Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Soluble ST2 Is Regulated by p75 Neurotrophin Receptor and Predicts Mortality in Diabetic Patients With Critical Limb Ischemia

doi: 10.1161/atvbaha.112.300497

Figure Lengend Snippet: Figure 1. p75 neurotrophin receptor (p75NTR) knockout normalizes postischemic neovascu larization and blood flow recovery in 3-month diabetic mice. A, Unilateral limb ischemia was performed in 3-month diabetic (Diab) and non diabetic (non diab) wild-type (WT) and p75KO mice. Representative color laser Doppler images of limb blood flow (BF) taken at 3 days postischemia are shown. Line graph shows the time course of postischemic foot BF recovery (calculated as the ratio between isch emic and contralater foot BF; n=12 mice per group). B and C, Column graphs show capil lary density in ischemic (14 days postsurgery) adductor (B) and gastrocnemious (C) muscles (n=6 per group). D and E, Column graphs show small arterioles (diameter <50 mm) den sities in ischemic adductors (D) and gastroc nemous (E) muscles (n=6 per group). *P<0.05 vs diabetic WT mice; #P<0.05 vs nondiabetic WT mice. Data represent mean±SEM.

Article Snippet: Sections were stained overnight with rabbit anti-mouse ST2 (Abnova), rabbit anti-human ST2 (Sigma), rabbit anti-mouse p75NTR (Abcam), rat anti-mouse CD31 (BD Biosciences) or appropriate IgG control (Dako), followed by incubation with ImmPRESS reagent horse anti-mouse Ig or anti-rabbit Ig (Vector Laboratories).

Techniques: Knock-Out, Muscles

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Soluble ST2 Is Regulated by p75 Neurotrophin Receptor and Predicts Mortality in Diabetic Patients With Critical Limb Ischemia

doi: 10.1161/atvbaha.112.300497

Figure Lengend Snippet: Figure 3. ST2/interleukin-33 (IL-33) characterization in skin wounds. A, Relative expression of ST2L, sST2, and IL-33 in skin wounds of diabetic and nondiabetic wild-type (WT) and p75KO mice. Results were normalized to 18S expression. Data represent mean±SEM, n=5. *P<0.05 vs diabetic WT mice; #P<0.05 vs nondiabetic WT mice; §P<0.05 vs nondiabetic p75KO mice. B, Representative Western blot analyses of ST2L and IL-33 in skin wounds of diabetic and nondiabetic WT and p75KO mice. Bar graphs show relative protein quantifica tion of ST2L and IL-33. Relative values are normalized by α/β Tubulin levels. Western blot data represent means± SD, n=3. *P<0.05 vs diabetic WT mice; #P<0.05 vs nondiabetic WT mice. C, Fluorescent immonocytochemistry for ST2 (green fluorescence), p75 neurotrophin receptor (p75NTR; red fluorescence), and CD31 (blue fluorescence) in skin wounds of diabetic WT mice. Magnfication, ×63. Scale bar, 50 μm.

Article Snippet: Sections were stained overnight with rabbit anti-mouse ST2 (Abnova), rabbit anti-human ST2 (Sigma), rabbit anti-mouse p75NTR (Abcam), rat anti-mouse CD31 (BD Biosciences) or appropriate IgG control (Dako), followed by incubation with ImmPRESS reagent horse anti-mouse Ig or anti-rabbit Ig (Vector Laboratories).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Soluble ST2 Is Regulated by p75 Neurotrophin Receptor and Predicts Mortality in Diabetic Patients With Critical Limb Ischemia

