Structured Review

Cell Signaling Technology Inc phosphorylated ikkβ
(+)-Vitisin (Vt-A) concentration-dependently repressed RANKL-induced phosphorylation of <t>IKKβ,</t> degradation of IκB and nuclear translocation of NF-κB, respectively. Protein samples were prepared after 15 min (for IKK and IκB) or 1 h (for NF-κB) of RANKL stimulation then analyzed by Western blotting. Results are expressed as the mean ± SEM for each group from four to five separate experiments. Relative protein level was normalized by histone (for NF-κB), total IKK (for phosphorylated IKK) or β-actin (for IκB), respectively. *p
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1) Product Images from "(+)-Vitisin A Inhibits Osteoclast Differentiation by Preventing TRAF6 Ubiquitination and TRAF6-TAK1 Formation to Suppress NFATc1 Activation"

Article Title: (+)-Vitisin A Inhibits Osteoclast Differentiation by Preventing TRAF6 Ubiquitination and TRAF6-TAK1 Formation to Suppress NFATc1 Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089159

(+)-Vitisin (Vt-A) concentration-dependently repressed RANKL-induced phosphorylation of IKKβ, degradation of IκB and nuclear translocation of NF-κB, respectively. Protein samples were prepared after 15 min (for IKK and IκB) or 1 h (for NF-κB) of RANKL stimulation then analyzed by Western blotting. Results are expressed as the mean ± SEM for each group from four to five separate experiments. Relative protein level was normalized by histone (for NF-κB), total IKK (for phosphorylated IKK) or β-actin (for IκB), respectively. *p
Figure Legend Snippet: (+)-Vitisin (Vt-A) concentration-dependently repressed RANKL-induced phosphorylation of IKKβ, degradation of IκB and nuclear translocation of NF-κB, respectively. Protein samples were prepared after 15 min (for IKK and IκB) or 1 h (for NF-κB) of RANKL stimulation then analyzed by Western blotting. Results are expressed as the mean ± SEM for each group from four to five separate experiments. Relative protein level was normalized by histone (for NF-κB), total IKK (for phosphorylated IKK) or β-actin (for IκB), respectively. *p

Techniques Used: Concentration Assay, Translocation Assay, Western Blot

2) Product Images from "RasGRP1 Is an Essential Signaling Molecule For Development of B1a Cells With Autoantigen Receptors"

Article Title: RasGRP1 Is an Essential Signaling Molecule For Development of B1a Cells With Autoantigen Receptors

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1502132

RasGRP1 is dispensable for BCR signaling outcomes beyond p-ERK in B1 cells (A) RasGRP1 is dispensable for BCR-stimulated p-JNK. B1a cells were sorted from wild type (WT) and RasGRP1-deficient (KO) mice and were stimulated with anti-Ig (15 μg/ml) (αIg) for 5 and 10 min. p-JNK was evaluated by immunoblot. Membranes were stripped and reprobed with JNK-specific antibody as a loading control. (B) RasGRP1 is dispensable for BCR-stimulated p-p38. Peritoneal lymphocytes from wild type and RasGRP1-deficient mice were unstimulated (US) (WT: red line; KO: blue line) or stimulated (WT: green line; KO: orange line) with anti-Ig (αIg) (15 μg/ml) for 1 min. p-p38 in B1 cells (CD19+CD43+) was evaluated by intracellular immunofluorence staining and flow cytometry. (C) Constitutive expression of NFATc1 is intact in RasGRP1-deficient B1 cells. NFATc1 protein expression was evaluated in WT B1 cells, WT B2 cells, KO B1 cells, and KO B2 cells by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. (D) BCR-stimulated NFATc1 protein expression is intact in RasGRP1-deficient B1 cells. WT B1 cells (left upper panels), WT B2 cells (left lower panels), KO B1 cells (right upper panels), and KO B2 cells (right lower panels) were stimulated with anti-Ig (15 μg/ml) (αIg) for 0, 1, or 2 days. NFATc1 protein expression was evaluated by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. Results represent one of three comparable experiments.
Figure Legend Snippet: RasGRP1 is dispensable for BCR signaling outcomes beyond p-ERK in B1 cells (A) RasGRP1 is dispensable for BCR-stimulated p-JNK. B1a cells were sorted from wild type (WT) and RasGRP1-deficient (KO) mice and were stimulated with anti-Ig (15 μg/ml) (αIg) for 5 and 10 min. p-JNK was evaluated by immunoblot. Membranes were stripped and reprobed with JNK-specific antibody as a loading control. (B) RasGRP1 is dispensable for BCR-stimulated p-p38. Peritoneal lymphocytes from wild type and RasGRP1-deficient mice were unstimulated (US) (WT: red line; KO: blue line) or stimulated (WT: green line; KO: orange line) with anti-Ig (αIg) (15 μg/ml) for 1 min. p-p38 in B1 cells (CD19+CD43+) was evaluated by intracellular immunofluorence staining and flow cytometry. (C) Constitutive expression of NFATc1 is intact in RasGRP1-deficient B1 cells. NFATc1 protein expression was evaluated in WT B1 cells, WT B2 cells, KO B1 cells, and KO B2 cells by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. (D) BCR-stimulated NFATc1 protein expression is intact in RasGRP1-deficient B1 cells. WT B1 cells (left upper panels), WT B2 cells (left lower panels), KO B1 cells (right upper panels), and KO B2 cells (right lower panels) were stimulated with anti-Ig (15 μg/ml) (αIg) for 0, 1, or 2 days. NFATc1 protein expression was evaluated by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. Results represent one of three comparable experiments.

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing

3) Product Images from "Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation"

Article Title: Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2013/509482

Effects of CZE on RANKL-induced intracellular signaling and the expression of transcription factors in osteoclasts. BMMs were treated with M-CSF and RANKL in the presence or absence of CZE (25 μ g/mL) for the indicated time. Lysate (30 μ g) was subjected to SDS-PAGE and analyzed by immunoblotting. (a) MAPK (ERK, JNK, and p38) activation was measured by using their respective antibodies. (b)-(c) The expression of c-Fos and NFATc1 was detected by anti-c-Fos and NFATc1 antibody, respectively. Fold change normalized by actin is presented in the right panel. Data are representatively obtained from three independent experiments and are expressed as the mea n ± SD. * P
Figure Legend Snippet: Effects of CZE on RANKL-induced intracellular signaling and the expression of transcription factors in osteoclasts. BMMs were treated with M-CSF and RANKL in the presence or absence of CZE (25 μ g/mL) for the indicated time. Lysate (30 μ g) was subjected to SDS-PAGE and analyzed by immunoblotting. (a) MAPK (ERK, JNK, and p38) activation was measured by using their respective antibodies. (b)-(c) The expression of c-Fos and NFATc1 was detected by anti-c-Fos and NFATc1 antibody, respectively. Fold change normalized by actin is presented in the right panel. Data are representatively obtained from three independent experiments and are expressed as the mea n ± SD. * P

Techniques Used: Expressing, SDS Page, Activation Assay, Microelectrode Array

4) Product Images from "Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis"

Article Title: Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.4480

Effects of PEE on RANKL-induced intracellular signaling. Bone marrow-derived macrophages were treated with or without PEE (25 µg/ml) in the presence of RANKL and M-CSF for the indicated durations (0, 1, 2, 3 and 4 days). Whole cell extracts were subjected to western blot analysis. (A) Phospho-CREB, (B) NFATc1 and (C) c-fos were detected with the specific antibodies. β-actin was used as a loading control. PEE, Peucedanum japonicum Thunb. ethanol extract; RANKL, receptor activator of nuclear factor κB ligand; M-CSF, macrophage colony-stimulating factor; CREB, cAMP response element-binding protein; NFATc1, nuclear factor of activated T cells, cytoplasmic 1.
Figure Legend Snippet: Effects of PEE on RANKL-induced intracellular signaling. Bone marrow-derived macrophages were treated with or without PEE (25 µg/ml) in the presence of RANKL and M-CSF for the indicated durations (0, 1, 2, 3 and 4 days). Whole cell extracts were subjected to western blot analysis. (A) Phospho-CREB, (B) NFATc1 and (C) c-fos were detected with the specific antibodies. β-actin was used as a loading control. PEE, Peucedanum japonicum Thunb. ethanol extract; RANKL, receptor activator of nuclear factor κB ligand; M-CSF, macrophage colony-stimulating factor; CREB, cAMP response element-binding protein; NFATc1, nuclear factor of activated T cells, cytoplasmic 1.

Techniques Used: Derivative Assay, Western Blot, Binding Assay

5) Product Images from "Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action"

Article Title: Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18061142

Melatonin showed little effect on RANKL-induced MAPKs activation but significantly reduced NFATc1 expression. ( A ) BMMs were cultured with osteoclastogenic medium (30 ng/mL M-CSF + 100 ng/mL RANKL) for the indicated days in the absence or presence of melatonin (500 µM). The mRNA expressions of NFATc1, c-Fos, TRAP, CTR (calcitonin receptor), and CTK (cathepsin K) were determined by RT-PCR analyses. TRAP, CTR, and CTK were served as a osteoclastic differentiation marker; ( B ) BMMs were cultured as in panel ( A ) and the NFATc1 expression ( upper panel) and c-Fos expression ( lower panel) were examined by immunoblotting; ( C ) BMMs were serum starved for 5 h, pretreated with vehicle or melatonin (500 µM) for 2 h, and stimulated with RANKL (100 ng/mL) for the indicated times. Cell lysates were subjected to Western blotting using phospho-specific antibodies against ERK, JNK, and p38. The equal loadings of samples were probed by ERK, JNK, and p38 antibodies, respectively; ( D ) BMMs were cultured as in panel ( A ) and the protein expression of RANK was determined by immunoblotting. Numbers represent the relative band intensity of indicated bands normalized to that of control (actin or respective un-phosphorylated MAPKs) by densitometry. * p
Figure Legend Snippet: Melatonin showed little effect on RANKL-induced MAPKs activation but significantly reduced NFATc1 expression. ( A ) BMMs were cultured with osteoclastogenic medium (30 ng/mL M-CSF + 100 ng/mL RANKL) for the indicated days in the absence or presence of melatonin (500 µM). The mRNA expressions of NFATc1, c-Fos, TRAP, CTR (calcitonin receptor), and CTK (cathepsin K) were determined by RT-PCR analyses. TRAP, CTR, and CTK were served as a osteoclastic differentiation marker; ( B ) BMMs were cultured as in panel ( A ) and the NFATc1 expression ( upper panel) and c-Fos expression ( lower panel) were examined by immunoblotting; ( C ) BMMs were serum starved for 5 h, pretreated with vehicle or melatonin (500 µM) for 2 h, and stimulated with RANKL (100 ng/mL) for the indicated times. Cell lysates were subjected to Western blotting using phospho-specific antibodies against ERK, JNK, and p38. The equal loadings of samples were probed by ERK, JNK, and p38 antibodies, respectively; ( D ) BMMs were cultured as in panel ( A ) and the protein expression of RANK was determined by immunoblotting. Numbers represent the relative band intensity of indicated bands normalized to that of control (actin or respective un-phosphorylated MAPKs) by densitometry. * p

Techniques Used: Activation Assay, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Marker, Western Blot

6) Product Images from "Pharmacological inhibition of tankyrase induces bone loss in mice by increasing osteoclastogenesis"

Article Title: Pharmacological inhibition of tankyrase induces bone loss in mice by increasing osteoclastogenesis

Journal: Bone

doi: 10.1016/j.bone.2017.10.017

Osteoblast-promoting effect of tankyrase inhibitors. Primary calvaria cells isolated from WT pups were cultured in the presence of IWR-1 (3 μM), G007-LK (0.3 μM), or ICG001 (3 μM) in OIM. Nuclear and cytoplasmic protein samples were collected at indicated time points and subjected to Western blot analysis of the indicated proteins. (A) Western blot analysis of SH3BP2 and AXIN in the cytoplasmic fraction after culture for 10 days. (B) Western blot analysis of β-catenin in the nuclear fraction after culture for 7 days. (C) Western blot analysis of ABL, TAZ, and RUNX2 in the nuclear fraction after culture for 7 days. (D) The intensities of the bands were quantified, and the ratio of RUNX2 to NUP98 was calculated and normalized to that of non-treated cells.
Figure Legend Snippet: Osteoblast-promoting effect of tankyrase inhibitors. Primary calvaria cells isolated from WT pups were cultured in the presence of IWR-1 (3 μM), G007-LK (0.3 μM), or ICG001 (3 μM) in OIM. Nuclear and cytoplasmic protein samples were collected at indicated time points and subjected to Western blot analysis of the indicated proteins. (A) Western blot analysis of SH3BP2 and AXIN in the cytoplasmic fraction after culture for 10 days. (B) Western blot analysis of β-catenin in the nuclear fraction after culture for 7 days. (C) Western blot analysis of ABL, TAZ, and RUNX2 in the nuclear fraction after culture for 7 days. (D) The intensities of the bands were quantified, and the ratio of RUNX2 to NUP98 was calculated and normalized to that of non-treated cells.

