Journal: Cancer gene therapy

Article Title: Tumor-associated GM-CSF overexpression induces immunoinhibitory molecules via STAT3 in myeloid-suppressor cells infiltrating liver metastases.

doi: 10.1038/cgt.2016.19

Figure Lengend Snippet: Figure 5. Signal transducer and activator of transcription factor 3 (STAT3) and Janus-activated kinase 2 (JAK2) blockade inhibits indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1) expression in liver myeloid-derived suppressor cells (L-MDSCs). L-MDSCs were isolated from the tumor-bearing livers and cultured for 48 h in the presence of Stattic (100 μM) and AZD1480 (10 μM) to inhibit STAT3 and JAK2, respectively. Untreated L-MDSCs were used as positive IDO and PD-L1 expression controls. Cells were isolated and stained for surface PD-L1 and intracellular IDO. Isotype controls were used to determine non-specific background staining and to set the threshold for positive IDO and PD-L1 expression. Immunophenotyping results of a representative experiment are shown as histograms (a, top) and averages of IDO+ and PD-L1+ cells (a, bottom). IDO/PD-L1 co-expression was evaluated in the same samples (b, top) and the extent of IDO/PD-L1 downregulation averaged (b, bottom). The results are representative of six independent experiments. Error bars are based on s.e.m. values. P-values were calculated using a two-tailed t-test.

Article Snippet: L-MDSC STAT3 inhibition was carried out ex vivo by treating the cells with Stattic (100 μM, Santa Cruz Biotechnology, Dallas, TX, USA) or AZD1480 (10 μM, Santa Cruz); Janus-activated kinase 2 (JAK2) was targeted with BSK805 (5, 10 and 50 μM, Santa Cruz).

Techniques: Expressing, Derivative Assay, Isolation, Cell Culture, Staining, Two Tailed Test

Journal: Cancer gene therapy

Article Title: Tumor-associated GM-CSF overexpression induces immunoinhibitory molecules via STAT3 in myeloid-suppressor cells infiltrating liver metastases.

doi: 10.1038/cgt.2016.19

Figure Lengend Snippet: Figure 6. Signal transducer and activator of transcription factor 3 (STAT3) and Janus-activated kinase 2 (JAK2) blockade abrogates granulocyte- macrophages colony-stimulating factor (GM-CSF)-driven indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1) expression in liver myeloid-derived suppressor cells (L-MDSCs). L-MDSCs were purified from the tumor-bearing livers and cultured in the presence or absence of recombinant mouse GM-CSF (20 ng ml−1). STAT3 inhibitor Stattic and JAK2 inhibitors AZD1480 and BSK805 were added to GM-CSF- treated L-MDSCs at 100, 10 and 5 μM, respectively. An additional treatment of GM-CSF was added to L-MDSCs after 24 h of culture. After 48 h of incubation, L-MDSCs were pelleted and stained for surface PD-L1 and intracellular IDO as described in Materials and Methods. Isotype controls were used to determine non-specific background staining for IDO and PD-L1 and to set the threshold for positive expression. The extent of IDO and PD-L1 downregulation induced by STAT3 and JAK2 blockade is shown in histograms (a). Averages for IDO and PD-L1 expression were calculated using L-MDSCs isolated from four mice (b). IDO/PD-L1 co-expression was determined in the same samples (c), and averages of L-MDSCs from four mice were calculated (d). Bioluminescence plate-based assay was used to measure the immunosuppressive effect of L-MDSCs on chimeric antigen receptor T (CAR-T) cell cytotoxic function. Bioluminescence produced by CEA+ target cells was measured in the presence of CAR-T cells specific for CEA with or without L-MDSCs pooled from four mice (e). In addition, MDSC were pretreated with Stattic before being used in the assay. GM-CSF was supplemented to some wells. The bar chart represents the average of three technical replicates per treatment (f). The assay was replicated twice. Error bars represent s.e.m. values. P-values were calculated using a two-tailed t-test.