doi: 10.1161/atvbaha.112.300497

Figure Lengend Snippet: Figure 4. p75 neurotrophin receptor (p75NTR) regulates the expression of ST2. A, Representative Western blot bands for p75NTR, ST2, and interleukin (IL)-33 proteins of human umbilical vein endothelial cells (HUVECs) treated with treated for 16 hours with either tumor necrosis factor-α (TNF-α) at the concentration of 10 ng/mL or phorbol-12-myristate-13-acetateate (PMA) at the concentration of 1 μmol/L for 16 hours (data are quantified in Supplementary Figure 9A). B, Detection of sST2 by ELISA assay in the medium of HUVECs treated as reported above. *P<0.05 vs dimethyl sulfoxide (DMSO); #P<0.05 vs PBS. C, HUVECs were transfected with small interfering RNA (siRNA) oligos for p75NTR or control oligos and treated with TNF-α for 16 hours. Representative Western blot bands for p75NTR and ST2 (data are quantified in Figure IXB in the online-only Data Supplement). D, Detection of sST2 by ELISA assay in the medium of HUVECs after p75NTR silencing and TNF-α treatment. ELISA data represent mean±SEM, *P<0.05 vs control; #P<0.05 vs control+TNF-α. (n=3). E, Representative Western blot analyses for p75NTR, phospho–c-Jun N-terminal kinase (p-JNK), JNK, phospho−mitogen-activated protein kinase (p-p38MAPK), p38MAPK, phospho–activating transcription factor 2 (p-ATF-2), ATF-2, and ST2 proteins of HUVECs cells infected with adenovirus (Ad).Null or Ad.p75NTR (data are quantified in Figure XIE in the online-only Data Supple ment). F, Representative Western blot bands for ATF-2, c-Jun, and ST2 proteins of HUVECs transfected with ATF-2 siRNA oligos and subsequently infected with Ad.Null or Ad.p75NTR (data are quantified in Figure XIF in the online-only Data Supplement). G, Representa tive Western blot bands for c-Jun and ST2 proteins of HUVECs cells transfected with c-Jun siRNA oligos and subsequently infected with Ad.Null or Ad.p75NTR (data are quantified in Figure XIG in the online-only Data Supplement). H, Detection of sST2 by ELISA assay in the medium of HUVECs after ATF-2, c-Jun silenc ing, p75NTR or null transduction. ELISA data represent mean±SEM. *P<0.05 vs Ad.Null+control; #P<0.05 vs control+Ad.p75NTR (n=3).

Article Snippet: Sections were stained overnight with rabbit anti-mouse ST2 (Abnova), rabbit anti-human ST2 (Sigma), rabbit anti-mouse p75NTR (Abcam), rat anti-mouse CD31 (BD Biosciences) or appropriate IgG control (Dako), followed by incubation with ImmPRESS reagent horse anti-mouse Ig or anti-rabbit Ig (Vector Laboratories).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Small Interfering RNA, Control, Infection, Transduction

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 1. p75NTR is a marker for human muscle satellite cells and regenerating fibers. (A) p75NTR immunoreactivity in a satellite cell of adult skeletal muscle. (B) Confocal imaging for p75NTR and CD56/neural cell adhesion molecule (NCAM) on satellite cells in vivo. Laminin staining was used to define myofibers. In (A) and (B), arrows indicate positively stained satellite cells. (C) Percen- tages of CD56/NCAM-positive satellite cells expressing p75NTR in human adult skeletal muscle. Circles represent distinct tissue samples; horizontal bar represents mean. (D) Quantification of CD56/NCAM-positive regenerating myofibers. Black bars indicate average values for each group, dots represent distinct tissue samples. (E, F) p75NTR expression (F) on CD56/NCAM-positive (E) regenerating fibers. Stainings were performed on serial sections from a dermatomyositis (DM) muscle. Arrows indicate positively stained fibers. Scale bars = (A, B) 5 Km; (E, F) 10 Km. ***p G 0.001 versus control. CTRL, control; PM, polymyositis; IBM, inclusion body myositis; BMD, Becker muscular dystrophy.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Marker, Imaging, In Vivo, Staining, Expressing, Control

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 2. p75NTR is expressed in cultured muscle precursor cells and transiently upregulated upon differentiation. (A) Double immunofluorescence for p75NTR and CD56/NCAM in cultured myoblasts. (B) Upregulation of p75NTR mRNA levels upon dif- ferentiation. Quantitative real-time PCR for p75NTR was performed for each time point. For statistics, p75NTR transcript levels at distinct time points were compared with baseline (Day 0). (C) Flow cytometry experiments showing percentages of p75NTR- positive mononucleated myoblasts after exposure to differentiation medium. (D) Immunofluorescence for p75NTR and dystrophin in myotubes (Day 11). (E) Kinetics of p75NTR expression on myotubes and correlation with dystrophin induction. Costaining for p75NTR and dystrophin was performed on differentiating cultures. The percentages of p75NTR (square) or dystrophin (circle) or p75NTR-positive dystrophin-positive (triangle) myotubes were measured at different time points. Experiments were performed in triplicate, and at least 100 myotubes per replicate were counted. Similar observations were obtained in at least 3 independent experiments in at least 2 primary cell lines. Error bars represent SD. Scale bars = (A, D) 30 Km. *p G 0.05, ***p G 0.001.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Expressing