Techniques Used: Isolation, Cell Culture, Western Blot

7) Product Images from "Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation"

Article Title: Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021170

TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.
Figure Legend Snippet: TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.

Techniques Used:

8) Product Images from "Calcineurin signaling and PGC-1? expression are suppressed during muscle atrophy due to diabetes"

Article Title: Calcineurin signaling and PGC-1? expression are suppressed during muscle atrophy due to diabetes

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamcr.2010.03.019

GSK-3β signaling is unchanged in 21day STZ-treated rat muscle (A) The phosphorylation (i.e., inactivation) of GSK-3β on Ser-9 in gastrocnemius muscles was examined by Western blot analysis using antibodies that detect phospho-S9 and total GSK-3β. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.31 (B) The phosphorylation of glycogen synthase on Ser-641 also was examined as a downstream target of GSK-3β activity by Western blot analysis using antibodies that detect total and phospho-Ser-641 glycogen synthase. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.69 (C) Western blot analysis of Akt phosphorylation was performed using antibodies that detect total and phospho-Ser-473 Akt. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM: P
Figure Legend Snippet: GSK-3β signaling is unchanged in 21day STZ-treated rat muscle (A) The phosphorylation (i.e., inactivation) of GSK-3β on Ser-9 in gastrocnemius muscles was examined by Western blot analysis using antibodies that detect phospho-S9 and total GSK-3β. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.31 (B) The phosphorylation of glycogen synthase on Ser-641 also was examined as a downstream target of GSK-3β activity by Western blot analysis using antibodies that detect total and phospho-Ser-641 glycogen synthase. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.69 (C) Western blot analysis of Akt phosphorylation was performed using antibodies that detect total and phospho-Ser-473 Akt. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM: P

Techniques Used: Western Blot, Activity Assay

9) Product Images from "Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy"

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy

Journal: Scientific Reports

doi: 10.1038/s41598-018-37091-8

The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p
Figure Legend Snippet: The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p

Techniques Used: Expressing, Incubation, Western Blot

10) Product Images from "Type I Phosphotidylinosotol 4-Phosphate 5-Kinase ? Regulates Osteoclasts in a Bifunctional Manner *"

Article Title: Type I Phosphotidylinosotol 4-Phosphate 5-Kinase ? Regulates Osteoclasts in a Bifunctional Manner *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.446054

PIP5KIγ deficiency regulates RANKL-induced signaling in osteoclast precursors. A , serum- and cytokine-starved WT and PIP5KIγ −/− (KO) macrophages were exposed to M-CSF (100 ng/ml). Phosphorylated and total AKT, ERK, and JNK were immunoblotted with time. Total relevant proteins serve as loading control. B , serum- and cytokine-starved WT and PIP5KIγ −/− (KO) macrophages were exposed to RANKL (100 ng/ml) Phosphorylated and total AKT, JNK, IkB, and p38 were immunoblotted with time. Total relevant proteins serve as loading controls. C , PIP5KIγ −/− liver cells, transduced with HA-PIP5KIγ2, were stimulated by RANKL (100 ng/ml) and M-CSF (100 ng/ml) for as long as 15 min. HA immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb. D , WT and PIP5KIγ −/− BMMs, stained with Flur-4, were stimulated with RANKL (200 ng/ml). [Ca 2+ ] i was imaged prior to con and 8 min after RANKL exposure. Scale bar , 25 μm. E , average increase in [Ca 2+ ] i during stimulation with RANKL in WT and PIP5KIγ-deficient BMMs.
Figure Legend Snippet: PIP5KIγ deficiency regulates RANKL-induced signaling in osteoclast precursors. A , serum- and cytokine-starved WT and PIP5KIγ −/− (KO) macrophages were exposed to M-CSF (100 ng/ml). Phosphorylated and total AKT, ERK, and JNK were immunoblotted with time. Total relevant proteins serve as loading control. B , serum- and cytokine-starved WT and PIP5KIγ −/− (KO) macrophages were exposed to RANKL (100 ng/ml) Phosphorylated and total AKT, JNK, IkB, and p38 were immunoblotted with time. Total relevant proteins serve as loading controls. C , PIP5KIγ −/− liver cells, transduced with HA-PIP5KIγ2, were stimulated by RANKL (100 ng/ml) and M-CSF (100 ng/ml) for as long as 15 min. HA immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb. D , WT and PIP5KIγ −/− BMMs, stained with Flur-4, were stimulated with RANKL (200 ng/ml). [Ca 2+ ] i was imaged prior to con and 8 min after RANKL exposure. Scale bar , 25 μm. E , average increase in [Ca 2+ ] i during stimulation with RANKL in WT and PIP5KIγ-deficient BMMs.

Techniques Used: Transduction, Staining

11) Product Images from "Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/? gene expression via acetylation of nuclear factor of activated T cells c1"

Article Title: Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/? gene expression via acetylation of nuclear factor of activated T cells c1

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr162

Proposed model of the interaction of calcineurin-NFATc1, MEK1–ERK1/2 and p300signaling leading to slow skeletal muscle fiber-specific gene expression. Stimuli that increase [Ca 2+ ] i result in translocation of dephosphorylated NFAT to the nucleus where it participates in mediating Ca 2+ -inducible gene expression of slow fiber genes, such as MyHCI/β. This induction of MyHCI/β can be further stimulated by Ca 2+ -mediated activation of the MEK1–ERK1/2 pathway resulting in the phosphorylation of transcriptional coactivator p300, leading to its recruitment to NFATc1 bound to the MyHCI/β promoter. Once recruited, phosphorylated p300 acetylates NFATc1 and enhances NFATc1–DNA binding. As a consequence, p300 enhances gene expression of MyHCI/β mediated by increased intracellular Ca 2+ -concentration. CaM, calmodulin; CKI/II, casein kinase I/II; CsA, cyclosporine A; GSK3β, glycogensynthase kinase-3β; MCIP (myocyte-enriched calcineurin interacting protein).
Figure Legend Snippet: Proposed model of the interaction of calcineurin-NFATc1, MEK1–ERK1/2 and p300signaling leading to slow skeletal muscle fiber-specific gene expression. Stimuli that increase [Ca 2+ ] i result in translocation of dephosphorylated NFAT to the nucleus where it participates in mediating Ca 2+ -inducible gene expression of slow fiber genes, such as MyHCI/β. This induction of MyHCI/β can be further stimulated by Ca 2+ -mediated activation of the MEK1–ERK1/2 pathway resulting in the phosphorylation of transcriptional coactivator p300, leading to its recruitment to NFATc1 bound to the MyHCI/β promoter. Once recruited, phosphorylated p300 acetylates NFATc1 and enhances NFATc1–DNA binding. As a consequence, p300 enhances gene expression of MyHCI/β mediated by increased intracellular Ca 2+ -concentration. CaM, calmodulin; CKI/II, casein kinase I/II; CsA, cyclosporine A; GSK3β, glycogensynthase kinase-3β; MCIP (myocyte-enriched calcineurin interacting protein).

Techniques Used: Expressing, Translocation Assay, Activation Assay, Binding Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay

MEK1–ERK1/2 synergy with NFATc1 depends on transcriptional coactivator p300. C2C12 cells were transiently transfected with wildtype −2.4 kb MyHCI/β (−2.4 I/βwt) promoter construct and cotransfected ( A ) with or ( B ) without expression vectors coding for wild-type NFATc1 (NFATc1wt), or constitutively nuclear NFATc1ΔSRR, or empty vector. Cells were additionally cotransfected with expression vectors for wild-type p300 (p300wt), or acetyltransferase-deficient p300 (p300DY), or MEK1ca or MEK1dn, or empty vector. In ( B ) mutant (NFAT binding site) −2.4 kb MyHCI/β (−2.4 I/βNFATmut) promoter construct was also transfected. Cells were grown for 24 h in GM and then for 2 days in DM with or without U0126 (10 µM), or Ca 2+ -ionophore A23187 (0.1 µM). ( C ) Western blot analysis of NFATc1 expression in Ca 2+ -ionophore-treated C2C12 cells transfected with p300wt, p300DY or the empty vector, using an anti-NFATc1 antibody. The blot was reprobed with anti-α-tubulin antibody as loading control. ( D ) Western blot analysis of p300 expression in non-transfected C2C12 myotubes, or cells transfected with a pool of double-stranded 20–25-nt siRNA that specifically target mouse p300 (siRNA p300), or with non-specific doublestranded control siRNA (siRNA control), using an anti-p300 antibody. The blot was reprobed with anti-histone H3 antibody as loading control. ( E ) C2C12 cells were transiently transfected with a −2.4-kb MyHCI/β promoter construct and cotransfected with NFATc1ΔSRR or empty vector. After grown for 24 h in GM, cells were cotransfected with p300 siRNA (siRNA p300) or a non-specific control siRNA (siRNA control), and then grown for 2 d in DM. The promoter activity in ( A ), ( B ) and ( E ) is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.
Figure Legend Snippet: MEK1–ERK1/2 synergy with NFATc1 depends on transcriptional coactivator p300. C2C12 cells were transiently transfected with wildtype −2.4 kb MyHCI/β (−2.4 I/βwt) promoter construct and cotransfected ( A ) with or ( B ) without expression vectors coding for wild-type NFATc1 (NFATc1wt), or constitutively nuclear NFATc1ΔSRR, or empty vector. Cells were additionally cotransfected with expression vectors for wild-type p300 (p300wt), or acetyltransferase-deficient p300 (p300DY), or MEK1ca or MEK1dn, or empty vector. In ( B ) mutant (NFAT binding site) −2.4 kb MyHCI/β (−2.4 I/βNFATmut) promoter construct was also transfected. Cells were grown for 24 h in GM and then for 2 days in DM with or without U0126 (10 µM), or Ca 2+ -ionophore A23187 (0.1 µM). ( C ) Western blot analysis of NFATc1 expression in Ca 2+ -ionophore-treated C2C12 cells transfected with p300wt, p300DY or the empty vector, using an anti-NFATc1 antibody. The blot was reprobed with anti-α-tubulin antibody as loading control. ( D ) Western blot analysis of p300 expression in non-transfected C2C12 myotubes, or cells transfected with a pool of double-stranded 20–25-nt siRNA that specifically target mouse p300 (siRNA p300), or with non-specific doublestranded control siRNA (siRNA control), using an anti-p300 antibody. The blot was reprobed with anti-histone H3 antibody as loading control. ( E ) C2C12 cells were transiently transfected with a −2.4-kb MyHCI/β promoter construct and cotransfected with NFATc1ΔSRR or empty vector. After grown for 24 h in GM, cells were cotransfected with p300 siRNA (siRNA p300) or a non-specific control siRNA (siRNA control), and then grown for 2 d in DM. The promoter activity in ( A ), ( B ) and ( E ) is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Mutagenesis, Binding Assay, Western Blot, Activity Assay

