA-TLR4 cells. b Transcriptional activity of STAT3 in A375 NC and A375 CA-TLR4 cells. c Morphological images of A375 NC and A375 CA-TLR4 cells. Cells expressing green fluorescent protein (GFP) displayed green fluorescence. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. d Representative images (left panel) and weights (right panel) of A375 NC and A375 CA-TLR4 tumors ( n = 7, two independent experiments). e Immunoblot analyses of total STAT3 and phosphorylated STAT3 (p-STAT3) in A375 NC and A375 CA-TLR4 tumors. Representative immunoblotting results are shown in the left panel. Protein levels of p-STAT3 and STAT3 in A375 CA-TLR4 tumors relative to that in A375 NC tumors (regarded as 1) are shown in the right panel. GAPDH was used as a loading control. In a , c – e , data are shown as the mean ± SD of three independent experiments. * P < 0.05 vs. A375 NC group. f Representative IHC staining results of Ki-67, CD31, E-cadherin, N-cadherin and vimentin in A375 NC and A375 CA-TLR4 tumors. Scale bar: 200 μm.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Activity Assay, Expressing, Fluorescence, Staining, Control, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Representative images (left panel) and weights (right panel) of B16 NC and B16 STAT3β tumors ( n = 7, two independent experiments). b Immunoblot analyses of phosphorylated STAT3 (p-STAT3) in B16 NC and B16 STAT3β tumors. Representative immunoblotting results are shown in the left panel. Relative protein levels of p-STAT3 in tumors are shown in the right panel. The protein level of p-STAT3 in B16 NC tumors without LPS stimulation was regarded as 1. c Representative IHC staining results of Ki-67, CD31, E-cadherin, N-cadherin and vimentin in B16 NC and B16 STAT3β tumors. Scale bar: 200 μm. d Flow cytometric analyses of splenic immune cells and tumor-infiltrating immune cells in the melanoma-bearing mice. Representative flow cytometry plots are shown in the left panels. Percentages of splenic MDSCs and CD8 T cells, and tumor-infiltrating CD8 T and NK cells are shown in the right panels. Data are shown as the mean ± SD. * P < 0.05 vs. the B16 NC tumor group without LPS stimulation. # P < 0.05, ## P < 0.01 vs. the LPS-stimulated B16 NC tumor group.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: a Immunoblot analyses of TLR4, total STAT3 and p-STAT3 proteins in parthenolide (PTL)- and TAK-242-treated melanoma cells. Cells were treated with parthenolide or TAK-242 for 24 h. Representative results of three independent experiments are shown. GAPDH was used as a loading control. b Pharmacological inhibition of the TLR4/STAT3 pathway inhibit proliferation of melanoma cells. Cells were treated with parthenolide or TAK-242 for 7 days and stained with crystal violet. Photographs of stained cells are shown in the left panel. Quantitative results are shown in the right panel. The area of cell colonies in the control group was regarded as 100%. * P < 0.05, ** P < 0.01, vs. the individual control group. c Parthenolide suppresses tumor growth in melanoma-bearing mice. Representative images of B16 tumors are shown in the left panel. Weights of tumors are shown in the right panel. * P < 0.05 vs. vehicle (PBS)-treated control group. d B16 tumor volumes. * P < 0.05 vs. vehicle-treated control group at each time point. e Body weights of B16 tumor-bearing mice. Data are shown as the mean ± SD, n = 5. f Parthenolide inhibits the TLR4/STAT3 pathway in tumor tissues from melanoma-bearing mice. Immunoblot analyses of TLR4, STAT3 and phosphorylated STAT3 (p-STAT3) in B16 tumors. Representative immunoblotting results are shown in the left panel. Relative protein levels of p-STAT3, STAT3 and TLR4 are shown in the right panel. Data are presented as mean ± SD of three independent experiments. * P < 0.05 vs. vehicle-treated control group.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: Western Blot, Control, Inhibition, Staining

Journal: Cell Death & Disease

Article Title: Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression

doi: 10.1038/s41419-020-2440-1

Figure Lengend Snippet: TLR4 signaling activates STAT3 through MYD88 and TRIF, and subsequently upregulates a series of STAT3 target genes to promote melanoma progression.

Article Snippet: Immunohistochemistry (IHC) staining assays using anti-human TLR4 antibody (SCBT: sc-293072; CA, USA) and anti-human phospho-STAT3 (Y705) antibody (CST: #9145; MA, USA) were performed following standard protocols provided by Servicebio, Inc. (Wuhan Servicebio Technology Co., Ltd., Hubei, China) and scanned using an automatic digital slide scanner (Pannoramic MIDI II, 3D Histech, Ltd., Budapest, Hungary).

Techniques: