Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression promotes NF-CAF transition through the JAK2-STAT3 pathway. a & b , Representative images and quantitative analysis of western blotting showing that EREG overexpression in NFs and EREG interference in CAFs were successful. Moreover, the expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression and downregulated after EREG interference. GAPDH was used as a loading control. c & d & e , Both phosphorylated forms of JAK2 and STAT3 and the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly augmented after EREG overexpression. The change in expression of major participants of other pathways after EREG overexpression was not as significant. GAPDH was used as a loading control. f & g & h , Representative images and quantification of western blotting showing that expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression but was reduced after treatment with the JAK2 inhibitor AG490, indicating that AG490 antagonizes EREG-mediated NF activation. CAF markers, as well as (p-)JAK2 and (p-)STAT3 expression and p-JAK2/JAK2 and p-STAT3/STAT3 ratios decreased after EREG interference in CAFs but were restored by IL-6 treatment. GAPDH was used as a loading control. i , ELISA showing the IL-6 level in the CM of NFs/CAFs after the indicated treatment. J&K, Representative images and quantification of immunohistochemical staining in clinical samples revealed that high EREG expression was correlated with high phospho-JAK2 and phospho-STAT3 expression, while low EREG expression was correlated with low phospho-JAK2 and phospho-STAT3 expression. Magnification: 200×. *: p < 0.05, **: p < 0.01, ***: p < 0.001

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, Western Blot, Over Expression, Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression in fibroblasts promotes tumor growth in vivo. a , Photographs of tumor formation in nude mice and tumor xenografts 4 weeks after inoculation. b & c , Tumor growth curves measured after injection of HSC3 cells or conditioned fibroblasts as indicated. The tumor volume was calculated every 7 days until termination. d - g , RT-PCR analysis of Ereg, Jak2, Stat3 and Il6 expression in tissues of resected tumors, revealing successful overexpression/interference of Ereg and associated upregulation or downregulation of Jak2, Stat3 and Il6 . h , Immunohistochemical staining showed that tumors that developed from EREG-overexpressing NFs had a higher level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors that developed from control NFs. Tumors that developed from siEREG-transfected CAFs showed a lower level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors developed from siNC-transfected CAFs. S: stroma, T: tumor. Magnification: 200×. i & j , Western blotting showed that CAFs significantly promote EMT in vivo compared with NFs, with decreased E-cadherin expression and increased N-cadherin and vimentin expression. This EMT-promoting ability was attenuated after EREG knockdown. On the other hand, EREG overexpression in NFs gave NFs a CAF-like EMT-promoting ability. k , Increased EREG expression in NFs led to acquisition of the CAF phenotype in a JAK2-STAT3-dependent way and supported OSCC invasion through the promotion of EMT in tumor cells

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, In Vivo, Injection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Immunohistochemical staining, Staining, Control, Transfection, Western Blot, Knockdown

Journal: International immunopharmacology

Article Title: Identification of therapeutic targets and immune landscape in glioblastoma through crosstalk with glioma-associated mesenchymal stem cells.

doi: 10.1016/j.intimp.2025.114228

Figure Lengend Snippet: Fig. 10. FLNC enhances glioma cell resistance to staurosporine. (A, B) CCK8 assay assessed the effect of FLNC overexpression on the resistance of U87 and U251 cell lines to staurosporine. (C, D) CCK-8 assay demonstrated the impact of FLNC knockdown on the resistance of U87 and U251 cell lines to staurosporine. (E-H) Colony formation assay evaluated the influence of FLNC overexpression on the colony-forming ability of U87 and U251 cells under staurosporine treatment (20 nM), with representative images (E, G) (Scale bar = 50 μm) and statistical results (F, H). (I, J) In U87 and U251 cells, FLNC overexpression/knockdown modulated the inhibitory effect of staurosporine (5 nM) on the Erk and JAK2/STAT3 pathways. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The membranes were blocked with 5 % non-fat milk at room temperature for 2 h and then incubated overnight at 4 ◦C with primary antibodies, including FLNC (28492–1- AP, Proteintech), Erk1/2 (11257–1-AP, Proteintech), Phospho-ERK1/2 (28733–1-AP, Proteintech), JAK2 (#3230, CST), Phospho-JAK2 (#3774, CST), STAT3 (#12640, CST), Phospho-STAT3 (#9145, CST), and GAPDH (60004–1-Ig, Proteintech).