MEK1–ERK1/2 signaling indirectly regulates NFATc1-p300 complex formation at the NFAT site via p300. EMSAs demonstrate that NFATc1–p300 DNA binding complex stability depends on MEK1–ERK1/2 signaling. Radiolabeled oligonucleotide probes containing the −439/−432-bp NFAT binding site from the MyHCI/β promoter were incubated ( A ) with nuclear extracts (NEs) from C2C12 myotubes grown for 3 days in DM or ( B ) with in vitro translated NFATc1 or unprogrammed rabbit reticulocyte lysate (RL), with or without NEs from C2C12 myotubes grown for 3 days in DM. In ( A ) cells were transfected 24 h prior NE preparation with or without constitutively nuclear NFATc1ΔSRR expression vector alone, or were cotransfected with expression vectors for MEK1ca, or MEK1dn or p300wt, or p300DY, or empty vector. In addition, cells were grown with or without U0126 (10 µM). In supershift experiments, preimmune serum (PI), or anti-NFATc1, or anti-p300 antibodies (Ab) were added. A 200-fold excess of unlabeled wild-type competitor DNA (competitor +) was used in ( B ) for determination of specific protein–DNA binding reaction (lane 5). After the incubation, samples were fractionated on 5% polyacrylamide gels. Complexes are indicated by an arrowhead. SS: supershift, with ( A ) arrowhead 1 indicating the supershifted NFATc1ΔSRR–probe complex, and arrowhead 2 indicating the NFATc1ΔSRR/p300–probe complex and ( B ) arrowhead 1 indicating the supershifted NFATc1–probe complex, and arrowhead 2 indicating the NFATc1/p300–probe complex. Probe: bottom of the gel indicates excess probe. WB: western blot analysis of transfected NFATc1ΔSRR in NEs by probing aliquots with an anti-NFATc1 antibody.
Figure Legend Snippet: MEK1–ERK1/2 signaling indirectly regulates NFATc1-p300 complex formation at the NFAT site via p300. EMSAs demonstrate that NFATc1–p300 DNA binding complex stability depends on MEK1–ERK1/2 signaling. Radiolabeled oligonucleotide probes containing the −439/−432-bp NFAT binding site from the MyHCI/β promoter were incubated ( A ) with nuclear extracts (NEs) from C2C12 myotubes grown for 3 days in DM or ( B ) with in vitro translated NFATc1 or unprogrammed rabbit reticulocyte lysate (RL), with or without NEs from C2C12 myotubes grown for 3 days in DM. In ( A ) cells were transfected 24 h prior NE preparation with or without constitutively nuclear NFATc1ΔSRR expression vector alone, or were cotransfected with expression vectors for MEK1ca, or MEK1dn or p300wt, or p300DY, or empty vector. In addition, cells were grown with or without U0126 (10 µM). In supershift experiments, preimmune serum (PI), or anti-NFATc1, or anti-p300 antibodies (Ab) were added. A 200-fold excess of unlabeled wild-type competitor DNA (competitor +) was used in ( B ) for determination of specific protein–DNA binding reaction (lane 5). After the incubation, samples were fractionated on 5% polyacrylamide gels. Complexes are indicated by an arrowhead. SS: supershift, with ( A ) arrowhead 1 indicating the supershifted NFATc1ΔSRR–probe complex, and arrowhead 2 indicating the NFATc1ΔSRR/p300–probe complex and ( B ) arrowhead 1 indicating the supershifted NFATc1–probe complex, and arrowhead 2 indicating the NFATc1/p300–probe complex. Probe: bottom of the gel indicates excess probe. WB: western blot analysis of transfected NFATc1ΔSRR in NEs by probing aliquots with an anti-NFATc1 antibody.

Techniques Used: Binding Assay, Incubation, In Vitro, Transfection, Expressing, Plasmid Preparation, Western Blot

The MEK1-ERK1/2 pathway is not affecting NFATc1 subcellular localization. ( A ) Western blot analysis of the localization of endogenous NFATc1 (empty vector) and transfected constitutively nuclear NFATc1ΔSRR in C2C12 myotubes. C2C12 cells transfected with NFATc1ΔSRR or the empty vector were grown for 24 h in GM and for 2 days in DM in the presence or absence (Control) of Ca 2+ -ionophore A23187 (0.1 µM) and/or U0126 (10 µM). After cell fractionation, aliquots of the pellet (nuclear, N) and supernatant (cytoplasmic, C) fraction were analyzed by western blotting using anti-NFATc1 and anti-p53 antibodies. Detection of p53 serves as a control for nuclear localization. ( B ) Immunofluorescence analysis of the localization of endogenous NFATc1 in C2C12 myotubes. Cells transfected with MEK1ca expression vector were grown for 24 h in GM and then for 3 days in DM, untransfected C2C12 myotubes for 4 days in DM (Control) or for 2 days in DM and then for additional 2 days in DM in the presence of Ca 2+ -ionophore (0.1 µM) and/or U0126 (10 µM). Cells were stained with an anti-NFATc1-antibody. NFATc1 was visualized by a FITC-labeled secondary antibody. Fluorescence was detected by using an inverted fluorescence photomicroscope at a magnification of ×400.
Figure Legend Snippet: The MEK1-ERK1/2 pathway is not affecting NFATc1 subcellular localization. ( A ) Western blot analysis of the localization of endogenous NFATc1 (empty vector) and transfected constitutively nuclear NFATc1ΔSRR in C2C12 myotubes. C2C12 cells transfected with NFATc1ΔSRR or the empty vector were grown for 24 h in GM and for 2 days in DM in the presence or absence (Control) of Ca 2+ -ionophore A23187 (0.1 µM) and/or U0126 (10 µM). After cell fractionation, aliquots of the pellet (nuclear, N) and supernatant (cytoplasmic, C) fraction were analyzed by western blotting using anti-NFATc1 and anti-p53 antibodies. Detection of p53 serves as a control for nuclear localization. ( B ) Immunofluorescence analysis of the localization of endogenous NFATc1 in C2C12 myotubes. Cells transfected with MEK1ca expression vector were grown for 24 h in GM and then for 3 days in DM, untransfected C2C12 myotubes for 4 days in DM (Control) or for 2 days in DM and then for additional 2 days in DM in the presence of Ca 2+ -ionophore (0.1 µM) and/or U0126 (10 µM). Cells were stained with an anti-NFATc1-antibody. NFATc1 was visualized by a FITC-labeled secondary antibody. Fluorescence was detected by using an inverted fluorescence photomicroscope at a magnification of ×400.

Techniques Used: Western Blot, Plasmid Preparation, Transfection, Cell Fractionation, Immunofluorescence, Expressing, Staining, Labeling, Fluorescence

MEK1–ERK1/2 signaling increases Ca 2+ -ionophore-induced NFATc1 transcriptional activity depending on a specific NFAT binding site. C2C12 cells were transiently transfected with a −2.4-kb wild-type MyHCI/β (−2.4 I/βwt) promoter construct or a −2.4-kb MyHCI/β promoter construct mutated in the −439/−432-bp NFAT binding site (−2.4 I/βmut) alone, or were cotransfected with expression vectors for NFATc1, or MEK1ca, or empty vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM). The promoter activity is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.
Figure Legend Snippet: MEK1–ERK1/2 signaling increases Ca 2+ -ionophore-induced NFATc1 transcriptional activity depending on a specific NFAT binding site. C2C12 cells were transiently transfected with a −2.4-kb wild-type MyHCI/β (−2.4 I/βwt) promoter construct or a −2.4-kb MyHCI/β promoter construct mutated in the −439/−432-bp NFAT binding site (−2.4 I/βmut) alone, or were cotransfected with expression vectors for NFATc1, or MEK1ca, or empty vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM). The promoter activity is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.

Techniques Used: Activity Assay, Binding Assay, Transfection, Construct, Expressing, Plasmid Preparation

MEK1–ERK1/2 signaling increases Ca 2+ -ionophore-induced slow MyHCI/β promoter activation in C2C12 myotubes. C2C12 cells were transiently transfected with a −2.4-kb MyHCI/β or with a −2.8-kb MyHCIId/x promoter luciferase reporter construct alone or cotransfected with expression vectors for constitutively active MEK1 (MKK1ca), or dominant negative MEK1 (MEK1dn), or empty vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM) and/or U0126 (10 µM). The promoter activity is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.
Figure Legend Snippet: MEK1–ERK1/2 signaling increases Ca 2+ -ionophore-induced slow MyHCI/β promoter activation in C2C12 myotubes. C2C12 cells were transiently transfected with a −2.4-kb MyHCI/β or with a −2.8-kb MyHCIId/x promoter luciferase reporter construct alone or cotransfected with expression vectors for constitutively active MEK1 (MKK1ca), or dominant negative MEK1 (MEK1dn), or empty vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM) and/or U0126 (10 µM). The promoter activity is expressed as relative light units per unit β-galactosidase (RLU/β-Gal). The data represent the mean ± SD of triplicate data points.

Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Expressing, Dominant Negative Mutation, Plasmid Preparation, Activity Assay

NFATc1 is acetylated by p300 in a MEK1–ERK1/2-dependent manner. ( A ) Analysis of p300 phosphorylation by IP and western blot. C2C12 cells were transfected with MEK1ca, or MEK1dn, or empty vector. After 24 h in GM cells were further grown in DM. Two days after transfection cells were pretreated with or without U0126 (10 µM) for 30 min, followed by treatment with Ca 2+ -ionophore A23187 (0.1 µM) for 3 h. Protein from cell lysates was immunoprecipitated (IP) with anti-p300 antibody. Phosphorylation of p300 was analyzed using an anti-phospho-serine (p-Ser) MAPK/CDK antibody, and p300 protein expression was detected by reprobing with an anti-p300 antibody. ( B ) In vitro kinase assay with p300wt or p300SA3 incubated with recombinant activated ERK2. The phosphorylation level of p300 was analyzed by western blot using anti-phospho-Ser/Thr-Pro antibodies. The input of proteins was analyzed with anti-p300 and anti-ERK2 antibodies. Analysis of NFATc1 lysine acetylation and ERK1/2 activation in ( C ) and ( D ) HEK 293 cells; ( E ) C2C12 myotubes; and ( F ) mouse soleus muscle by western blot (WB). In (C–E) HEK 293 or C2C12 cells were transiently transfected with or without NFATc1-c-Myc expression vector alone, or with p300wt, or p300DY, or CBP, or MEK1ca, or MEK1dn, or empty vector. HEK 293 cells were then grown for 2 days in GM, C2C12 cells for 24 h in GM and for 2 days in DM. HDAC inhibitors (300 nM TSA and 5 mM NIA) were added 24 h before lysis in (C) and (D) and to the lysis buffer in (E). C2C12 myotubes were grown in the presence or absence of Ca 2+ -ionophore (0.1 µM) and/or U0126 (10 µM) for 12 h before lysis. (F) Isolated mouse soleus muscles were electrostimulated (30 min, 15 Hz; + stimul) or not stimulated (− stimul). Eight solei were pooled per each group and proteins were immunoprecipitated (IP) from nuclear extracts with anti-NFATc1 antibodies. Expression of Myc-tagged NFATc1 was monitored by an anti-c-Myc antibody, and NFATc1 lysine acetylation was detected by using an anti acetyl-lysine antibody (K-Ac). Expression of endogenous NFATc1 was analyzed by reprobing with an anti-NFATc1 antibody. Expression of phosphorylated ERK1 and 2 (p-ERK1 and 2) was analyzed using anti-phospho-ERK1/2 antibodies, and expression of total ERK1 and 2 with anti-ERK1/2 antibodies, directly analyzed from cell lysates in (F). Exposure times for enhanced chemiluminescence detection with the LAS-3000 imaging system in (D) were 10 s or 1 min. Detection of α-tubulin served as a loading control. Molecular weights are indicated. ( G ) Band intensities from western blot analysis as shown in (F) were normalized to α-tubulin and presented as mean of 2 and single values (open circle).
Figure Legend Snippet: NFATc1 is acetylated by p300 in a MEK1–ERK1/2-dependent manner. ( A ) Analysis of p300 phosphorylation by IP and western blot. C2C12 cells were transfected with MEK1ca, or MEK1dn, or empty vector. After 24 h in GM cells were further grown in DM. Two days after transfection cells were pretreated with or without U0126 (10 µM) for 30 min, followed by treatment with Ca 2+ -ionophore A23187 (0.1 µM) for 3 h. Protein from cell lysates was immunoprecipitated (IP) with anti-p300 antibody. Phosphorylation of p300 was analyzed using an anti-phospho-serine (p-Ser) MAPK/CDK antibody, and p300 protein expression was detected by reprobing with an anti-p300 antibody. ( B ) In vitro kinase assay with p300wt or p300SA3 incubated with recombinant activated ERK2. The phosphorylation level of p300 was analyzed by western blot using anti-phospho-Ser/Thr-Pro antibodies. The input of proteins was analyzed with anti-p300 and anti-ERK2 antibodies. Analysis of NFATc1 lysine acetylation and ERK1/2 activation in ( C ) and ( D ) HEK 293 cells; ( E ) C2C12 myotubes; and ( F ) mouse soleus muscle by western blot (WB). In (C–E) HEK 293 or C2C12 cells were transiently transfected with or without NFATc1-c-Myc expression vector alone, or with p300wt, or p300DY, or CBP, or MEK1ca, or MEK1dn, or empty vector. HEK 293 cells were then grown for 2 days in GM, C2C12 cells for 24 h in GM and for 2 days in DM. HDAC inhibitors (300 nM TSA and 5 mM NIA) were added 24 h before lysis in (C) and (D) and to the lysis buffer in (E). C2C12 myotubes were grown in the presence or absence of Ca 2+ -ionophore (0.1 µM) and/or U0126 (10 µM) for 12 h before lysis. (F) Isolated mouse soleus muscles were electrostimulated (30 min, 15 Hz; + stimul) or not stimulated (− stimul). Eight solei were pooled per each group and proteins were immunoprecipitated (IP) from nuclear extracts with anti-NFATc1 antibodies. Expression of Myc-tagged NFATc1 was monitored by an anti-c-Myc antibody, and NFATc1 lysine acetylation was detected by using an anti acetyl-lysine antibody (K-Ac). Expression of endogenous NFATc1 was analyzed by reprobing with an anti-NFATc1 antibody. Expression of phosphorylated ERK1 and 2 (p-ERK1 and 2) was analyzed using anti-phospho-ERK1/2 antibodies, and expression of total ERK1 and 2 with anti-ERK1/2 antibodies, directly analyzed from cell lysates in (F). Exposure times for enhanced chemiluminescence detection with the LAS-3000 imaging system in (D) were 10 s or 1 min. Detection of α-tubulin served as a loading control. Molecular weights are indicated. ( G ) Band intensities from western blot analysis as shown in (F) were normalized to α-tubulin and presented as mean of 2 and single values (open circle).