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Human Neurotrophin Receptor p75NTR Defines Differentiation-Oriented Skeletal Muscle Precursor Cells: Implications for Muscle Regeneration

doi: 10.1097/nen.0b013e3182084391

Figure Lengend Snippet: FIGURE 4. p75NTR regulates myogenesis and dystrophin expression in myotubes. (A) Fusion index in a blocking experi- ment with anti-p75NTR antibody or isotype control adminis- tered during cell differentiation. (B) Extent of myogenesis in p75NTRlow, p75NTRhigh, and p75NTRhigh myoblasts treated with anti-p75NTR blocking antibody. In (A) and (B), fusion was evaluated at Day 6 after differentiation induction. (C) p75NTR silencing in myoblasts and effects on myogenesis. Silencing efficiency and relative fusion index were evaluated at Day 8 after differentiation induction. p75NTR mRNA (first panel) and protein (second panel) levels in p75NTR-silenced myotubes were significantly lower than in control myotubes. p75NTR RNA interference decreased cell differentiation (third panel). (D) p75NTR silencing did not alter myoblast proliferation. Small- interfering RNA administration was performed at Day 0; cells were maintained in growth medium for a few days and counted. (E) Cell cycle analysis was detected by propidium iodide incor- poration in silenced myoblasts. Evaluation was performed at Day 3 after transfection with p75NTR or control siRNA. Cell cycle entry in p75NTR-silenced cells was unchanged. (F) Quantifica- tion of dystrophin, A-dystroglycan, and dysferlin-positive myo- tubes in differentiated cultures at Day 8 after differentiation induction. Shown experiments were performed in triplicate, and at least 100 myotubes per replicate were counted. Similar observations were obtained in at least 3 independent experi- ments. Error bars represent SD. Results in (A, C, D, and E) were confirmed in 2 primary cell lines. **p G 0.01, ***p G 0.001; ns, not significant.

Article Snippet: Immunofluorescence Six-micrometer-thick tissue sections were fixed with methanol and blocked and stained with the following primary antibodies: mouse monoclonal anti-human p75NTR (clone 74902; R&D Systems, Milan, Italy); mouse monoclonal antihuman CD56 (clone 555514; BD Biosciences); rabbit polyclonal anti-human dystrophin and rat polyclonal anti-human laminin (both provided by Dr. Mora); mouse monoclonal anti-human MyoG (clone F5D; Dako, Milan, Italy); mouse monoclonal anti-human dysferlin (clone HAM1; Novocastra, Florence, Italy); and mouse monoclonal anti-human Adystroglycan (clone 43DAG1/8D5; Novocastra).