Techniques: CCK-8 Assay, Over Expression, Knockdown, Colony Assay

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Metabolic parameters

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques:

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Expression and phosphorylation of JAK2 in corpus cavernosum. (A) Representative western blot results for JAK2 and phosphorylated JAK2. (B,C) Relative expression of phosphorylated JAK2 and JAK2 compared with β‐actin; data are shown as the fold changes over the control group; n = 4 for each group. (D) Representative images of immunohistochemistry detecting JAK2 expression (200×). *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Immunohistochemistry

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Erectile function of mice. (A) Representative recordings of intracavernosal pressure (ICP) and arterial pressure (AP) during 1‐min electrical stimulation at 5.0 V. (B) The ratio of maximal intracavernosal pressure (MIP) to mean systemic arterial blood pressure (MAP) for each group; n = 10 for each group. *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Control

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Oxidative stress level in corpus cavernosum. (A) Representative western blot results for NOX1, NOX2, and NOX4. (B–D) Relative expression of NOX1, NOX2, and NOX4 compared with β‐actin; data are shown as the fold changes over the control group; n = 4 for each group. (E) MDA concentration in corpus cavernosum was measured with an MDA assay kit; n = 4 for each group. *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus. NOX: NADPH oxidase. MDA: malondialdehyde

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Western Blot, Expressing, Control, Concentration Assay, Multiple Displacement Amplification

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: NO‐cGMP pathway in corpus cavernosum. (A) NO concentration in corpus cavernosum measured with a NO assay kit; n = 4 for each group. (B) Concentration of cGMP in corpus cavernosum measured with an enzyme‐linked immunosorbent assay kit; n = 4 for each group. *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus. cGMP: cyclic guanosine monophosphate

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Apoptosis in corpus cavernosum. (A,B) Representative images of immunofluorescence detecting endothelium and smooth muscle with antibodies against CD‐31 or α‐SMA (green, 200×); nuclei were stained with DAPI (blue). (C,D) Percentage of endothelium and smooth muscle content detected with immunofluorescence; n = 4 for each group. (E) Representative western blot results for cleaved caspase‐3 and caspase‐3. (F,G) Relative expression of cleaved caspase‐3 and caspase‐3 compared with β‐actin detected with western blot; data are shown as the fold changes over the control group; n = 4 for each group. (H) Caspase‐3 activity detected with a caspase‐3 activity assay kit; n = 4 for each group. (I) Representative images of terminal deoxynucleotidyl transferase 2′‐deoxyuridine 5′‐triphosphate nick end labeling (TUNEL) staining (200×); apoptotic cells were stained brown and non‐apoptotic cells were blue. (J) Apoptosis index in the corpus cavernosum detected with TUNEL staining; n = 4 for each group. *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control, Activity Assay, Caspase-3 Activity Assay, End Labeling, TUNEL Assay

Journal: Andrology

Article Title: JAK2 deficiency improves erectile function in diabetic mice through attenuation of oxidative stress, apoptosis, and fibrosis

doi: 10.1111/andr.13061

Figure Lengend Snippet: Fibrosis in corpus cavernosum. (A) Representative western blot results for TGF‐β1, phosphorylated Smad2/3, Smad2/3, and Collagen IV. (B–E) Relative expression of TGF‐β1, phosphorylated Smad2/3, Smad2/3, and Collagen IV compared with β‐actin detected with western blot; data are shown as the fold changes over the control group; n = 4 for each group. (F) Representative images of Masson's trichrome staining (200×); smooth muscle was stained red and collagen was blue. (G) Ratio of collagen to smooth muscle detected with Masson's trichrome staining; n = 4 for each group. *: p < 0.05 compared with control group. #: p < 0.05 compared with JAK2 −/− group. §: p < 0.05 compared with DM group. DM: diabetes mellitus. TGF‐β1: transforming growth factor beta 1

Article Snippet: After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated overnight with primary antibodies against the following proteins at 4°C: β‐actin (1:500; Boster, BM0627, Wuhan, China), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1, 1:500; Abcam, ab55831, Cambridge, UK), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Abcam, ab154244), phospho‐JAK2 (Tyr1007/1008; 1:1000; CST, 3776, Danvers, MA, USA), JAK2 (1:1000; CST, 3230), caspase3 (1:1000; CST, 9665), cleaved caspase3 (1:1000; CST, 9664), TGF‐β1 (1:500; Abcam, ab64715), phospho‐Smad2/3 (1:1000; CST, 8828), Smad2/3 (1:1000; CST, 8685), and Collagen IV (1:500; Abcam, ab6586).

Techniques: Western Blot, Expressing, Control, Staining