Journal: Molecular cancer therapeutics

Article Title: Enzalutamide Induced Feed-Forward Signaling Loop Promotes Therapy-Resistant Prostate Cancer Growth Providing an Exploitable Molecular Target for Jak2 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0508

Figure Lengend Snippet: A, CWR22Pc, LAPC4 and ENZ-R cells were cultured with ENZ or Jak2 inhibitor AZD1480 alone or in combination for 12 days at indicated concentrations. Expression levels of active Stat5 were determined by immunoprecipitation (IP) of Stat5 followed by immunoblotting (WB) for pStat5a/b and total Stat5. Whole cell lysates (WCL) were immunoblotted for pStat3, Stat3 and Actin. B, Genetic knockdown of Jak2 blocks ENZ-induced Stat5 phosphorylation in PC cells. Jak2 was suppressed by lentiviral Jak2 shRNA vs. shCtrl in PC cells for 3 days followed by ENZ or vehicle for 7 days. Active Stat5, Stat5 and Jak2 levels were evaluated by IP and WB, as depicted. C, CWR22Pc and LAPC-4 cells were treated with ENZ or vehicle for 6 h at the indicated concentrations. Control cells were stimulated with prolactin (Prl) (10 nM) for 20 min as control for cytokine-induced Jak2 phosphorylation. Alternatively, PC cells were treated with ENZ or vehicle for 12 days or stimulated with Prl for 20 min at the indicated concentrations. Stat5 and Jak2 were IP:ed and immunoblotted (WB) for pStat5, pJak2, total Stat5, and total Jak2. WCLs were immunoblotted for Actin. D, AR is required for ENZ induction of Jak2 activation. AR was suppressed in CWR22Pc cells by lentiviral AR shRNA (shAR) vs. shCtrl for 3 days followed by ENZ for 6 h. In parallel experiments, CWR22Pc cell were cultured with 1.5 μM DHT for 6 h or 7 days. ENZ induction of Jak2-Stat5 activation requires a cytokine receptor shown by treatment of CWR22Pc cells with ENZ alone or in combination with a prolactin receptor antagonist LFA102 for 6 h or 7 days at the indicated concentrations. E, ENZ induction of Jak2-Stat5 activation requires ongoing protein synthesis in PC cells. CWR22Pc cells were treated with cyclohexamide (CHX) (35 μM) for 24 h followed by ENZ treatment (40 μM) for 6 h. F, Phosphatase inhibitor Vanadate (VAN) abolished ENZ induction of Jak2 phosphorylation. CWR22Pc cells were treated with ENZ (6 h) or VAN (2 h) or vehicle alone or pre-treated with VAN (2 h) followed by ENZ (6 h). G, ENZ-induced Jak2 phosphorylation is decreased by depletion of either PTPƐ or SHP-2 phosphatases. CWR22Pc and LAPC4 cells were infected with lentivirus expressing shRNA targeting PTPƐ, SHP-2, PTP1B or shCtrl for 3 days followed by ENZ or vehicle treatment for 6 h at the indicated concentrations. IPs and WBs in were conducted as described in A.

Article Snippet: ENZ and AZD1480 were purchased from MedChem Express, MG132 and cyclohexamide (CHX) from Calbiochem, LFA102 ( 43 ) from Novartis, sodium orthovanadate from Sigma-Aldrich and IST5-002 was provided by Fox Chase Chemical Diversity Center.

Techniques: Cell Culture, Expressing, Immunoprecipitation, Western Blot, Knockdown, Phospho-proteomics, shRNA, Control, Activation Assay, Infection

Journal: Molecular cancer therapeutics

Article Title: Enzalutamide Induced Feed-Forward Signaling Loop Promotes Therapy-Resistant Prostate Cancer Growth Providing an Exploitable Molecular Target for Jak2 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0508