Techniques: Expressing, Blocking Assay, Control, Cell Differentiation, Small Interfering RNA, Cell Cycle Assay, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: The expression of NR4A1 is induced in osteoarthritis. (A,B) The detection of NR4A1 and p65 expression in human osteoarthritis cartilage and normal cartilage through western-blot (A) and RT-PCR analysis (B) . (C,D) The effect of NF-κB signal inhibitor JSH23 on IL-1β induced NR4A1 expression in rat chondrocytes: (C) RT-PCR analysis; (D) Western-blot analysis. # p < 0.05, compared with the negative control group. * p < 0.05, compared with the IL-1β group.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: NR4A1 could inhibit NF-κB signal and chondrocyte inflammation associated gene expression. (A–D) The overexpression experiment: The RT-PCR analysis (A) and western-blot analysis (B) of the effect of NR4A1 overexpression on IL-1β induced COX-2, iNOS, MMP3, MMP9, MMP13 expression in rat chondrocytes. (C) Quantity analysis of the protein bands in panel (B) . (D) Luciferase reporter gene analysis to detect the transcriptional activity of NF-κB pathway. (E–H) The siRNA experiment: The RT-PCR analysis (E) and western-blot analysis (F) of the effect of NR4A1 knockdown on IL-1β induced COX-2, iNOS, MMP3, MMP9, MMP13 expression in rat chondrocytes. (G) Quantity analysis of the protein bands in panel (F) . (H) Luciferase reporter gene analysis to detect the transcriptional activity of NF-κB pathway. * p < 0.05.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: The NR4A1 is relatively inhibited in the central area of OA. (A) Human normal and OA samples. The upper row: SO staining, scale bar = 1 mm. The Lower row: Immunohistochemistry (IHC) against NR4A1, scale bar = 50 μm. (B) Rat OA models. Yellow dotted box: relatively normal area in OA joint. Purple dotted box: the central area of OA joint. Scale bar = 100 μm.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: NR4A1 was relatively inactivated in osteoarthritis through HDACs mediated transcriptional suppression and MAKP dependent phosphorylation. (A,B) The expression of NR4A1 in conditions of chronic NF-κB signal activation. (A) The mRNA level. (B) The protein level of NR4A1, phosphorylated NR4A1, acetyl H4 lys8 and acetyl H3 lys27. Chondrocytes treated with 10 ng/ml IL-1β for different time were collected for the detection. nc, negative control. (C) The dynamic expression of HDACs under chronic NF-κB signal activation; * p < 0.05 compared with negative control at the same time point. (D) The effect of HDAC inhibitor SAHA (5 μM) on the NR4A1 expression, acetyl H4 lys8 and acetyl H3 lys27 after 72 h IL-1β treatment. (E) The effect of p38 inhibitor SB203580, JNK inhibitor SP600125 and ERK inhibitor FR180204 on NR4A1 phosphorylation. (F) The NR4A1 and phosphorylated NR4A1 level in human osteoarthritis and normal cartilage tissues. (G) The effect of p38 inhibitor SB203580, JNK inhibitor SP600125 and ERK inhibitor FR180204 on NR4A1 subcellular location.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Activation Assay, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: Cytosporone B reactivated NR4A1 and attenuated chondrocyte inflammation and OA development in vitro and in vivo . (A,B) The effect of cytosporone B on IL-1β induced COX2, iNOS, MMP3, MMP9 and MMP13 expression. (A) Protein level. (B) mRNA level. (C) The effect of cytosporone B on NR4A1 phosphorylation, NF-κB signal activation and MAPK pathway activation. (D) SO staining of sham surgery and rat knee joint OA model treated with 1 μM cytosporone B or vehicle. Scale bar = 500 μm (upper), 250 μm (lower). (E) The effect of 1 μM cytosporone B on NR4A1 subcellular location. (F) OARSI scores of each group. * p < 0.05.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques: In Vitro, In Vivo, Expressing, Activation Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reactivation of NR4A1 Restrains Chondrocyte Inflammation and Ameliorates Osteoarthritis in Rats

doi: 10.3389/fcell.2020.00158

Figure Lengend Snippet: The schematic diagram of the regulatory role of NR4A1 in chondrocyte inflammation and osteoarthritis. NR4A1 acts as an endogenous inhibitor of p65 and suppresses chondrocyte inflammation. However, NR4A1 was relatively inactivated in osteoarthritis through HDACs mediated transcriptional suppression and MAPK dependent phosphorylation. Cytosporone B can reactivate NR4A1 and attenuate chondrocyte inflammation.

Article Snippet: NR4A1 siRNA were purchased from Genepharma Technology Co., Ltd. (Shanghai, China).

Techniques:

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Roles of NR4A1 and NR4A2 in endometriotic cell growth and apoptosis. IHEEC and IHESC cells were transfected with siNR4A1 and siNR4A2, and effects on protein levels (A) and cell viability (B) were determined as outlined in the Methods. Apoptosis was analyzed 72 hours after transfection of IHEEC and IHESC cells with siRNAs targeting NR4A1 (siNR4A1) or NR4A2 (siNR4A2). Representative flow cytometry dot plots showing apoptosis profiles in IHEEC and IHESC are presented (C, D). In each plot: the upper left quadrant (Q1) represents necrotic cells (PI-positive only), the upper right quadrant (Q2) shows late apoptotic cells (Annexin V-FITC and PI double-positive), the lower right quadrant (Q3) indicates early apoptotic cells (Annexin V-FITC–positive only), and the lower left quadrant (Q4) represents viable cells (double-negative). Quantification of early apoptotic (E) and late apoptotic (F) cell populations is shown. Statistical significance was determined as follows: * P < .05, ** P < .01, *** P < .001. Abbreviations: FITC, fluorescein isothiocyanate; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2; PI, propidium iodide; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Transfection, Flow Cytometry, Small Interfering RNA