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, In Vitro, Kinase Assay, Incubation, Recombinant, Activation Assay, Lysis, Isolation, Imaging

12) Product Images from "Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms"

Article Title: Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms

Journal: Oncogene

doi: 10.1038/onc.2013.132

Allografts of NFATc1-induced tumors showed continuous dependency on NFATc1 for tumor progression and survival but not on T cell functions Ovarian tumors from mutants were isolated and cultured ( A ). Most of the cultured cells expressed NFATc1 ( B ). Cultured tumor cells were injected subcutaneously into the lower flanks of nude mice. 100% of the Dox-treated recipients developed tumors, whereas none of the untreated mice did ( C ). Termination of Dox treatment resulted in significant decrease of tumor size. Such decrease was reverted again if Dox treatment was restarted ( D ). Statistical analysis (two-tailed t -test) showed a significant increase in tumor volume when Dox was continuously provided up to 64 days (1940.54±714.34 mm 3 , P=0.001, n=4). Conversely, a significant decrease in tumor volume (40.80±81.60 mm 3 ) was observed when Dox was interrupted from day 76 to day 99 (P=0.001, n=4). After Dox was restarted on day 99, tumor volume increased again and reached 702.34±523.30 mm 3 (P=0.04, n=4) on day 120 ( E ). Tumor size was measured at the end of each interval. Immunostaining of tumors from nude mice indicated that the majority of tumor cells were β-Gal + ( F ). The allografts in the nude mice resembled the original tumors in overall morphology, cellular composition ( G ), and the substantial levels of proliferation and Stat3 activation in NFATc1 − cells ( H - I ). By immunostaining with an anti CD3 antibody, T cells were seen in primary ovarian tumor but not in the grafts grown in nude mice ( J-K ). Extensive IL6 expression was noticed in the grafts in the nude mice ( L ).
Figure Legend Snippet: Allografts of NFATc1-induced tumors showed continuous dependency on NFATc1 for tumor progression and survival but not on T cell functions Ovarian tumors from mutants were isolated and cultured ( A ). Most of the cultured cells expressed NFATc1 ( B ). Cultured tumor cells were injected subcutaneously into the lower flanks of nude mice. 100% of the Dox-treated recipients developed tumors, whereas none of the untreated mice did ( C ). Termination of Dox treatment resulted in significant decrease of tumor size. Such decrease was reverted again if Dox treatment was restarted ( D ). Statistical analysis (two-tailed t -test) showed a significant increase in tumor volume when Dox was continuously provided up to 64 days (1940.54±714.34 mm 3 , P=0.001, n=4). Conversely, a significant decrease in tumor volume (40.80±81.60 mm 3 ) was observed when Dox was interrupted from day 76 to day 99 (P=0.001, n=4). After Dox was restarted on day 99, tumor volume increased again and reached 702.34±523.30 mm 3 (P=0.04, n=4) on day 120 ( E ). Tumor size was measured at the end of each interval. Immunostaining of tumors from nude mice indicated that the majority of tumor cells were β-Gal + ( F ). The allografts in the nude mice resembled the original tumors in overall morphology, cellular composition ( G ), and the substantial levels of proliferation and Stat3 activation in NFATc1 − cells ( H - I ). By immunostaining with an anti CD3 antibody, T cells were seen in primary ovarian tumor but not in the grafts grown in nude mice ( J-K ). Extensive IL6 expression was noticed in the grafts in the nude mice ( L ).

Techniques Used: Isolation, Cell Culture, Injection, Mouse Assay, Two Tailed Test, Immunostaining, Activation Assay, Expressing

13) Product Images from "A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase"

Article Title: A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase

Journal: Scientific Reports

doi: 10.1038/srep22389

Effect of the Ewha-18278 on the RANKL-induced signaling pathways. ( A ) BMMs were pretreated with Ewha-18278 for 1 hour, the cells were stimulated with RANKL for the indicated times. The cell lysates were subjected to immunoblot analysis. RANKL signaling pathways assessed by phosphorylation of ERK, p38, JNK, and IκBα. Immunoblots were stripped and then reprobed with total ERK, p38, JNK, and IκBα. ( B , C ) BMMs were treated with Ewha-18278 in the presence of M-CSF and RANKL for the indicated periods. Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels of NFATc1 ( B ) and Atp6v0d2 ( C ) genes. The relative expression levels were normalized to the level of actin mRNA as an internal control gene. * P
Figure Legend Snippet: Effect of the Ewha-18278 on the RANKL-induced signaling pathways. ( A ) BMMs were pretreated with Ewha-18278 for 1 hour, the cells were stimulated with RANKL for the indicated times. The cell lysates were subjected to immunoblot analysis. RANKL signaling pathways assessed by phosphorylation of ERK, p38, JNK, and IκBα. Immunoblots were stripped and then reprobed with total ERK, p38, JNK, and IκBα. ( B , C ) BMMs were treated with Ewha-18278 in the presence of M-CSF and RANKL for the indicated periods. Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels of NFATc1 ( B ) and Atp6v0d2 ( C ) genes. The relative expression levels were normalized to the level of actin mRNA as an internal control gene. * P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing

14) Product Images from "Anti-Osteoporotic Effects of Polysaccharides Isolated from Persimmon Leaves via Osteoclastogenesis Inhibition"

Article Title: Anti-Osteoporotic Effects of Polysaccharides Isolated from Persimmon Leaves via Osteoclastogenesis Inhibition

Journal: Nutrients

doi: 10.3390/nu10070901

Effects of PLE0 on RANKL-induced activation of MAPKs and NF-kB. RANKL (100 ng/mL)-stimulated BMMs were incubated for the indicated periods of time with or without PLE0 (25 μg/mL). Whole-cell extracts were subjected to Western blot analysis with specific antibodies as indicated. p-JNK, phospho-JNK; p-ERK, phospho-ERK; p-p38, phospho-p38; p-p65, phospho-p-p65.
Figure Legend Snippet: Effects of PLE0 on RANKL-induced activation of MAPKs and NF-kB. RANKL (100 ng/mL)-stimulated BMMs were incubated for the indicated periods of time with or without PLE0 (25 μg/mL). Whole-cell extracts were subjected to Western blot analysis with specific antibodies as indicated. p-JNK, phospho-JNK; p-ERK, phospho-ERK; p-p38, phospho-p38; p-p65, phospho-p-p65.

Techniques Used: Activation Assay, Incubation, Western Blot

15) Product Images from "Spi-C positively regulates RANKL-mediated osteoclast differentiation and function"

Article Title: Spi-C positively regulates RANKL-mediated osteoclast differentiation and function

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-020-0427-8

A proposed model for the role of Spi-C during RANKL-mediated osteoclast differentiation and function. Spi-C is a transcription factor induced by the RANKL-mediated JNK pathway. The activation of p38 MAPK and PI3K by RANKL regulates the nuclear translocation of Spi-C to induce osteoclast marker gene expression, thus increasing osteoclast formation and bone resorption function.
Figure Legend Snippet: A proposed model for the role of Spi-C during RANKL-mediated osteoclast differentiation and function. Spi-C is a transcription factor induced by the RANKL-mediated JNK pathway. The activation of p38 MAPK and PI3K by RANKL regulates the nuclear translocation of Spi-C to induce osteoclast marker gene expression, thus increasing osteoclast formation and bone resorption function.

Techniques Used: Activation Assay, Translocation Assay, Marker, Expressing

Decrease in RANKL-mediated NF-κB and MAPK activation by Spi-C depletion. BMMs were transduced with a control shRNA lentiviral vector (shCon) or Spi-C shRNA expression lentiviral vector (shSpi-C), serum-starved, and incubated with RANKL (200 ng/ml) for the indicated times. Then, the cells were subjected to western blot analysis with antibodies specific to p-ERK1/2, ERK1/2, p-JNK, JNK1/2, p-p38, p38, IκBα, Spi-C, and actin. Actin was used as a loading control (right panels). The protein bands were quantified using densitometry, and the levels of p-ERK1/2, p-JNK and p-p38 were normalized to the levels of ERK1/2, JNK1/2 and p38, respectively (left panels). ** p
Figure Legend Snippet: Decrease in RANKL-mediated NF-κB and MAPK activation by Spi-C depletion. BMMs were transduced with a control shRNA lentiviral vector (shCon) or Spi-C shRNA expression lentiviral vector (shSpi-C), serum-starved, and incubated with RANKL (200 ng/ml) for the indicated times. Then, the cells were subjected to western blot analysis with antibodies specific to p-ERK1/2, ERK1/2, p-JNK, JNK1/2, p-p38, p38, IκBα, Spi-C, and actin. Actin was used as a loading control (right panels). The protein bands were quantified using densitometry, and the levels of p-ERK1/2, p-JNK and p-p38 were normalized to the levels of ERK1/2, JNK1/2 and p38, respectively (left panels). ** p

Techniques Used: Activation Assay, Transduction, shRNA, Plasmid Preparation, Expressing, Incubation, Western Blot

16) Product Images from "Spi-C positively regulates RANKL-mediated osteoclast differentiation and function"

Article Title: Spi-C positively regulates RANKL-mediated osteoclast differentiation and function

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-020-0427-8

A proposed model for the role of Spi-C during RANKL-mediated osteoclast differentiation and function. Spi-C is a transcription factor induced by the RANKL-mediated JNK pathway. The activation of p38 MAPK and PI3K by RANKL regulates the nuclear translocation of Spi-C to induce osteoclast marker gene expression, thus increasing osteoclast formation and bone resorption function.
Figure Legend Snippet: A proposed model for the role of Spi-C during RANKL-mediated osteoclast differentiation and function. Spi-C is a transcription factor induced by the RANKL-mediated JNK pathway. The activation of p38 MAPK and PI3K by RANKL regulates the nuclear translocation of Spi-C to induce osteoclast marker gene expression, thus increasing osteoclast formation and bone resorption function.