Figure Lengend Snippet: A, The experimental design for a sequential treatment of PC cells with AR inhibition followed by genetic or pharmacological knockdown of Stat5. B, Stat5 inhibitors alone or in combination with ENZ are more effective than ENZ alone in suppressing PC cell viability. Sequential therapy with ENZ followed by IST5-002 decreases viability of PC cells surviving ENZ treatment. CWR22Pc cells were treated at the start of the experiment (day 0) with vehicle, ENZ (40 μM) for 5 days, IST5-002 (12.5 μM) alone or in combination, as indicated or left untreated (MOCK). For sequential treatment, cells were treated at the start of the experiment (day 0) with ENZ (40 μM) followed by treatment with vehicle (ENZ>vehicle) or IST5-002 (12.5 μM) (ENZ>IST5-002) for 5 or 10 days. The fractions of viable cells are indicated by columns with SDs. Activation of Stat5 was determined by IP of followed by WB for pStat5 and total Stat5. WCLs were immunoblotted for Actin. C, Fluorescence-activated cell sorting (FACS) analysis of cell cycle distribution at the indicated timepoints. D, Pictures of PC cells stained with crystal violet after the treatments, as described in B. E, Combination of ENZ with AZD1480 is more effective than ENZ alone in decreasing viability of PC cells surviving ENZ treatment. Sequential treatment of CWR22Pc cells with ENZ (40 μM) for 5 days followed by AZD1480 (0.8 μM) for 5 or 10 days suppresses viability of PC cells surviving ENZ treatment. CWR22Pc cells were treated and analyzed for viable cells as described in A. F, FACS analysis of cell cycle distribution at the indicated timepoints. G, Crystal violet staining of the parallel wells. H, IST5-002 and AZD1480 decreased viability of both parental CWR22Pc cells and ENZ-R cells (AR-F876L) after 5 and 10 days of treatment. ENZ decreased viability of parental cells but did not affect viability of ENZ-R cells after 5 or 10 days of treatment. The fractions of viable cells are indicated by columns with SDs.

Article Snippet: ENZ and AZD1480 were purchased from MedChem Express, MG132 and cyclohexamide (CHX) from Calbiochem, LFA102 ( 43 ) from Novartis, sodium orthovanadate from Sigma-Aldrich and IST5-002 was provided by Fox Chase Chemical Diversity Center.

Techniques: Inhibition, Knockdown, Activation Assay, Fluorescence, FACS, Staining

Journal: Molecular cancer therapeutics

Article Title: Enzalutamide Induced Feed-Forward Signaling Loop Promotes Therapy-Resistant Prostate Cancer Growth Providing an Exploitable Molecular Target for Jak2 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0508

Figure Lengend Snippet: A, Inhibition of Stat5 by IST5-002 suppressed androgen-sensitive CWR22Pc xenograft tumor growth more effectively than bicalutamide (BIC) or ENZ, as shown by tumor growth curves (upper panel) and representative tumor images (lower panel). CWR22Pc cells were inoculated subcutaneously (s.c.) into flanks of castrated athymic nude mice supplied with sustained-release DHT pellets. Mice were surgically castrated or treated daily with vehicle, BIC (30 mg/kg), ENZ (30 mg/kg) or IST5-002 (IST5) (50 mg/kg), and tumor growth rates were calculated for each treatment group and are presented as fold changes in tumor volume (volume at timepoint/volume at treatment start). B, Experimental design for the sequential therapy of the xenograft tumors in vivo. A two-phase in vivo experiment using vehicle or ENZ as first-line therapy (phase I, 13 days) and vehicle, ENZ, AZD1480, IST5-002 or ENZ+IST5-002 as second-line therapy (phase II, 18 days). On day 31, mice were sacrificed and CWR22Pc xenograft tumors collected for analyses. C-D, Sequential, second-line therapy with IST5-002 (IST5) or ENZ+IST5-002 (IST5) effectively suppressed the growth of androgen-sensitive (left panel) (C) and ENZ-resistant (right panel) (D). CWR22Pc xenograft tumors, as shown by tumor growth curves (upper panels) and representative tumor images (lower panels). CWR22Pc cells were inoculated s.c. into flanks of castrated athymic nude mice supplied with sustained release DHT pellets. Mice were treated with vehicle or ENZ (30 mg/kg) for 13 days. On day 13, mice were randomly distributed into subgroups and treated daily for an additional 18 days with vehicle, ENZ (30 mg/kg), AZD1480 (30 mg/kg), IST5-002 (IST5) (50 mg/kg) or ENZ (30 mg/kg)+IST5-002 (IST5) (50 mg/kg). Tumor growth rates are calculated and presented as described in (A). E, To test the efficacy of a second-line therapy with combined ENZ and IST5-002 (IST5) vs. ENZ monotherapy in clinical PCs, seven localized PCs obtained from radical prostatectomies were cultured ex vivo in tumor explant cultures in the presence of vehicle (8 days), ENZ (40 μM) alone (8 days) or ENZ (40 μM) as first-line therapy (4 days) followed by ENZ (40 μM) and IST5-002 (IST5) (25 μM) as a second-line therapy (4 days). Second-line therapy with combined ENZ and IST5-002 (IST5) induced cell death in clinical PCs more effectively than ENZ alone, as demonstrated by extensive loss of viable acinar epithelium. The number of viable epithelial cells in the explants at the end of the cultures were counted and presented as percentage per epithelial cells prior to culture. Representative histologies are shown (image magnification 40X; scale bar: 50μm). F, Levels of nuclear Stat5 in the PC explants were determined by immunostaining using biotin-streptavidin-amplified peroxidase-antiperoxidase immunodetection at the end of the cultures and expressed as percentages of Stat5 positive cells per 100 epithelial cells in explants for each treatment group (12-20 explants) (image magnification 40X; scale bar: 50μm).