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs on proliferation and migration of IHEEC and IHESC cells. The structure of the DIM-3,5 analogs (A) and corresponding dissociation constants (Kd) values of selected DIM-3,5 analogs for NR4A1 and NR4A2 are shown (B). IC 50 values for IHEEC and IHESC cells at 48 hours, determined from XTT assays (C). IHEEC and stromal IHESC cells were treated with selected DIM-3,5 analogs, and cell proliferation was assessed using XTT assays, as described in the Methods section (D, E). IHEEC (F) and IHESC (G) cells were transfected with oligonucleotides targeting NR4A1 or NR4A2 or treated with DIM-3,5 analogs (6.5 µM) for 24 hours, and effects on cell migration were determined in a scratch assay as outlined in the Materials and Methods. Data are presented as means ± SD for 3 replicate determinations, and statistical differences ( P < .05) of treated vs controls (DMSO) are indicated (*). Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Migration, Transfection, Wound Healing Assay

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A knockdown on EGFR and C-PARP expression in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) and oligonucleotides that target NR4A1 and NR4A2 (B). Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were also treated with DIM-3,5 ligands (C) and transfected with siNR4A1 or siNR4A2 (D), and whole-cell lysates were obtained and analyzed by Western blots as outlined in the Methods. Protein expression levels were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). The same lysates from IHEEC cells were used for the blots shown in and , and the same lysates from IHESC cells were used for the blots shown in and ; therefore, the β-actin loading controls are identical. Data are presented as mean ± SD. * P < .05. Abbreviations: C-PARP, cleaved poly(ADP-ribose) polymerase; DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Knockdown, Expressing, Control, Western Blot, Transfection

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A1/NR4A2 knockdown on EMT-related markers expression in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) for 24 hours and transfected with siNR4A1 or siNR4A2 (B) for 72 hours. Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 analogs (C) for 24 hours, and transfected with siNR4A1 or siNR4A2 (D) for 72 hours, and whole-cell lysates were analyzed by Western blots. Protein expression levels of EMT markers were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; EMT, epithelial-to-mesenchymal transition; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs NR4A1 and NR4A2 knockdown on β1-integrin, estrogen receptor β, and mTOR signaling in IHEEC and IHESC cells. IHEEC cells were treated with DIM-3,5 analogs for 24 hours (A) and transfected with siRNAs targeting NR4A1 (siNR4A1) or NR4A2 (siNR4A2) (B) for 72 hours. Whole-cell lysates were then collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 analogs for 24 hours (C) and transfected with siNR4A1 or siNR4A2 for 72 hours (D). Whole-cell lysates were analyzed by Western blots. Protein expression levels were quantified by measuring relative band intensities compared to control siRNA-treated cells (set at 1.0) and normalized to β-actin (A–D). Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; mTOR, mechanistic target of rapamycin; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Knockdown, Transfection, Western Blot, Expressing, Control, Small Interfering RNA

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A1/NR4A2 knockdown on fibrosis markers in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) and transfected with siNR4A1 or siNR4A2 (B). Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 compound for 24 hours (C) and transfected with siNR4A1 or siNR4A2 for 72 hours (D), and whole-cell lysates were analyzed by Western blots. Protein expression levels were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). The same lysates from IHESC cells were used for the blots shown in and , as well as and ; therefore, the β-actin loading controls are identical. Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Knockdown, Control, Transfection, Western Blot, Expressing

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Suppression of ectopic lesion growth in a mouse model of endometriosis by DIM-3,5-Cl 2 treatment. Mice with surgically induced endometriosis were treated once daily with DIM-3,5-Cl 2 (2.5 mg/kg) or vehicle control for 34 days (A). Following treatment, ectopic lesions were harvested, and their volumes were calculated using the formula: 0.5 × Length × Width 2 (B). Luciferase activity of ectopic lesions was monitored throughout the treatment period using an in vivo imaging system. Luciferase signal intensity for each lesion was quantified and plotted in the corresponding graph (C). Proliferation in ectopic lesions was assessed by Ki-67 IHC (D). The levels of NR4a1 and Nr4a2 in epithelial and stromal compartments were determined by IHC (F, G), and H-scores were calculated using QuPath software . Apoptosis was evaluated by TUNEL assay (E), and the percentage of TUNEL-positive cells in ectopic lesions was quantified using QuPath. H-scores for Nr4a1 and Nr4a2 expression in each cell compartment were also determined using QuPath. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; IHC, immunohistochemistry; N.S., not significant; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Control, Luciferase, Activity Assay, In Vivo Imaging, Software, TUNEL Assay, Expressing, Immunohistochemistry, End Labeling

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1 is significantly upregulated while proteins in LKB1/AMPK signaling is downregulated in H/R-treated BRL-3A cells. (a-b) Expression of NR4A1 in BRL-3A cells treated with H/R was detected using western blotting and RT-qPCR. (c) The expression or proteins in LKB1/AMPK signaling in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control.