Article Snippet: L-MDSC STAT3 inhibition was carried out ex vivo by treating the cells with Stattic (100 μM, Santa Cruz Biotechnology, Dallas, TX, USA) or AZD1480 (10 μM, Santa Cruz); Janus-activated kinase 2 (JAK2) was targeted with BSK805 (5, 10 and 50 μM, Santa Cruz).

Techniques: Expressing, Derivative Assay, Cell Culture, Recombinant, Incubation, Staining, Isolation, Produced, Two Tailed Test

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Malignant astrocytic tumor progression potentiated by JAK-mediated recruitment of myeloid cells

doi: 10.1158/1078-0432.CCR-16-1508

Figure Lengend Snippet: (A) Schematic of RCAS low grade murine glioma bearing animals post irradiation (2wks) and GFP+ bone marrow transplantation, treated with AZD1480. (B) Representative image of T2-weighted 7T MRI of vehicle control (n=7) and AZD1480 treated (n=7) animals at 10 weeks. Animals were imaged around 7 weeks. (C) Representative photomicrographs of tumor cross sections from mice treated with vehicle control (n=5) and AZD1480 (n=5). Quantification of Average number of GFP cells per high power field in bar graph format, (n=10),Scale bar 50μm, **p=0.004. (D)Representative photomicrographs of tumor cross sections from mice treated with vehicle control (n=5) and AZD1480 (n=5) co-stained with GFP+ and CD11b+. Quantification of average number of cells per high power field in bar graph format labeled with GFP+, CD11b+ and double labeled with GFP+/CD11b+, (n=10), Scale Bar 50μm, *p<0.05.

Article Snippet: AZD1480 was purchased from Chemietek.

Techniques: Irradiation, Transplantation Assay, Staining, Labeling

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Malignant astrocytic tumor progression potentiated by JAK-mediated recruitment of myeloid cells

doi: 10.1158/1078-0432.CCR-16-1508

Figure Lengend Snippet: (A) Animal schematic depicting experimental design to examine effect of AZD1480 (treatment for 3 weeks) on tumor progression. Treatment with AZD1480 was stopped at 6 weeks (B) Representative images of T2-weighted 7T MRI of vehicle control (n=9) and AZD1480(n=9) treated animals at 6 weeks. Representative images of H&E staining of tumor sections from control (n=8) and AZD1480 (n=8) treated animals at 6 weeks, Scale Bar 100 μm. (C) Representative flow cytometry graphs of CD11b+/GR1+ from blood (n=3/group) and bone marrow (n=7/group) of vehicle control and AZD1480 treated tumor bearing mice at 6 weeks, *p=0.0386 blood, p=0.0556 BM (D) Kaplan-Meier symptom free survival curve for RCAS mice treated for 3 weeks only with vehicle control (n=9) and AZD1480,(n=9) *** P<0.0001.(E) Kaplan-Meier symptom free survival curve for RCAS mice treated indefinitely with vehicle control(n=6) and AZD1480(n=6), ***p<0.0001.

Article Snippet: AZD1480 was purchased from Chemietek.