Article Snippet: ENZ and AZD1480 were purchased from MedChem Express, MG132 and cyclohexamide (CHX) from Calbiochem, LFA102 ( 43 ) from Novartis, sodium orthovanadate from Sigma-Aldrich and IST5-002 was provided by Fox Chase Chemical Diversity Center.

Techniques: Inhibition, In Vivo, Cell Culture, Ex Vivo, Immunostaining, Amplification, Immunodetection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: IL6-STAT3-C/EBPβ-IL6 positive feedback loop in tumor-associated macrophages promotes the EMT and metastasis of lung adenocarcinoma

doi: 10.1186/s13046-024-02989-x

Figure Lengend Snippet: IL-6 promotes LUAD progression by activating the JAK2/STAT3 pathway in M2-like macrophages. a GSVA analysis of differential pathways between AMs, TAMs in early and advanced LUAd. b Effects of IL-6 stimulation on common pathway expression in TAMs detected by western blot analysis. c Expression of the JAK2/STAT3 pathway with IL-6-stimulated, IL-6-stimulated with the addition of IL-6-NA in M2-like macrophages were analyzed by western blot. d Effect of AZD1480 and WP1066 on JAK2/STAT3 pathway expression in THP1 derived M2-like macrophages after IL-6 stimulation were analyzed by western blot. e Cell migration and invasion ability of LUAD cells alone, co-culture with M2-like macrophages or co-culture with M2-like macrophages followed by addition of AZD1480 and WP1066 was determined by the transwell assay. f RT-qPCR and Elisa analysis showed IL-6 expression in M2-like macrophages after IL-6 stimulation or IL-6 stimulation with the addition of AZD1480 and WP1066 (Left: Elisa results. Right: RT-qPCR results). g IL-6 expression after STAT3 overexpression or knockdown was analyzed by western blot. h RT-qPCR and Elisa analysis showed IL-6 expression in M2-like macrophages after STAT3 overexpression or knockdown

Article Snippet: JAK2 inhibitor, AZD-1480 5 μM (TargetMol, T3069, MA, USA).

Techniques: Analysis, Expressing, Western Blot, Derivative Assay, Migration, Co-culture Assay, Transwell Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Real-time Polymerase Chain Reaction

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: Biochemistry and Biophysics Reports

Article Title: Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

doi: 10.1016/j.bbrep.2020.100832

Figure Lengend Snippet: JAK2 inhibitors inhibit proliferation, while JAK1/3 inhibitors do not. A-D: SeAx cells were seeded at a cell density of 50,000 cells per well in a 96 wells plate. After incubation for 48 h with AZD1480, decernotinib, ruxolitinib and tofacitinib, viability was quantitated. E: IC 50 values were determined for AZD1480 and ruxolitinib. Error bars represent SD of three separate experiments. ****P < 0.0001 versus AZD1480.

Article Snippet: JAK inhibitors (JAKi) AZD1480 (Tocris, Bristol, UK), decernotinib (MedChem Express, Monmouth Junction, NJ), ruxolitinib (Selleck, Houston, TX) and tofacitinib (Pfizer, Capelle a/d Ijssel, The Netherlands) were dissolved in DMSO to a concentration of 10 mM.

Techniques: Incubation

Journal: Biochemistry and Biophysics Reports

Article Title: Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

doi: 10.1016/j.bbrep.2020.100832

Figure Lengend Snippet: Cucurbitacin I inhibits STAT3 and STAT5 activation, while cucurbitacin E only inhibits STAT3 activation. A: Protein lysates of SeAx cells after treatment with AZD1480, cucurbitacin E, cucurbitacin I and Stattic were visualized to show the levels of STAT3, activated STAT3, STAT5A/B and activated STAT5. B-G: Protein levels were quantified, corrected for GAPDH loading control, and normalized to the respective medium control. Total STAT3 (B), pSTAT3 (C), pSTAT3/total STAT3 levels (D) and total STAT5 (E), pSTAT5 (F) and pSTAT5/total STAT5 levels (G) were determined. Error bars represent SD of either two (STAT3) or three (STAT5) separate experiments. *P = 0.05. ****P < 0.0001.

Article Snippet: JAK inhibitors (JAKi) AZD1480 (Tocris, Bristol, UK), decernotinib (MedChem Express, Monmouth Junction, NJ), ruxolitinib (Selleck, Houston, TX) and tofacitinib (Pfizer, Capelle a/d Ijssel, The Netherlands) were dissolved in DMSO to a concentration of 10 mM.