Techniques Used: Activation Assay, Translocation Assay, Marker, Expressing

Decrease in RANKL-mediated NF-κB and MAPK activation by Spi-C depletion. BMMs were transduced with a control shRNA lentiviral vector (shCon) or Spi-C shRNA expression lentiviral vector (shSpi-C), serum-starved, and incubated with RANKL (200 ng/ml) for the indicated times. Then, the cells were subjected to western blot analysis with antibodies specific to p-ERK1/2, ERK1/2, p-JNK, JNK1/2, p-p38, p38, IκBα, Spi-C, and actin. Actin was used as a loading control (right panels). The protein bands were quantified using densitometry, and the levels of p-ERK1/2, p-JNK and p-p38 were normalized to the levels of ERK1/2, JNK1/2 and p38, respectively (left panels). ** p
Figure Legend Snippet: Decrease in RANKL-mediated NF-κB and MAPK activation by Spi-C depletion. BMMs were transduced with a control shRNA lentiviral vector (shCon) or Spi-C shRNA expression lentiviral vector (shSpi-C), serum-starved, and incubated with RANKL (200 ng/ml) for the indicated times. Then, the cells were subjected to western blot analysis with antibodies specific to p-ERK1/2, ERK1/2, p-JNK, JNK1/2, p-p38, p38, IκBα, Spi-C, and actin. Actin was used as a loading control (right panels). The protein bands were quantified using densitometry, and the levels of p-ERK1/2, p-JNK and p-p38 were normalized to the levels of ERK1/2, JNK1/2 and p38, respectively (left panels). ** p

Techniques Used: Activation Assay, Transduction, shRNA, Plasmid Preparation, Expressing, Incubation, Western Blot

17) Product Images from "Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6"

Article Title: Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20205196

PPOA- N -Ac-2-Cl attenuates osteoclastogenesis via inhibition of MAPK and IκBa/p65 (NF-κB) signaling pathways. ( A – C ) BMMs were stimulated with 100 ng/mL RANKL for 0, 5, 10, 20, 30, or 60 min after pretreatment with 6 μM PPOA- N -Ac-2-Cl or DMSO for 2 h, and the phosphorylation of MAPKs, Akt, IκBa, and p65 was analyzed by immunoblotting. The densitometry graphs for the blots are shown in Figure S3 . β-actin was used as the loading control.
Figure Legend Snippet: PPOA- N -Ac-2-Cl attenuates osteoclastogenesis via inhibition of MAPK and IκBa/p65 (NF-κB) signaling pathways. ( A – C ) BMMs were stimulated with 100 ng/mL RANKL for 0, 5, 10, 20, 30, or 60 min after pretreatment with 6 μM PPOA- N -Ac-2-Cl or DMSO for 2 h, and the phosphorylation of MAPKs, Akt, IκBa, and p65 was analyzed by immunoblotting. The densitometry graphs for the blots are shown in Figure S3 . β-actin was used as the loading control.

Techniques Used: Inhibition

18) Product Images from "Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass"

Article Title: Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1511285112

( A ) Representative immunofluorescence images of NFATc1 (red) in WT and Tmem178 −/− BMMs cultured with 10 ng/mL M-CSF and 50 ng/mL RANKL for the indicated days. DAPI (blue) was used as nuclear stain. ( B ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT and Tmem178 −/− preOCs for the indicated time points. Lamin B is shown as loading control, representative of three experiments. ( C ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT cells expressing empty vector pMX or Tmem178-HA for the indicated time points. Histone 3 is shown as loading control, representative of three experiments.
Figure Legend Snippet: ( A ) Representative immunofluorescence images of NFATc1 (red) in WT and Tmem178 −/− BMMs cultured with 10 ng/mL M-CSF and 50 ng/mL RANKL for the indicated days. DAPI (blue) was used as nuclear stain. ( B ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT and Tmem178 −/− preOCs for the indicated time points. Lamin B is shown as loading control, representative of three experiments. ( C ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT cells expressing empty vector pMX or Tmem178-HA for the indicated time points. Histone 3 is shown as loading control, representative of three experiments.

Techniques Used: Immunofluorescence, Cell Culture, Staining, Western Blot, Translocation Assay, Expressing, Plasmid Preparation

19) Product Images from "Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation"

Article Title: Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2014.282996

Activities of Akt or ERK1/2 signalling in WT and Epac1KO in response to chronic CB treatment
Figure Legend Snippet: Activities of Akt or ERK1/2 signalling in WT and Epac1KO in response to chronic CB treatment

Techniques Used:

20) Product Images from "Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition"

Article Title: Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

Journal: PLoS ONE

doi: 10.1371/journal.pone.0128263

Effects of CB and DEX on phosphorylation of FOXO1, FOXO3a, and S6K1. (A) Phosphorylation of FOXO1 at serine 259 in the CB, DEX, and CB+DEX groups was similarly and significantly greater than that in the Control (** P
Figure Legend Snippet: Effects of CB and DEX on phosphorylation of FOXO1, FOXO3a, and S6K1. (A) Phosphorylation of FOXO1 at serine 259 in the CB, DEX, and CB+DEX groups was similarly and significantly greater than that in the Control (** P

Techniques Used:

21) Product Images from "Blocking the ZZ Domain of Sequestosome1/p62 Suppresses Myeloma Growth and Osteoclast Formation In Vitro and Induces Dramatic Bone Formation in Myeloma-Bearing Bones In Vivo"

Article Title: Blocking the ZZ Domain of Sequestosome1/p62 Suppresses Myeloma Growth and Osteoclast Formation In Vitro and Induces Dramatic Bone Formation in Myeloma-Bearing Bones In Vivo

Journal: Leukemia

doi: 10.1038/leu.2015.229

A Binding domains of p62. Sequestosome 1 (p62) is an adapter protein without intrinsic enzymatic activity that serves as a platform for the formation of many of the signaling complexes that result in NFκB and p38 MAPK activation in the marrow microenvironment of MM patients. The ZZ domain of p62 is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα. SH2 = Src homology 2, aPKCs = atypical protein kinase C, PB1 = Phox and Bem1p, RIP1 = receptor-interacting protein-1, TRAF6 = TNF receptor-associated factor 6, TBS = TRAF6 binding site, UBA = ubiquitin-associated domain. 1B: Structure of XRK3F2 .
Figure Legend Snippet: A Binding domains of p62. Sequestosome 1 (p62) is an adapter protein without intrinsic enzymatic activity that serves as a platform for the formation of many of the signaling complexes that result in NFκB and p38 MAPK activation in the marrow microenvironment of MM patients. The ZZ domain of p62 is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα. SH2 = Src homology 2, aPKCs = atypical protein kinase C, PB1 = Phox and Bem1p, RIP1 = receptor-interacting protein-1, TRAF6 = TNF receptor-associated factor 6, TBS = TRAF6 binding site, UBA = ubiquitin-associated domain. 1B: Structure of XRK3F2 .

Techniques Used: Binding Assay, Activity Assay, Activation Assay

22) Product Images from "Ibrutinib blocks Btk-dependent NF-ĸB and NFAT responses in human macrophages during Aspergillus fumigatus phagocytosis"

Article Title: Ibrutinib blocks Btk-dependent NF-ĸB and NFAT responses in human macrophages during Aspergillus fumigatus phagocytosis

Journal: Blood

doi: 10.1182/blood-2017-12-823393

Endosomally driven Btk responses during human macrophage infection with A fumigatus are required for optimal fungal growth control. (A) TLR9 engagement and BTK phosphorylation are required for NFAT activation in response to A fumigatus in hMDMS. hMDMs were pretreated with Ibrutinib (1 µM), the TLR9-blocking nucleotide ODN2088 (10 µM), or ODN nucleotide control (10 µM) for 1 hour. Macrophages were then infected with A fumigatus swollen conidia (MOI = 1) for 1 hour. Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-NFATc1 and Histone H3 antibodies. (B-C) BTK mediates an endosomal NF-ĸB activation pathway in human macrophages. (B) Monocyte-derived macrophages were pretreated with Scramble or BTK-targeting siRNA (100 nM) for 72 hours and additionally with cytochalasin D (10 µM) or vehicle. Macrophages were stimulated with eGFP A fumigatus swollen conidia (MOI = 1) for 1 hour, and NFATc1 and NFKB translocation were measured by confocal microscopy. Nuclear translocation was quantified by calculating the percent overlap of the nuclear DAPI and transcription factor–linked fluorophore channels. Data were calculated from 7 fields of view taken at random per biological repeat. Mean and standard deviation of 3 biological repeats are represented. (C) Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-BTK and anti–β-actin antibodies. Statistical analysis was performed using paired Student t tests: ns, not significant; * P
Figure Legend Snippet: Endosomally driven Btk responses during human macrophage infection with A fumigatus are required for optimal fungal growth control. (A) TLR9 engagement and BTK phosphorylation are required for NFAT activation in response to A fumigatus in hMDMS. hMDMs were pretreated with Ibrutinib (1 µM), the TLR9-blocking nucleotide ODN2088 (10 µM), or ODN nucleotide control (10 µM) for 1 hour. Macrophages were then infected with A fumigatus swollen conidia (MOI = 1) for 1 hour. Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-NFATc1 and Histone H3 antibodies. (B-C) BTK mediates an endosomal NF-ĸB activation pathway in human macrophages. (B) Monocyte-derived macrophages were pretreated with Scramble or BTK-targeting siRNA (100 nM) for 72 hours and additionally with cytochalasin D (10 µM) or vehicle. Macrophages were stimulated with eGFP A fumigatus swollen conidia (MOI = 1) for 1 hour, and NFATc1 and NFKB translocation were measured by confocal microscopy. Nuclear translocation was quantified by calculating the percent overlap of the nuclear DAPI and transcription factor–linked fluorophore channels. Data were calculated from 7 fields of view taken at random per biological repeat. Mean and standard deviation of 3 biological repeats are represented. (C) Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-BTK and anti–β-actin antibodies. Statistical analysis was performed using paired Student t tests: ns, not significant; * P

Techniques Used: Infection, Activation Assay, Blocking Assay, SDS Page, Western Blot, Derivative Assay, Translocation Assay, Confocal Microscopy, Standard Deviation

23) Product Images from "STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex"

Article Title: STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-017-0048-5

STAC2 inhibits the RANKL-stimulated activation of MAPK and NF-κB. a , b BMMs were transduced with the pMX-puro retrovirus (EV) or retrovirus expressing Flag-STAC2 (STAC2). The transduced BMMs were then serum-starved and incubated with RANKL (200 ng/ml) ( a ) or M-CSF (100 ng/ml) ( b ) for the indicated times, and subjected to western blot analysis with antibodies to p-JNK, JNK, p-p38, p38, p-ERK, ERK, IκBα, Flag, and tubulin. Data are representative of at least three independent experiments a , b
Figure Legend Snippet: STAC2 inhibits the RANKL-stimulated activation of MAPK and NF-κB. a , b BMMs were transduced with the pMX-puro retrovirus (EV) or retrovirus expressing Flag-STAC2 (STAC2). The transduced BMMs were then serum-starved and incubated with RANKL (200 ng/ml) ( a ) or M-CSF (100 ng/ml) ( b ) for the indicated times, and subjected to western blot analysis with antibodies to p-JNK, JNK, p-p38, p38, p-ERK, ERK, IκBα, Flag, and tubulin. Data are representative of at least three independent experiments a , b

Techniques Used: Activation Assay, Transduction, Expressing, Incubation, Western Blot

24) Product Images from "Massive elimination of multinucleated osteoclasts by eupatilin is due to dual inhibition of transcription and cytoskeletal rearrangement"

Article Title: Massive elimination of multinucleated osteoclasts by eupatilin is due to dual inhibition of transcription and cytoskeletal rearrangement

Journal: Bone Reports

doi: 10.1016/j.bonr.2015.10.003

Dephosphorylation of cofilin and rapid downregulation of TESK1 and LIMK2 in response to eupatilin. (A) Cell lysates were prepared in the presence or absence of eupatilin. Western blot analysis was performed with a phosphor-specific cofilin, total cofilin, or β-actin antibody. (B–C) Mouse BMMs were pre-treated with eupatilin (50 μM) or control for 1 h and differentiation was driven by addition of RANKL (100 ng/mL) for the indicated time points. mRNAs were then extracted and expression of TESK1 and LIMK2 mRNA levels were analyzed with real-time RT-PCR. The mRNA levels at time zero, namely 1 h after eupatilin or control pre-treatment, were indicated with arrows for comparison.
Figure Legend Snippet: Dephosphorylation of cofilin and rapid downregulation of TESK1 and LIMK2 in response to eupatilin. (A) Cell lysates were prepared in the presence or absence of eupatilin. Western blot analysis was performed with a phosphor-specific cofilin, total cofilin, or β-actin antibody. (B–C) Mouse BMMs were pre-treated with eupatilin (50 μM) or control for 1 h and differentiation was driven by addition of RANKL (100 ng/mL) for the indicated time points. mRNAs were then extracted and expression of TESK1 and LIMK2 mRNA levels were analyzed with real-time RT-PCR. The mRNA levels at time zero, namely 1 h after eupatilin or control pre-treatment, were indicated with arrows for comparison.