Techniques: Staining, Flow Cytometry

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Receptor tyrosine kinases (RTKs) expression profiles of myofascial trigger points (MTrPs) tissue derived from upper trapezius muscle of myofascial pain syndrome (MPS) patients and control groups. ( A ) The upregulated RTKs members were validated by microarray analysis. MPS group = 11, Con group = 7. Platelet-derived growth factor receptor-α (PDGFR-α): Con, 879 ± 83.08; 95% CI, 802.2–955.8 versus MPS, 1,060 ± 96.84; 95% CI, 994.9–1,125; units, fluorescent value; mean ± SD; P < 0.001. PDGFR-β: Con, 338.8 ± 47.96; 95% CI, 294.5–383.2 versus MPS, 308.4 ± 51.67; 95% CI, 273.7–343.1; units, fluorescent value; mean ± SD; P > 0.05. ( B ) Representative microscopic images showing morphology of muscle fibers in different groups. The morphology of MTrPs showed that annular or enlarged muscle fibers ( yellow arrows ) of different sizes with centralized nuclei in cross-sectional spaces under microscopy ( yellow arrows ). Scale bars, 20 μm. ( C ) The relationship between pain intensity and expression level of phosphorylated PDGFR-α (p-PDGFR-α) was characterized by a significant positive correlation (r = 0.711; n = 11; P < 0.05). ( D ) Results from enzyme-linked immunosorbent assay (ELISA) showed that the level of serum platelet-derived growth factor-AA (PDGF-AA) was increased. Con, 3.74 ± 0.82; 95% CI, 2.96–4.5; versus MPS, 5.97 ± 0.98; 95% CI, 5.31–6.6; units, ng/ml; mean ± SD; P < 0.001. ( E ) Results from immunohistochemistry (IHC) showed that the expression of PDGF-AA ( yellow arrows ) was upregulated at MTrPs. Scale bars, 20 μm. Con, 1.51 ± 0.33; 95% CI, 1.16–1.85; versus MPS, 10.2 ± 1.57; 95% CI, 8.55–11.85; units, integrated optical density (IOD); mean ± SD; P < 0.001. ( F ) The cross-sectional area of muscle fibers was increased in MPS group. Con, 995.2 ± 166.5; 95% CI, 902.9–1,087; versus MPS, 1,398 ± 124.2; 95% CI, 1,330–1,467; units, μm 2 ; mean ± SD; P < 0.001. ( G ) Representative fluorescence microscopic images showing expression of p-PDGFR-α ( green ) in different groups. Scale bars, 20 μm. Con, 1.00 ± 0.10; 95% CI, 0.89–1.11; versus MPS, 1.44 ± 0.20; 95% CI, 1.23–1.64; units, mean intensity; mean ± SD; P < 0.001. * P < 0.05; ** P < 0.01; *** P < 0.001. ALK, anaplastic lymphoma kinase; EphA, ephrin receptor A; EphB, ephrin receptor B; LTK, leukocyte tyrosine kinase; TRKB, tyrosine kinase receptor B; ZAP70, zeta-chain-associated protein kinase 70.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Expressing, Derivative Assay, Control, Microarray, Microscopy, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Fluorescence