Article Snippet: Small interference RNAs (siRNAs) carrying NR4A1 (siRNA-NR4A1-1 and siRNA-NR4A1-2) and its negative control (siRNA-NC) were purchased from RiboBio.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1 inhibits the activation of LKB1/AMPK pathway in BRL-3A cells treated with H/R. (a-b) siRNA NR4A1-mediated transfection efficiency was determined by western blotting and RT-qPCR. (c) Effect of NR4A1 knockdown on the phosphorylation levels of the LKB1/AMPK signaling in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control; ###P < 0.001 vs. H/R+ siRNA-NC. (d-e) Western blot and RT-qPCR were used for detecting the transfection efficiency of NR4A1 overexpressed plasmid. ***P < 0.001 vs. ov-NC. (f). Effect of NR4A1 overexpression on the phosphorylation levels of the LKB1/AMPK signaling pathway in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ Ov-NC.

Article Snippet: Small interference RNAs (siRNAs) carrying NR4A1 (siRNA-NR4A1-1 and siRNA-NR4A1-2) and its negative control (siRNA-NC) were purchased from RiboBio.

Techniques: Activation Assay, Transfection, Western Blot, Quantitative RT-PCR, Knockdown, Phospho-proteomics, Control, Plasmid Preparation, Over Expression

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown attenuates oxidative stress and inflammation of BRL-3A cells exposed to H/R through LKB1/AMPK pathway. (a) Cell viability was determined by CCK-8 assay. (b-c) Intracellular ROS level was examined by DCFH-DA staining. (d-e) Measurement of MDA and SOD by respective kits. (f-i) Expression levels of TNF-α, IL-1β, IL-6, and MCP-1 mRNA in BRL-3A cells treated with H/R was determined by RT-qPCR. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Small interference RNAs (siRNAs) carrying NR4A1 (siRNA-NR4A1-1 and siRNA-NR4A1-2) and its negative control (siRNA-NC) were purchased from RiboBio.

Techniques: Knockdown, CCK-8 Assay, Staining, Expressing, Quantitative RT-PCR, Control

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown ameliorates BRL-3A cell apoptosis induced by H/R through LKB1/AMPK pathway. (a) Representative dot plots from flow cytometry analysis. (b) Cell apoptosis was determined by flow cytometry analysis. (c) Expression of apoptosis-related proteins (Bcl-2, Bax, caspase3, cleaved caspase3, PARP, and cleaved RAPR) were detected by western blotting. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; Δ P<0.05, ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Small interference RNAs (siRNAs) carrying NR4A1 (siRNA-NR4A1-1 and siRNA-NR4A1-2) and its negative control (siRNA-NC) were purchased from RiboBio.

Techniques: Knockdown, Flow Cytometry, Expressing, Western Blot, Control

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown promotes autophagy of BRL-3A cells treated with H/R through LKB1/AMPK pathway. Western blot was used to evaluate the expression of autophagy-related proteins. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Small interference RNAs (siRNAs) carrying NR4A1 (siRNA-NR4A1-1 and siRNA-NR4A1-2) and its negative control (siRNA-NC) were purchased from RiboBio.

Techniques: Knockdown, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: Macrophage memories of early-life injury drive neonatal nociceptive priming

doi: 10.1016/j.celrep.2024.114129

Figure Lengend Snippet: (A) Example sorting of peritoneal macrophages from indicated conditions. (B) PCA from ATAC-seq datasets. (C) PCA form the RNA-seq dataset. (D) The top 100 differentially accessible chromatin regions in macrophages in P35 naive cells compared to P35 primed cells (age matched). (E) The top 100 differentially expressed genes (DEGs) for the same conditions. (F) A table of factors that had differentially accessible regions and DEGs. NGFR (p75NTR) is indicated. (G and H) ATAC-seq (G) indicating higher reads of NGFR (* p = 0.001 vs. controls, one-way ANOVA, Tukey’s, n = 3–4/group) and (H) transcripts per million data for RNA-seq (* p = 0.008 vs. controls). Mean ± SEM.