Techniques Used: De-Phosphorylation Assay, Western Blot, Expressing, Quantitative RT-PCR

25) Product Images from "FAM19A5, a brain-specific chemokine, inhibits RANKL-induced osteoclast formation through formyl peptide receptor 2"

Article Title: FAM19A5, a brain-specific chemokine, inhibits RANKL-induced osteoclast formation through formyl peptide receptor 2

Journal: Scientific Reports

doi: 10.1038/s41598-017-15586-0

FAM19A5 regulates RANKL-induced gene expression during osteoclastogenesis. ( A , C , D ) Mouse BMDMs were stimulated with FAM19A5 (2 μM) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 3 days. Cells were harvested for RNA preparation. qPCR was performed using specific primers for RANK , TRAF6 , OSCAR , TRAP , Blimp1 , c-FOS , NFATc1 , MafB , IRF-8 , DC-STAMP , OC-STAMP , Atp6v0d2 , and GAPDH . ( B ) Cells were harvested and Western blot analysis was conducted using anti-RANK, anti-TRAF6, anti-c-fos, anti-NFATc1, and β-actin antibodies. Data are presented as means ± SE (n = 3). Data are representative of three independent experiments ( A , C , D ). *p
Figure Legend Snippet: FAM19A5 regulates RANKL-induced gene expression during osteoclastogenesis. ( A , C , D ) Mouse BMDMs were stimulated with FAM19A5 (2 μM) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 3 days. Cells were harvested for RNA preparation. qPCR was performed using specific primers for RANK , TRAF6 , OSCAR , TRAP , Blimp1 , c-FOS , NFATc1 , MafB , IRF-8 , DC-STAMP , OC-STAMP , Atp6v0d2 , and GAPDH . ( B ) Cells were harvested and Western blot analysis was conducted using anti-RANK, anti-TRAF6, anti-c-fos, anti-NFATc1, and β-actin antibodies. Data are presented as means ± SE (n = 3). Data are representative of three independent experiments ( A , C , D ). *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

26) Product Images from "Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl"

Article Title: Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl

Journal: Cancer cell

doi: 10.1016/j.ccr.2010.04.025

Inhibition of calcineurin-NFAT by CsA sensitizes Ph + leukemia cells to Bcr-Abl inhibition A) K562 cells were treated with imatinib and CsA alone or in combination as indicated (increasing CsA concentrations of 0, 1, 2.5, and 5 μM are indicated by the triangles) for 72 hr and viable cells were counted by flow cytometry. B) K562 cells were treated with dasatinib and CsA as in A for 48 hr and viable cells were counted. C) K562 cells were infected with an NFAT-luciferase reporter. After 32 hr, the cells were treated with the indicated concentrations of CsA alone or together with ionomycin at 1 μg/ml for 16 hr after which cells were harvested and assayed for luciferase activity. D) K562 cells were treated with imatinib and CsA as indicated. After 24 hr, the cells were harvested and lysates subjected to western blot analysis for phosphorylated (p) Bcr-Abl, CrkL, STAT5 and total phosphotyrosine or EIF-4E (loading control). E) KBM7 CML cells were treated with imatinib and CsA as in A for 48 hr and viable cells were counted. F) SUP-B15 Ph + ALL cells were treated with imatinib and CsA as in A .
Figure Legend Snippet: Inhibition of calcineurin-NFAT by CsA sensitizes Ph + leukemia cells to Bcr-Abl inhibition A) K562 cells were treated with imatinib and CsA alone or in combination as indicated (increasing CsA concentrations of 0, 1, 2.5, and 5 μM are indicated by the triangles) for 72 hr and viable cells were counted by flow cytometry. B) K562 cells were treated with dasatinib and CsA as in A for 48 hr and viable cells were counted. C) K562 cells were infected with an NFAT-luciferase reporter. After 32 hr, the cells were treated with the indicated concentrations of CsA alone or together with ionomycin at 1 μg/ml for 16 hr after which cells were harvested and assayed for luciferase activity. D) K562 cells were treated with imatinib and CsA as indicated. After 24 hr, the cells were harvested and lysates subjected to western blot analysis for phosphorylated (p) Bcr-Abl, CrkL, STAT5 and total phosphotyrosine or EIF-4E (loading control). E) KBM7 CML cells were treated with imatinib and CsA as in A for 48 hr and viable cells were counted. F) SUP-B15 Ph + ALL cells were treated with imatinib and CsA as in A .

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Infection, Luciferase, Activity Assay, Western Blot

27) Product Images from "A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase"

Article Title: A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase

Journal: Scientific Reports

doi: 10.1038/srep22389

Effect of the Ewha-18278 on the RANKL-induced signaling pathways. ( A ) BMMs were pretreated with Ewha-18278 for 1 hour, the cells were stimulated with RANKL for the indicated times. The cell lysates were subjected to immunoblot analysis. RANKL signaling pathways assessed by phosphorylation of ERK, p38, JNK, and IκBα. Immunoblots were stripped and then reprobed with total ERK, p38, JNK, and IκBα. ( B , C ) BMMs were treated with Ewha-18278 in the presence of M-CSF and RANKL for the indicated periods. Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels of NFATc1 ( B ) and Atp6v0d2 ( C ) genes. The relative expression levels were normalized to the level of actin mRNA as an internal control gene. * P
Figure Legend Snippet: Effect of the Ewha-18278 on the RANKL-induced signaling pathways. ( A ) BMMs were pretreated with Ewha-18278 for 1 hour, the cells were stimulated with RANKL for the indicated times. The cell lysates were subjected to immunoblot analysis. RANKL signaling pathways assessed by phosphorylation of ERK, p38, JNK, and IκBα. Immunoblots were stripped and then reprobed with total ERK, p38, JNK, and IκBα. ( B , C ) BMMs were treated with Ewha-18278 in the presence of M-CSF and RANKL for the indicated periods. Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels of NFATc1 ( B ) and Atp6v0d2 ( C ) genes. The relative expression levels were normalized to the level of actin mRNA as an internal control gene. * P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing

28) Product Images from "Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis"

Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-019-0698-5

AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01
Figure Legend Snippet: AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

Techniques Used: Over Expression, In Vivo, Mouse Assay, Injection, Staining, Immunohistochemistry, Expressing, Imaging, Plasmid Preparation, Concentration Assay, Two Tailed Test

Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01
Figure Legend Snippet: Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

Techniques Used: Expressing, Invasion Assay, Inhibition, Western Blot, Plasmid Preparation, Staining, Mouse Assay, Injection, Two Tailed Test

Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature
Figure Legend Snippet: Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature

Techniques Used: Expressing, Activation Assay, Inhibition

AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α
Figure Legend Snippet: AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α

Techniques Used: Expressing, Immunohistochemistry, Staining

AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01
Figure Legend Snippet: AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

Techniques Used: Plasmid Preparation, Expressing, Over Expression, Western Blot, Invasion Assay, Two Tailed Test

AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01
Figure Legend Snippet: AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Two Tailed Test

29) Product Images from "CHD7 interacts with BMP R-SMADs to epigenetically regulate cardiogenesis in mice"

Article Title: CHD7 interacts with BMP R-SMADs to epigenetically regulate cardiogenesis in mice

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddt610

CHD7 interacts with SMAD1. ( A ) AH109 yeast cells harboring different plasmids were grown on selective plates. Growth indicates positive interaction between the bait and the prey. SMAD1 and the C-terminal region of mCHD7 (aa 2301–2646) interact
Figure Legend Snippet: CHD7 interacts with SMAD1. ( A ) AH109 yeast cells harboring different plasmids were grown on selective plates. Growth indicates positive interaction between the bait and the prey. SMAD1 and the C-terminal region of mCHD7 (aa 2301–2646) interact

Techniques Used:

Model of CHD7 recruitment to the enhancer of a BMP/SMAD target gene. Upon BMP stimulation, the SMAD1/5/8-SMAD4 complex is transferred into nucleus, where the complex binds to the SMAD-binding elements in the enhancers of BMP/SMAD target genes. The C-terminus
Figure Legend Snippet: Model of CHD7 recruitment to the enhancer of a BMP/SMAD target gene. Upon BMP stimulation, the SMAD1/5/8-SMAD4 complex is transferred into nucleus, where the complex binds to the SMAD-binding elements in the enhancers of BMP/SMAD target genes. The C-terminus

Techniques Used: Binding Assay

30) Product Images from "Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass"

Article Title: Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1511285112

( A ) Western blot analysis of RANKL-induced JNK, ERK, p38, and NF-κB in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments. ( B ) Western blot analysis of M-CSF-induced AKT and ERK activation in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments.
Figure Legend Snippet: ( A ) Western blot analysis of RANKL-induced JNK, ERK, p38, and NF-κB in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments. ( B ) Western blot analysis of M-CSF-induced AKT and ERK activation in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments.

Techniques Used: Western Blot, Activation Assay

31) Product Images from "Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation"

Article Title: Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2014.282996

Activities of CaMKII/HDAC4 or calcineurin/NFAT signaling in WT and Epac1KO in response to chronic CB treatment
Figure Legend Snippet: Activities of CaMKII/HDAC4 or calcineurin/NFAT signaling in WT and Epac1KO in response to chronic CB treatment

Techniques Used:

32) Product Images from "Non-canonical Wnt4 prevents skeletal aging and inflammation by inhibiting NF-κB"

Article Title: Non-canonical Wnt4 prevents skeletal aging and inflammation by inhibiting NF-κB

Journal: Nature medicine

doi: 10.1038/nm.3586

Wnt4 inhibits NF-κB by interfering with TAK1-TRAF6 binding. ( a ) Immunoblots showing the phosphorylation of Tak1, p65 and Iκbα in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. ( b ) Immunoblots showing p65 and Tata-binding protein (Tbp) in nuclear extracts of bone marrow macrophages treated with Rankl, rWnt4 and rWnt4 with Rankl. ( c ) Relative NF-κB-dependent luciferase reporter activities in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. ( d ) Immunoblots showing the Traf6-Tak1-Tab2 complex formation induced by Rankl in bone marrow macrophages. ( e ) Immunoblots showing the induction of Nfatc1 in bone marrow macrophages after treatment of Rankl, and rWnt4 with Rankl. ( f ) ChIP assays of the recruitment of p65 to the Nfatc1 promoter induced by Rankl. Anti-IgG and primers designed at 9 kb downstream of transcription start site (TSS) were used as negative control. ( g ) ChIP assays of Nfatc1 binding to the Nfatc1 promoter. ( h ) Immunoblots of β-catenin in cytosolic extract (CE) and nuclear extract (NE) of bone marrow macrophages treated with Wnt3a and Wnt4. ( i ) Relative Topflash luciferase activities in bone marrow macrophages treated with Wnt3a or Wnt4. ( j ) Real-time RT-PCR of Axin2 and Dkk1 in bone marrow macrophages treated with Wnt3a or Wnt4. n = 3; * P
Figure Legend Snippet: Wnt4 inhibits NF-κB by interfering with TAK1-TRAF6 binding. ( a ) Immunoblots showing the phosphorylation of Tak1, p65 and Iκbα in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. ( b ) Immunoblots showing p65 and Tata-binding protein (Tbp) in nuclear extracts of bone marrow macrophages treated with Rankl, rWnt4 and rWnt4 with Rankl. ( c ) Relative NF-κB-dependent luciferase reporter activities in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. ( d ) Immunoblots showing the Traf6-Tak1-Tab2 complex formation induced by Rankl in bone marrow macrophages. ( e ) Immunoblots showing the induction of Nfatc1 in bone marrow macrophages after treatment of Rankl, and rWnt4 with Rankl. ( f ) ChIP assays of the recruitment of p65 to the Nfatc1 promoter induced by Rankl. Anti-IgG and primers designed at 9 kb downstream of transcription start site (TSS) were used as negative control. ( g ) ChIP assays of Nfatc1 binding to the Nfatc1 promoter. ( h ) Immunoblots of β-catenin in cytosolic extract (CE) and nuclear extract (NE) of bone marrow macrophages treated with Wnt3a and Wnt4. ( i ) Relative Topflash luciferase activities in bone marrow macrophages treated with Wnt3a or Wnt4. ( j ) Real-time RT-PCR of Axin2 and Dkk1 in bone marrow macrophages treated with Wnt3a or Wnt4. n = 3; * P

Techniques Used: Binding Assay, Western Blot, Luciferase, Chromatin Immunoprecipitation, Negative Control, Quantitative RT-PCR