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Activation of platelet-derived growth factor receptor-α (PDGFR-α)–induced pain-like behaviors, Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) activation, and muscle contraction in normal rats. ( A ) Activation of PDGFR-α within gastrocnemius muscle decreased respectively mechanical withdrawal thresholds and the nest quality scores in Randall–Selitto and nest-building test. n = 10 rats per group for behavioral test and analysis. Randall–Selitto test on hour 2: wild-type (WT), 407.7 ± 4.35; 95% CI, 404.1–411.4; versus WT plus PDGF-A-chain, 351.8 ± 31.81; 95% CI, 325.2–378.4; units, g; mean ± SD; P < 0.001. Nest-building test: WT, 3.8 ± 0.92; 95% CI, 3.14–4.46; versus WT plus PDGF-A-chain, 2.7 ± 0.95; 95% CI, 2.02–3.38; units, scores; mean ± SD; P < 0.05. ( B and C ) Representative immunohistochemistry (IHC) images and Western blod (WB) results for showing expression of c-fos protein ( yellow arrows ) in L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 100 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantification were done for representative samples from each group. Three independent biologic replicate experiments were performed. IHC: WT, 3.86 ± 1.08; 95% CI, 2.72–4.90; versus WT plus PDGF-A-chain, 17.53 ± 2.05; 95% CI, 15.38–19.68; units, integrated optical density (IOD); mean ± SD; P < 0.001. Western blot (WB; normalized to GAPDH): WT, 0.21 ± 0.02; 95% CI, 0.19–0.22; versus WT plus PDGF-A-chain, 0.93 ± 0.46; 95% CI, 0.88–0.98; units, relative gray values; mean ± SD; P < 0.001. ( D ) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres and cross-sectional area of muscle fibers of different groups. Scale bars, 2 μm. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. Length of sarcomeres: WT, 2.13 ± 0.06; 95% CI, 2.10–2.16; versus WT plus PDGF-A-chain, 1.63 ± 0.05; 95% CI, 1.60–1.66; units, μm; mean ± SD; P < 0.001. Cross-sectional area of muscle fibers: WT, 1,572 ± 528; 95% CI, 1,280–1,865; versus WT plus PDGF-A-chain, 2,032 ± 246.7; 95% CI, 1,895–2,168; units, μm 2 ; mean ± SD; P < 0.01. ( E ) Activation of PDGFR-α increased protein levels of JAK2/STAT3 pathway, inflammatory factors, and contraction biomarkers in rats. WB results for proteins with similar molecular weight were from the same samples and run in parallel in different concentrations gels. Three independent biologic replicate experiments were performed. n = 6 values per group were included in the analysis. Descriptive statistics of WB can be found in the Supplemental Data ( https://links.lww.com/ALN/D632 ). Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; kD, kilodalton; MLCK, myosin light chain kinase; MTrPs, myofascial trigger points; p-MLC, phosphorylated myosin light chain; TNF-α, tumor necrosis factor-α.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Activation Assay, Derivative Assay, Randall–Selitto Test, Immunohistochemistry, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Molecular Weight