Article Snippet: siRNA against p75NTR – Mouse , Origene , SR414146.

Techniques: RNA Sequencing

Journal: Cell reports

Article Title: Macrophage memories of early-life injury drive neonatal nociceptive priming

doi: 10.1016/j.celrep.2024.114129

Figure Lengend Snippet: (A) Schematic of knocking out p75NTR from macrophages following neonatal incision. (B) Representative images of p75NTR labeling (green) co-stained with F4/80 (macrophages, white) in the injured hindpaw. Yellow arrows, double-positive staining. Scale bar, 25 μM. (C) There is a significant reduction (F = 4.012) in the percentage of double-positive cells in LysM;p75fl/fl animals compared to other genotypes (* p < 0.05 vs. controls (p75+/fl and p75 fl/fl) and LysM; p75+/fl, one way ANOVA, Tukey’s, n = 3–5/group). (D) We found no effect of tamoxifen prior to the second injury (No Tam vs. BL time points) on paw guarding. Following the second incision, there was robust paw guarding (F = 26.258) with a faster recovery in the LysM;p75NTRfl/fl mice (* p < 0.05 vs. BL, # p = 0.05 vs. BL p75 fl/fl, two-way RM ANOVA, Tukey’s). (E) No effect of tamoxifen on mechanical withdrawal thresholds was found prior to injury (No Tam vs. BL time points); however, there was an effect following a second incision (F = 40.516). Both groups displayed mechanical sensitivity on days 1, 3, and 7 (* p < 0.01 vs. BL). p75fl/fl controls continued to display significant sensitivities at day 14 while LysM; p75 fl/fl animals did not (p75fl/fl: * p = 0.002 vs. BL, LysM;p75fl/fl: p = 0.063 vs. BL). One day following the second incision, LysM;p75fl/fl animals were less hypersensitive than controls ( ^ p = 0.01 vs. controls). Two-way RM ANOVA, Tukey’s, n = 7/group. Colored horizontal lines indicate the duration of significance compared to BL for each group. Mean ± SEM.

Article Snippet: siRNA against p75NTR – Mouse , Origene , SR414146.

Techniques: Labeling, Staining

Journal: Cell reports

Article Title: Macrophage memories of early-life injury drive neonatal nociceptive priming

doi: 10.1016/j.celrep.2024.114129

Figure Lengend Snippet: (A) Example protein arrays of BMDM lysates with knockdown of p75NTR and stimulated with the indicated factors. (B) Heatmap indicating protein expression detected across each stimulus; n = 4/group. (C) Examples of significantly regulated proteins from the arrays (* p < 0.05 vs. siCON+Veh, ^ p < 0.05 vs. sip75+LPS, # p = 0.05 vs. sip75+Veh, ## p > 0.05 vs. sip75+Veh; one-way ANOVA, Tukey’s or ANOVA on ranks, Dunn’s; n = 4/group). (D–F) Representative images of iPSC-derived macrophages, sensory neurons, and Rhod-2 responses to ATP. Arrows, increased responses; arrowheads, new responses. Scale bar, 25 μm. (G) Example calcium transients from iPSC-derived sensory neurons in response to ATP, capsaicin, or KCl. Arrows indicate when the ATP or capsaicin was added, and the brackets indicate the part of the trace that was analyzed to calculate responses. scale bar, 20 μM. (H) Exposure of iPSC-derived sensory neurons to medium from macrophages with loss of p75NTR treated with NGF caused decreased responsiveness of neurons to ATP (F = 4.257) (* p < 0.04 vs. all other conditions, one-way ANOVA with Tukey’s post hoc, n = 15 cells per group). (I) No changes in capsaicin responses (F = 2.702) were observed (one way ANOVA). Mean ± SEM or percent change from controls with variance.

Article Snippet: siRNA against p75NTR – Mouse , Origene , SR414146.

Techniques: Knockdown, Expressing, Derivative Assay

Journal: Cell reports

Article Title: Macrophage memories of early-life injury drive neonatal nociceptive priming

doi: 10.1016/j.celrep.2024.114129

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: siRNA against p75NTR – Mouse , Origene , SR414146.

Techniques: Recombinant