Wnt4 attenuates osteoporosis induced by OVX. ( a,b ) µCT reconstruction ( a ) of metaphysis of distal femurs, as well as BMD and BV/TV ( b ) in WT vs Wnt4 mice at two months post OVX. Scale bars, 200 µm. ( c ) BFR measurement of calcein dual labeling in WT vs Wnt4 mice two months after OVX or sham operation. ( d, e ) Morphometric analysis of osteoblast counts ( d ) and osteoclast counts ( e ) in WT vs Wnt4 mice after OVX or sham operation. ( f ) TRAP staining of femur sections from WT and Wnt4 mice after OVX or sham operation. ( g–i ) ELISA of serum concentrations of Ocn ( g ), Trap5b ( h ), Il-6 and Tnf ( i ) in WT vs Wnt4 mice after OVX or sham operation. Scale bars, 30µm. ( j ) Immunostaining and quantification of active p65 in trabecular bone cells and surrounding bone marrow cells in WT and Wnt4 mice after OVX or sham operation. Scale bars, 30 µm. IOD, integral optical density. For b–e , and g–j , n = 8 for sham groups; n = 12 for OVX groups. * P
Figure Legend Snippet: Wnt4 attenuates osteoporosis induced by OVX. ( a,b ) µCT reconstruction ( a ) of metaphysis of distal femurs, as well as BMD and BV/TV ( b ) in WT vs Wnt4 mice at two months post OVX. Scale bars, 200 µm. ( c ) BFR measurement of calcein dual labeling in WT vs Wnt4 mice two months after OVX or sham operation. ( d, e ) Morphometric analysis of osteoblast counts ( d ) and osteoclast counts ( e ) in WT vs Wnt4 mice after OVX or sham operation. ( f ) TRAP staining of femur sections from WT and Wnt4 mice after OVX or sham operation. ( g–i ) ELISA of serum concentrations of Ocn ( g ), Trap5b ( h ), Il-6 and Tnf ( i ) in WT vs Wnt4 mice after OVX or sham operation. Scale bars, 30µm. ( j ) Immunostaining and quantification of active p65 in trabecular bone cells and surrounding bone marrow cells in WT and Wnt4 mice after OVX or sham operation. Scale bars, 30 µm. IOD, integral optical density. For b–e , and g–j , n = 8 for sham groups; n = 12 for OVX groups. * P

Techniques Used: Mouse Assay, Labeling, Staining, Enzyme-linked Immunosorbent Assay, Immunostaining

Wnt4 inhibits TNF-induced bone loss and NF-κB activation. ( a,b ) µCT reconstruction ( a ), BMD and BV/TV ( b ) of distal femoral metaphysis regions from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 200 µm. ( c ) Comparisons of MAR and BFR in TNFtg mice and TNFtg/Wnt4 mice. ( d,e ) Morphometric analysis of osteoblast counts ( d ) and osteoclast counts ( e ) in TNFtg mice and TNFtg/Wnt4 mice. ( f ) TRAP staining of osteoclasts surrounding trabecular bones in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40 µm. ( g–i ) ELISA of Ocn ( g ), Trap5b ( h ) and Il-6 ( i ) concentrations in serum collected from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. ( j ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bone in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40µm. TNF, TNFtg mice; T/W4, TNFtg/Wnt4 mice. For b–e , and g–j , n = 6 per group for WT and WNT4 mice; n = 8 per group for TNFtg and TNFtg/Wnt mice. * P
Figure Legend Snippet: Wnt4 inhibits TNF-induced bone loss and NF-κB activation. ( a,b ) µCT reconstruction ( a ), BMD and BV/TV ( b ) of distal femoral metaphysis regions from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 200 µm. ( c ) Comparisons of MAR and BFR in TNFtg mice and TNFtg/Wnt4 mice. ( d,e ) Morphometric analysis of osteoblast counts ( d ) and osteoclast counts ( e ) in TNFtg mice and TNFtg/Wnt4 mice. ( f ) TRAP staining of osteoclasts surrounding trabecular bones in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40 µm. ( g–i ) ELISA of Ocn ( g ), Trap5b ( h ) and Il-6 ( i ) concentrations in serum collected from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. ( j ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bone in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40µm. TNF, TNFtg mice; T/W4, TNFtg/Wnt4 mice. For b–e , and g–j , n = 6 per group for WT and WNT4 mice; n = 8 per group for TNFtg and TNFtg/Wnt mice. * P

Techniques Used: Activation Assay, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Immunostaining, Activity Assay

Wnt4 attenuates skeletal aging by inhibiting NF-κB. ( a–c ) µCT reconstruction ( a ), BMD and BV/TV ( b ), as well as H E staining ( c ) of distal femoral metaphysis regions from 6-, 18- and 24-months-old WT and Wnt4 mice. Scale bars, 200 µm ( a ); 300 µm ( c ). ( d ) Morphometric analysis of osteoblast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. ( e ) ELISA of Ocn concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. ( f ) Morphometric analysis of osteoclast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. ( g,h ) ELISA of Trap5b ( g ) and Il-6 ( h ) concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. ( i ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from 24-months-old WT and Wnt4 mice. Scale bars, 25 µm. For b , and d–i , n = 12 mice per group. * P
Figure Legend Snippet: Wnt4 attenuates skeletal aging by inhibiting NF-κB. ( a–c ) µCT reconstruction ( a ), BMD and BV/TV ( b ), as well as H E staining ( c ) of distal femoral metaphysis regions from 6-, 18- and 24-months-old WT and Wnt4 mice. Scale bars, 200 µm ( a ); 300 µm ( c ). ( d ) Morphometric analysis of osteoblast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. ( e ) ELISA of Ocn concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. ( f ) Morphometric analysis of osteoclast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. ( g,h ) ELISA of Trap5b ( g ) and Il-6 ( h ) concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. ( i ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from 24-months-old WT and Wnt4 mice. Scale bars, 25 µm. For b , and d–i , n = 12 mice per group. * P

Techniques Used: Staining, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Activity Assay

rWnt4 proteins attenuates established bone loss by inhibiting NF-κB. ( a–c ) µCT reconstruction ( a ), BMD and BV/TV ( b ), as well as H E staining ( c ) of distal femoral metaphysis regions from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 200 µm ( a ); 300 µm ( c ). ( d,e ) Morphometric analysis of osteoblast ( d ) and osteoclast ( e ) counts in distal femoral metaphysis from mice after sham operation, OVX and OVX with rWnt4 injection. ( f ) TRAP staining showing osteoclasts surrounding trabecular bones in mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30µm. ( g,h ) ELISA of Trap5b ( g ) and Ocn ( h ) concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. ( i ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30 µm. ( j ) ELISA of Il-6 and Tnf concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. n = 8 mice for sham group; n = 12 mice per group for mice receiving OVX and OVX with rWnt4 injection. * P
Figure Legend Snippet: rWnt4 proteins attenuates established bone loss by inhibiting NF-κB. ( a–c ) µCT reconstruction ( a ), BMD and BV/TV ( b ), as well as H E staining ( c ) of distal femoral metaphysis regions from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 200 µm ( a ); 300 µm ( c ). ( d,e ) Morphometric analysis of osteoblast ( d ) and osteoclast ( e ) counts in distal femoral metaphysis from mice after sham operation, OVX and OVX with rWnt4 injection. ( f ) TRAP staining showing osteoclasts surrounding trabecular bones in mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30µm. ( g,h ) ELISA of Trap5b ( g ) and Ocn ( h ) concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. ( i ) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30 µm. ( j ) ELISA of Il-6 and Tnf concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. n = 8 mice for sham group; n = 12 mice per group for mice receiving OVX and OVX with rWnt4 injection. * P

Techniques Used: Staining, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Immunostaining, Activity Assay

33) Product Images from "The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia"

Article Title: The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0599-2

Abcc8 −/− and Trpm4 −/− protects against TLR4-mediated NOS2 induction in murine microglia. a , b Immunoblots ( a ) and quantification ( b ) for NFATc1 (90 kDa isoform shown), NOS2 (130 kDa), pCaMKII (α/β, 50/60 kDa), CaMKII (α/β, 50/60 kDa), pCN (60 kDa), and CN (60 kDa), for tissue homogenates from the striatum of wild-type ( WT ), Abcc8 −/− and Trpm4 −/− mice, 24 h after injection of aCSF (5 μL) or LPS (5 μL; 0.1 μg/μL) into the striatum; gels were run separately for each protein analyzed; quantitative data were normalized to a loading control for total protein, HSC70 ; three replicates; ** p
Figure Legend Snippet: Abcc8 −/− and Trpm4 −/− protects against TLR4-mediated NOS2 induction in murine microglia. a , b Immunoblots ( a ) and quantification ( b ) for NFATc1 (90 kDa isoform shown), NOS2 (130 kDa), pCaMKII (α/β, 50/60 kDa), CaMKII (α/β, 50/60 kDa), pCN (60 kDa), and CN (60 kDa), for tissue homogenates from the striatum of wild-type ( WT ), Abcc8 −/− and Trpm4 −/− mice, 24 h after injection of aCSF (5 μL) or LPS (5 μL; 0.1 μg/μL) into the striatum; gels were run separately for each protein analyzed; quantitative data were normalized to a loading control for total protein, HSC70 ; three replicates; ** p

Techniques Used: Western Blot, Mouse Assay, Injection

Sur1-Trpm4 regulates NFATc1, CaMKII, and CN in murine N9 microglia. a , b Images ( a ) and quantification ( b ) of nuclear NFATc1 ( white ) in N9 microglia under control ( CTR ) conditions, following 15-min exposure to the Ca 2+ ionophore, A23187 (1 μM), used as positive control, and following 24-h exposure to LPS alone (1 μg/mL), LPS plus glibenclamide ( Glib ; 30 μM), LPS plus 9-phenanthrol ( 9Phe ; 5 μM), or LPS plus SKF-96395 ( SKF ; 7.5 μM); nuclei stained with DAPI ( blue ); typical nuclear diameter is 6–12 μm; quantitative data on specific nuclear labeling were normalized to the control; p
Figure Legend Snippet: Sur1-Trpm4 regulates NFATc1, CaMKII, and CN in murine N9 microglia. a , b Images ( a ) and quantification ( b ) of nuclear NFATc1 ( white ) in N9 microglia under control ( CTR ) conditions, following 15-min exposure to the Ca 2+ ionophore, A23187 (1 μM), used as positive control, and following 24-h exposure to LPS alone (1 μg/mL), LPS plus glibenclamide ( Glib ; 30 μM), LPS plus 9-phenanthrol ( 9Phe ; 5 μM), or LPS plus SKF-96395 ( SKF ; 7.5 μM); nuclei stained with DAPI ( blue ); typical nuclear diameter is 6–12 μm; quantitative data on specific nuclear labeling were normalized to the control; p

Techniques Used: Positive Control, Staining, Labeling

34) Product Images from "Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation"

Article Title: Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021170

TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.
Figure Legend Snippet: TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.

Techniques Used:

35) Product Images from "The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis *"

Article Title: The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.667535

Double inactivation of motif 2 and motif 3 does not affect the ability of RANK to activate the NF-κB, JNK, p38, and ERK pathways. BMMs expressing hFas-WT or hFas-M23 were treated with α-Fas (100 ng/ml) for 0, 5, or 10 min as indicated. The activation of the NF-κB, JNK, p38, and ERK pathways was assessed as phosphorylation of IκB, JNK, p38, and ERK by Western blot analysis. BMMs expressing hFas-WT were used as a positive control.
Figure Legend Snippet: Double inactivation of motif 2 and motif 3 does not affect the ability of RANK to activate the NF-κB, JNK, p38, and ERK pathways. BMMs expressing hFas-WT or hFas-M23 were treated with α-Fas (100 ng/ml) for 0, 5, or 10 min as indicated. The activation of the NF-κB, JNK, p38, and ERK pathways was assessed as phosphorylation of IκB, JNK, p38, and ERK by Western blot analysis. BMMs expressing hFas-WT were used as a positive control.