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Knockdown of platelet-derived growth factor receptor-α (PDGFR-α) or inhibition of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway ameliorates pain-like behaviors in myofascial trigger points (MTrPs) rats. ( A ) MTrPs model rats exhibits low-quality scores and mechanical withdrawal thresholds decreased. n = 10 rats per group for behavioral test and analysis. Nest-building test: wild-type (WT), 3.9 ± 1.00; 95% CI, 3.19–4.61; versus MTrPs, 2.2 ± 0.92; 95% CI, 1.54–2.86; units, scores; mean ± SD; P < 0.001. Randall–Selitto test in week 2: WT, 391.9 ± 17.43; 95% CI, 379.14–404.4; versus MTrPs, 360.8 ± 19.43; 95% CI, 346.9–374.7; units, g; mean ± SD; P < 0.001. ( B ) Western blot (WB) results showing the expression of phosphorylated PDGFR-α (p-PDGFR-α) within MTrPs in animal models. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): WT, 0.39 ± 0.03; 95% CI, 0.32–0.46; versus MTrPs, 0.91 ± 0.19; 95% CI, 0.43–1.39; units, relative gray values; mean ± SD; P < 0.01. ( C ) MTrPs rats model exhibits a highly similar morphology ( yellow arrows ) to MTrPs of MPS patients. ( D ) Representative immunohistochemistry (IHC) images for showing expression and localization ( yellow arrows ) of p-PDGFR-α in MTrPs of rats. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. IHC: WT, 2.11 ± 1.02; 95% CI, 1.04–3.18; versus MTrPs, 38.31 ± 7.64; 95% CI, 30.30–46.32; units, integrated optical density (IOD); mean ± SD; P < 0.001. Cross-sectional area: WT, 1,246 ± 223.2; 95% CI, 1,122–1,369; versus MTrPs, 2,636 ± 430.6; 95% CI, 2,398–2,875; units, μm 2 ; mean ± SD; P < 0.001. ( E and F ) Representative behavioral images showing knockdown of PDGFR-α or inhibition of JAK2/STAT3 pathway increased nest scores and mechanical withdrawal thresholds of MTrPs rats. n = 10 rats per group for behavioral test and analysis. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 1.90 ± 0.88; 95% CI, 1.24–2.53; versus NC-scramble, 1.80 ± 0.79; 95% CI, 1.24–2.36; versus knockdown-PDGFR-α, 2.9 ± 0.99; 95% CI, 2.19–3.61; units, scores; mean ± SD; P < 0.05; saline, 1.70 ± 0.67; 95% CI, 1.22–2.18; versus AZD1480, 2.60 ± 0.97; 95% CI, 1.91–3.29; units, scores; mean ± SD; P < 0.05. Randall–Selitto test on hour 2: vector, 296.3 ± 32.22; 95% CI, 273.2–319.3; versus NC-scramble, 296.2 ± 23.34; 95% CI, 279.5–312.9; versus knockdown-PDGFR-α, 343.8 ± 29.59; 95% CI, 322.6–364.9; units, g; mean ± SD; P < 0.01. Saline, 295.2 ± 12.05; 95% CI, 285.2–305.3; versus AZD1480, 375.1 ± 10.57; 95% CI, 366.3–384.0; units, g; mean ± SD; P < 0.001. ( G ) Schematic representation of virus infection in MTrPs. Scale bar, 20 μm. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and WB analysis were used to validate the efficiency of PDGFR-α knockdown. n = 6 rats per group. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): vector, 0.50 ± 0.07; 95% CI, 0.43–0.58; versus NC-scramble, 0.58 ± 0.07; 95% CI, 0.51–0.66; versus knockdown-PDGFR-α, 0.13 ± 0.03; 95% CI, 0.11–0.16; units, relative gray values; mean ± SD; P < 0.01; RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.08; 95% CI, 0.92–1.08; versus NC-scramble, 0.94 ± 0.11; 95% CI, 0.83–1.06; versus knockdown-PDGFR-α, 0.38 ± 0.15; 95% CI, 0.22–0.54; units, 2 -ΔΔCT ; mean ± SD; P < 0.001. ( H ) Representative IHC images for showing expression of c-fos protein ( yellow arrows ) in L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. Experiments were repeated at least three times, and quantitation was done for representative samples from each group. n = 6 values per group were included in the analysis. IHC: WT, 5.61 ± 3.23; 95% CI, 2.22–8.99; versus MTrPs, 81.48 ± 4.69; 95% CI, 76.56–86.41; versus knockdown-PDGFR-α, 30.71 ± 7.56; 95% CI, 22.77–38.64; versus AZD1480, 24.14 ± 9.77; 95% CI, 13.89–34.40; units, IOD; mean ± SD; P < 0.001. * P < 0.05; ** P < 0.01; *** P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HE, hematoxylin-eosin staining; NC, negative control.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Knockdown, Derivative Assay, Inhibition, Randall–Selitto Test, Western Blot, Expressing, Immunohistochemistry, Injection, Plasmid Preparation, Saline, Virus, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Quantitation Assay, Staining, Negative Control