Techniques: Activation Assay, Control

Journal: Journal of Cell Communication and Signaling

Article Title: PKCι induces differential phosphorylation of STAT3 to modify STAT3-related signaling pathways in pancreatic cancer cells

doi: 10.1007/s12079-023-00780-9

Figure Lengend Snippet: The differential phosphorylation of STAT3 at Y705 and S727 residues caused by STAT3 inhibitors Stattic, AZD 1480, and MEK inhibitor Trametinib in PDAC. a and b Immunoblot detection of pY705, pS727 STAT3, total STAT3 and GAPDH levels in MiaPaCa treated with various concentrations of a AZD1480, Stattic, and b Trametinib. c and d Comparison of the relative optical intensity of c pY705 and d pS727 STAT3 bands shown in a and b. The optical density of pY705 and pS727 STAT3 bands was normalized by total STAT3. The relative optical intensity of protein bands in control vehicle-treated samples was set to 1 (n = 3, *p < 0.05 compared with control). e and f Immunoblot analyses of pY705, pS727 STAT3, total STAT3, and GAPDH levels in PANC-1 treated with various concentrations of e AZD1480, Stattic, and f Trametinib. g and h Comparison of the relative optical intensity of (g) pY705 and (h) pS727 STAT3 bands shown in e and f. The optical density of pY705 and pS727 STAT3 bands was normalized by total STAT3. The relative optical density of control protein bands was set to 1 (n = 3, *p < 0.05 compared with control)

Article Snippet: Stattic, AZD1480 and Trametinib were all from APExBIO (TX, USA).

Techniques: Western Blot, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: PKCι induces differential phosphorylation of STAT3 to modify STAT3-related signaling pathways in pancreatic cancer cells

doi: 10.1007/s12079-023-00780-9

Figure Lengend Snippet: Inhibition of PKCι with ATM enhances the apoptotic effects exerted by STAT3 inhibitors or MEK inhibitor via modulating differential phosphorylation of STAT3. a and g Immunoblot analyses of the protein levels of pY705, pS727 STAT3, total STAT3, and GAPDH in a MiaPaCa and g PANC-1 treated with 50 nM ATM or 2 μM Stattic alone, or ATM combined with Stattic. b and h Comparison of the relative optical density of pY705, pS727 STAT3 bands in a and g. The optical density of pY705 and pS727 STAT3 bands was normalized by total STAT3. The relative optical density of control protein bands was set to 1 (n = 3, *p < 0.05 compared with control. **p < 0.05 compared with samples treated with ATM. ***p < 0.05 compared with samples treated with Stattic). c and i Immunoblot analyses of pY705, pS727 STAT3, total STAT3, and GAPDH levels in c MiaPaCa and i PANC-1 treated with 50 nM ATM or 1 μM AZD1480 alone, or ATM along with AZD1480. d and j The relative optical density of pY705, pS727 STAT3 bands shown in c and i normalized by total STAT3. The relative optical intensity of control protein bands was set to 1 (n = 3, *p < 0.05 compared with control. **p < 0.05 compared with samples treated with ATM. ***p < 0.05 compared with samples treated with AZD1480). e and k Immunoblot detection of the expression of pY705, pS727 STAT3, total STAT3, and GAPDH in e MiaPaCa and k PANC-1 treated with 50 nM ATM or 10 nM Trametinib alone, or ATM plus Trametinib. f and l The relative optical density of pY705, and pS727 STAT3 bands present in e and k normalized by total STAT3. The relative optical density of control protein bands was set to 1 (n = 3, *p < 0.05 compared with control. **p < 0.05 compared with samples treated with ATM. ***p < 0.05 compared with samples treated with Trametinib). m and p The apoptotic population in m MiaPaCa or p PANC-1 treated with vehicle solvent (DMSO), 50 nM ATM, 2 μM Stattic, or ATM combined with Stattic. Experiments were conducted in triplicate and repeated three times with similar results. Data represent mean ± SD (*p < 0.05 compared with samples treated with ATM. **p < 0.05 compared with samples treated with Stattic). n and q The apoptotic population in n MiaPaCa or q PANC-1 treated with vehicle, 50 nM ATM, 1 μM AZD1480, or ATM plus AZD1480. Experiments were performed in triplicate and repeated three times with similar results. Data represent mean ± SD (*p < 0.05 compared with samples treated with ATM. **p < 0.05 compared with samples treated with AZD1480). o and r The apoptotic population in o MiaPaCa or r PANC-1 treated with vehicle, 50 nM ATM, 10 nM or 20 nM Trametinib, or ATM coupled with Trametinib. Experiments were performed in triplicate and repeated three times with similar results. Data represent mean ± SD (*p < 0.05 compared with samples treated with vehicle. **p < 0.05 compared with samples treated with ATM. ***p < 0.05 compared with samples treated with Trametinib)

Article Snippet: Stattic, AZD1480 and Trametinib were all from APExBIO (TX, USA).

Techniques: Inhibition, Western Blot, Comparison, Expressing, Solvent

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