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Platelet-derived growth factor receptor-α (PDGFR-α) depends on Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway to induce pain-like behaviors and muscle contraction in myofascial trigger points (MTrPs) rats. ( A ) Representative hematoxylin and eosin (HE) staining images showing knockdown of PDGFR-α or inhibition of JAK2/STAT3 pathway decreased the cross-sectional area as compared with controls. Scale bars, 20 μm. Top 15 cross-sectional area values were analyzed from 6 rats per group. Wild-type (WT), 1,324 ± 393.6; 95% CI, 1,106–1,524; versus MTrPs, 2,432 ± 344.9; 95% CI, 2,241–2,363; versus knockdown-PDGFR-α, 1,843 ± 356; 95% CI, 1,646–2,040; versus AZD1480, 2,015 ± 264.5; 95% CI, 1,869–2,162; units, μm 2 ; mean ± SD; P < 0.01; P < 0.001. ( B ) Knockdown of PDGFR-α or JAK2/STAT3 pathway within MTrPs suppresses inflammatory factors and contraction biomarkers expression. n = 8 rats per group. Three independent biologic replicate experiments were performed. Descriptive statistics of Western blot (WB) can be found in the Supplemental Data ( https://links.lww.com/ALN/D632 ). ( C ) Inhibition of JAK2/STAT3 pathway rescued the pain-like behaviors. Nest-building test: PDGF-A-chain-homodimer, 2.10 ± 0.99; 95% CI, 1.39–2.81; versus PDGF-A-chain-homodimer plus AZD1480, 3.3 ± 0.95; 95% CI, 2.62–3.98; units, scores; mean ± SD; P < 0.05. Randall–Selitto test on hour 2: PDGF-A-chain-homodimer, 308.3 ± 14.11; 95% CI, 298.2–318.4; versus PDGF-A-chain-homodimer plus AZD1480, 357.2 ± 9.51; 95% CI, 350.4–364; units, g; mean ± SD; P < 0.001. ( D ) Cross-sectional area of muscles fibers, PDGF-A-chain-homodimer, 2,032 ± 246.7; 95% CI, 1,895–2,168; versus PDGF-A-chain-homodimer plus AZD1480, 1,408 ± 164.7; 95% CI, 1,317–1,499; units, μm 2 ; mean ± SD; P < 0.001; and ( E ) contraction biomarkers expression resulting from PDGFR-α activation. n = 10 rats per group for behavioral test and analysis. Three independent biologic replicate experiments for WB were performed. Descriptive statistics of WB can be found in the Supplemental Data ( https://links.lww.com/ALN/D632 ). ( F ) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres of different groups. Scale bars, 2 μM. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. WT, 2.40 ± 0.05; 95% CI, 2.37–2.43; versus MTrPs, 1.54 ± 0.06; 95% CI, 1.50–1.58; versus knockdown-PDGFR-α, 1.68 ± 0.05; 95% CI, 1.65–1.70; versus AZD1480, 1.83 ± 0.04; 95% CI, 1.81–1.86; mean ± SD; P < 0.001; PDGF-A-chain-homodimer, 1.67 ± 0.04; 95% CI, 1.65–1.70; versus PDGF-A-chain-homodimer plus AZD1480, 1.76 ± 0.07; 95% CI, 1.71–1.80; units, μm; mean ± SD; P < 0.01. ( G ) Representative immunohistochemistry (IHC) staining images showing c-fos protein expression of L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. n = 6 values per group were included in the analysis. PDGF-A-chain-homodimer, 16.76 ± 2.21; 95% CI, 14.45–19.08; versus PDGF-A-chain-homodimer plus AZD1480, 9.25 ± 1.36; 95% CI, 7.83–10.68; units, integrated optical density (IOD); mean ± SD; P < 0.01. * P < 0.05; ** P < 0.01; *** P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; MLCK, myosin light chain kinase; p-MLC, phosphorylated myosin light chain; TNF-α, tumor necrosis factor-α.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Derivative Assay, Staining, Knockdown, Inhibition, Expressing, Western Blot, Randall–Selitto Test, Muscles, Activation Assay, Transmission Assay, Electron Microscopy, Immunohistochemistry

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Platelet-derived growth factor receptor-α (PDGFR-α) physically interacted with collagen type I α 1 (COL1A1) in myofascial trigger points (MTrPs) rats. ( A and B ) Representative immunohistochemistry (IHC) images and Western blot (WB) results for showing expression of COL1A1 of different groups. Scale bars, 100 μm. n = 8 rats per group and at least 4 images from 1 animal. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): wild-type (WT), 0.06 ± 0.01; 95% CI, 0.04–0.07; versus MTrPs, 1.05 ± 0.10; 95% CI, 0.94–1.15; units, relative gray values; mean ± SD; P < 0.001. ( C ) The interaction between PDGFR-α and COL1A1 identified was confirmed by immunoprecipitation in MTrPs rats. ( D ) Results of molecular docking showed that PDGFR-α forms interaction forces with amino acid sites such as LYS35-ASP846, GLU55-THR894, and HIS845-ARG34 with COL1A1. ( E ) Immunofluorescence (IF) results showed that PDGFR-α ( green ) colocalized with the COL1A1 ( red ) expression. n = 8 rats per group. For WB, n = 6 values per group were included in the analysis. Data are presented as mean ± SD. *** P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Derivative Assay, Immunohistochemistry, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence

Journal: Anesthesiology

Article Title: Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

doi: 10.1097/ALN.0000000000005167

Figure Lengend Snippet: Overexpression/knockdown of collagen type I α 1 (COL1A1) induced/ameliorated pain-like behaviors and increased/decreased inflammatory factors. ( A ) Overexpression of COL1A1 in rats exhibits low-quality scores and mechanical withdrawal thresholds decreased. n = 10 rats per group for behavioral test and analysis. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 3.90 ± 1.00; 95% CI, 3.19–4.61; versus overexpression-COL1A1, 2.40 ± 0.84; 95% CI, 1.80–3.00; units, scores; mean ± SD; P < 0.01. Randall–Selitto test: vector, 412.80 ± 16.02; 95% CI, 401.40–424.30; versus overexpression-COL1A1, 355.80 ± 28.76; 95% CI, 335.20–376.30; units, g; mean ± SD; P < 0.001. ( B ) Schematic representation of virus infection in rats. Scale bar, 20 μm. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was used to validate the efficiency of COL1A1 overexpression. n = 6 rats per group. RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.06; 95% CI, 0.94–1.07; versus overexpression-COL1A1, 2.81 ± 0.74; 95% CI, 2.03–3.59; units, 2 -ΔΔCT ; mean ± SD; P < 0.001. ( C ) Knockdown of COL1A1 in myofascial trigger points (MTrPs) rats increased nest scores and mechanical withdrawal thresholds. n = 10 rats per group for behavioral test. RT-qPCR was used to validate the efficiency of COL1A1 knockdown. n = 6 rats per group. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 1.80 ± 0.79; 95% CI, 1.24–2.56; versus NC-scramble, 2.10 ± 0.99; 95% CI, 1.39–2.81; versus knockdown-COL1A1, 3.10 ± 0.74; 95% CI, 2.57–3.63; units, scores; mean ± SD; P < 0.01; P < 0.05. Randall–Selitto test: vector, 306.40 ± 17.44; 95% CI, 293.90–318.90; versus NC-scramble, 302.40 ± 19.70; 95% CI, 288.30–316.50; versus knockdown-COL1A1, 341.20 ± 28.47; 95% CI, 320.80–361.50; units, g; mean ± SD; P < 0.01. RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.10; 95% CI, 0.90–1.10; versus NC-scramble, 1.07 ± 0.34; 95% CI, 0.71–1.43; versus knockdown-COL1A1, 0.35 ± 0.17; 95% CI, 0.17–0.53; units, 2 -ΔΔCT ; mean ± SD; P < 0.001. ( D ) Overexpression/knockdown of COL1A1 within MTrPs increased/decreased inflammatory factors and contraction biomarkers expression. n = 8 rats per group. Three independent biologic replicate experiments were performed. Western blot (WB) results for proteins with similar molecular weight were from the same samples and run in parallel in different concentrations gels. Descriptive statistics of WB can be found in the Supplemental Data ( https://links.lww.com/ALN/D632 ). ( E ) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres of different groups. Scale bars, 2 μm. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. Vector, 2.32 ± 0.04; 95% CI, 2.29–2.34; versus overexpression-COL1A1, 2.07 ± 0.05; 95% CI, 2.04–2.10; mean ± SD; P < 0.001; vector, 1.51 ± 0.03; 95% CI, 1.49–1.54; versus NC-scramble, 1.56 ± 0.05; 95% CI, 1.53–1.59; versus knockdown-COL1A1, 1.70 ± 0.06; 95% CI, 1.66–1.75; units, μm; mean ± SD; P < 0.001; P < 0.01. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; kD, kilodalton; MLCK, myosin light chain kinase; p-MLC: phosphorylated myosin light chain; NC, negative control; TNF-α, tumor necrosis factor-α.

Article Snippet: Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control.

Techniques: Over Expression, Knockdown, Injection, Plasmid Preparation, Randall–Selitto Test, Virus, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Molecular Weight, Transmission Assay, Electron Microscopy, Negative Control

Journal: OncoTargets and therapy

Article Title: Potential use of STAT3 inhibitors in targeted prostate cancer therapy: future prospects

doi: 10.2147/OTT.S32559

Figure Lengend Snippet: Novel Janus kinase (JAK) inhibitors in development: numerous JAK1 and JAK2 inhibitors are being developed in clinical trials for treatment of myeloproliferative diseases and solid malignancies

Article Snippet: AZD1480 is now being studied in phase I/II clinical trials in patients with myelofibrosis and phase I clinical trials, alone or with docetaxel (Sanofi-Aventis, Paris, France), in patients with advanced solid malignancies.

Techniques: Clinical Proteomics