Techniques Used: Expressing, Activation Assay, Western Blot, Positive Control

36) Product Images from "Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling *"

Article Title: Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.668939

Smad1/5 binds to NFATc1 P1 promoter, and BMP-2/Smad signaling induces NFATc1 nuclear translocation. A , NFATc1 5′ sequences flanking the P1 promoter harboring SBE are shown in red . Oligonucleotide spanning −70 to −47 bp ( green )
Figure Legend Snippet: Smad1/5 binds to NFATc1 P1 promoter, and BMP-2/Smad signaling induces NFATc1 nuclear translocation. A , NFATc1 5′ sequences flanking the P1 promoter harboring SBE are shown in red . Oligonucleotide spanning −70 to −47 bp ( green )

Techniques Used: Translocation Assay

37) Product Images from "Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation"

Article Title: Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2014.282996

Activities of CaMKII/HDAC4 or calcineurin/NFAT signaling in WT and Epac1KO in response to chronic CB treatment
Figure Legend Snippet: Activities of CaMKII/HDAC4 or calcineurin/NFAT signaling in WT and Epac1KO in response to chronic CB treatment

Techniques Used:

38) Product Images from "Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass"

Article Title: Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1511285112

( A ) Western blot analysis of RANKL-induced JNK, ERK, p38, and NF-κB in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments. ( B ) Western blot analysis of M-CSF-induced AKT and ERK activation in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments.
Figure Legend Snippet: ( A ) Western blot analysis of RANKL-induced JNK, ERK, p38, and NF-κB in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments. ( B ) Western blot analysis of M-CSF-induced AKT and ERK activation in WT and Tmem178 −/− BMMs ( Top ) and day 2 preOCs ( Bottom ), representative of three experiments.

Techniques Used: Western Blot, Activation Assay

39) Product Images from "Calcineurin signaling and PGC-1? expression are suppressed during muscle atrophy due to diabetes"

Article Title: Calcineurin signaling and PGC-1? expression are suppressed during muscle atrophy due to diabetes

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamcr.2010.03.019

GSK-3β signaling is unchanged in 21day STZ-treated rat muscle (A) The phosphorylation (i.e., inactivation) of GSK-3β on Ser-9 in gastrocnemius muscles was examined by Western blot analysis using antibodies that detect phospho-S9 and total GSK-3β. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.31 (B) The phosphorylation of glycogen synthase on Ser-641 also was examined as a downstream target of GSK-3β activity by Western blot analysis using antibodies that detect total and phospho-Ser-641 glycogen synthase. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.69 (C) Western blot analysis of Akt phosphorylation was performed using antibodies that detect total and phospho-Ser-473 Akt. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM: P
Figure Legend Snippet: GSK-3β signaling is unchanged in 21day STZ-treated rat muscle (A) The phosphorylation (i.e., inactivation) of GSK-3β on Ser-9 in gastrocnemius muscles was examined by Western blot analysis using antibodies that detect phospho-S9 and total GSK-3β. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.31 (B) The phosphorylation of glycogen synthase on Ser-641 also was examined as a downstream target of GSK-3β activity by Western blot analysis using antibodies that detect total and phospho-Ser-641 glycogen synthase. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM. n= 8/group; P =0.69 (C) Western blot analysis of Akt phosphorylation was performed using antibodies that detect total and phospho-Ser-473 Akt. Data are expressed as the mean ratio of phosphorylated protein to total protein ± SEM: P

Techniques Used: Western Blot, Activity Assay

40) Product Images from "STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex"

Article Title: STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-017-0048-5

STAC2 inhibits the RANKL-stimulated activation of MAPK and NF-κB. a , b BMMs were transduced with the pMX-puro retrovirus (EV) or retrovirus expressing Flag-STAC2 (STAC2). The transduced BMMs were then serum-starved and incubated with RANKL (200 ng/ml) ( a ) or M-CSF (100 ng/ml) ( b ) for the indicated times, and subjected to western blot analysis with antibodies to p-JNK, JNK, p-p38, p38, p-ERK, ERK, IκBα, Flag, and tubulin. Data are representative of at least three independent experiments a , b
Figure Legend Snippet: STAC2 inhibits the RANKL-stimulated activation of MAPK and NF-κB. a , b BMMs were transduced with the pMX-puro retrovirus (EV) or retrovirus expressing Flag-STAC2 (STAC2). The transduced BMMs were then serum-starved and incubated with RANKL (200 ng/ml) ( a ) or M-CSF (100 ng/ml) ( b ) for the indicated times, and subjected to western blot analysis with antibodies to p-JNK, JNK, p-p38, p38, p-ERK, ERK, IκBα, Flag, and tubulin. Data are representative of at least three independent experiments a , b

Techniques Used: Activation Assay, Transduction, Expressing, Incubation, Western Blot

Related Articles

Negative Control:

Article Title: Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression
Article Snippet: .. For immunoprecipitation, lysates were incubated overnight with the following antibodies at 4°C: histone H3 (1:100, Cell Signaling Technology, 2650), H3K4me3 (1:100, Cell Signaling Technology, 9727), H3K9/14ac (1:200, Millipore, 17-615 ) , H3K9me3 (1:200, Abcam, ab8898), γH2AX (1:50, Abcam, ab2893), and the negative control normal rabbit IgG (Millipore). ..

Immunoprecipitation:

Article Title: Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression
Article Snippet: .. For immunoprecipitation, lysates were incubated overnight with the following antibodies at 4°C: histone H3 (1:100, Cell Signaling Technology, 2650), H3K4me3 (1:100, Cell Signaling Technology, 9727), H3K9/14ac (1:200, Millipore, 17-615 ) , H3K9me3 (1:200, Abcam, ab8898), γH2AX (1:50, Abcam, ab2893), and the negative control normal rabbit IgG (Millipore). ..

Incubation:

Article Title: Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression
Article Snippet: .. For immunoprecipitation, lysates were incubated overnight with the following antibodies at 4°C: histone H3 (1:100, Cell Signaling Technology, 2650), H3K4me3 (1:100, Cell Signaling Technology, 9727), H3K9/14ac (1:200, Millipore, 17-615 ) , H3K9me3 (1:200, Abcam, ab8898), γH2AX (1:50, Abcam, ab2893), and the negative control normal rabbit IgG (Millipore). ..

Western Blot:

Article Title: The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells
Article Snippet: .. Western blot analysis was performed with anti-UBE2K (Cell Signaling, #8226, 1:1,000), anti-OCT4 (Stem Cell Technologies, #60093, 1:500), anti-SOX2 (Abcam, #97959, 1:1,000), anti-PAX6 (Stem Cell Technologies, #60094, 1:200), anti-Nestin (Stem Cell Technologies, #60091, 1:1,000), anti-MAP2 (Sigma, #1406, 1:1,000), anti-polyubiquitinylated conjugates (Enzo, PW8805-0500, 1:1,000), anti-ubiquitin (Merck Millipore, # 05-944, clone P4D1-A11, 1:1000), anti-H3K9me3 (Abcam, #8898, 1:1,000), anti-Histone H3 (Cell Signaling, #2650, 1:10,000), anti-H3K9me1 (Cell Signaling, #1418, 1:1,000), anti-H3K9me2 (Cell Signaling, #4658, 1:1,000), anti-H3K4me3 (Active Motif, #39916, 1:1,000), anti-H3K27me3 (Active Motif, #39155, 1:1,000), anti-H3K27ac (Active Motif, #39933, 1:1,000), anti-HTT (Cell Signaling, #5656, 1:1,000), anti-SETDB1 (Abcam, #107225, 1:500), anti-p53 (Cell Signaling, #9282, 1:2,000), anti-Histone H1 (Merck, 05-457, 1:1,000), anti-PSMD11 (Abcam, #99413, 1:1,000), anti-ß-actin (Abcam, #8226, 1:1,000) and α-tubulin (Sigma, T6199, 1:5,000). .. In vitro ubiquitination assays A concentration of 10 μg of purified human recombinant protein 6xHis::H3F3A (Prospec, PRO-1452) was mixed with 25 ng of E1 activating enzyme (Enzo Life Sciences, BML-UW9410-0050), 400 ng of UBE2K/E2-25K (R & D systems, E2-603), 2 μg of FLAG::ubiquitin (Sigma-Aldrich, U6253), ATP regeneration solution (Enzo Life Sciences, BML-EW9810-0100) and Ubiquitin Conjugation Reaction Buffer (Enzo Life Sciences, BML-KW9885-0001).

Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes
Article Snippet: .. Western analysis was performed using anti-AGO2 (1:1000; ab57 113; abcam), anti-GW182 (1:4000; A302-329A; Bethyl Labs), anti-tubulin (1:6000; T5201; Sigma), anti-calreticulin (1:1000; 2891S; Cell Signaling) or anti-histone H3 (1:10 000; 2650S; Cell Signaling) primary antibody and horseradish peroxidase-conjugated anti-mouse IgG (715-035-150) or anti-rabbit IgG (711-035-152) secondary antibody (1:5000–1:10 000; Jackson Immunolabs) as described earlier in the text. .. Co-immunoprecipitations Co-immunoprecipitation experiments were performed by mixing 30 µl of Protein G Plus/Protein A resin, 1.5 µg of antibody (anti-AGO2, ab57113, Abcam; anti-GW182, Bethyl Laboratories) and nuclear extract (0.5–1 mg of total protein) and incubating at 4°C for 4 h. Resin was washed 4x with IP wash buffer [20 mM Tris (pH 7.5), 0.4 M NaCl, 2 mM MgCl2 , 0.05% NP-40, 0.025% SDS] and co-purified proteins eluted by boiling resin in 25 µl 1× SDS loading buffer.

Article Title: FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1
Article Snippet: .. Western blot analysis was performed as we previously described ( , ) using primary antibodies against FASN (BD Biosciences; 610962), PARP-1 (Cell Signaling; 9542), histone H3 (Cell Signaling; 2650), NF-κB p65 (Santa Cruz; sc372), Ku70 (Santa Cruz; sc-1487), γ-H2AX (Millipore; 05636), and anti-PAR (Trevigen; 4335). .. Final images were captured using FluorChem HD2 imager, and staining intensity was quantified using the AlphaEaseFC program associated with the FluorChem HD2 imager (both from Alpha Innotech).

Article Title: ELYS regulates the localization of LBR by modulating its phosphorylation state
Article Snippet: .. The primary antibodies used for western blotting were the same as those used in immunofluorescence and were used at the same dilutions, with the additional antibodies: mouse anti-phosphorylated-histone-H3 at residue S10 (6G3, Cell Signaling Technology, MA), 1:2000; mouse anti-β-actin (A5441, Sigma-Aldrich), 1:3000; rabbit anti-GFP (598, MBL, Nagoya, Japan), 1:1000; mouse anti-GFP (11-814-460-001, Roche), 1:1000; goat anti-GFP (AB0020-200, SICGEN antibodies, Cantanhede, Portugal), 1:1000-2000; anti-PP1α (sc-6104, Santa Cruz Biotechnology), 1:2000; anti-PP1β (sc-6107, Santa Cruz Biotechnology), 1:2000; anti-PP1γ (sc-6109, Santa Cruz Biotechnology), 1:10,000; rabbit anti-NUP107 (A301-787A, Bethyl Laboratories, Montgomery, TX), 1:1000; rabbit anti-HP-1 (C7F11, Cell Signaling Technology), 1:500; and rabbit anti-histone-3 (ab1791, Abcam), 1:4000. .. The mAb414 antibody was used (MMS-120P, Covance) to detect NUP62 at a 1:3000 dilution.

Chromatin Immunoprecipitation:

Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource
Article Snippet: .. The following antibodies were used for ChIP: histone H3 (Cell Signalling Technology, #2650; 4 μl per IP), H3K36me3 (abcam, #ab9050; 2 μg per IP), H3K9me3 (abcam, #ab8898; 2 μg per IP), normal rabbit IgG (Cell Signaling Technology, #2727; 2 μl per IP). .. Primers used for ChIP-qPCR analysis are presented in .

Article Title: Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation *
Article Snippet: .. The following antibodies used in chromatin immunoprecipitation assay were commercially available: anti-RNAPII (H224, sc-9001x; Santa Cruz), anti-RNAPII C-terminal domain phospho-S2 (ab5095; Abcam), anti-RNAPII C-terminal domain phospho S5 (ab5131; Abcam), anti-TFIIB (sc-225x; Santa Cruz), anti-histone H3 (2650; Cell Signaling), anti-histone H3 acetyl K9 (ab4441; Abcam), anti-histone H3 trimethyl K4 (ab8580; Abcam), anti-histone H3 trimethyl K36 (ab9050; Abcam), and anti-α-tubulin (CP06; CalBiochem). .. Anti-NELF-A, -B, -C, and -E antibodies used in the Western blot analysis and ChIP assay have been previously described ( , ).

Immunofluorescence:

Article Title: ELYS regulates the localization of LBR by modulating its phosphorylation state
Article Snippet: .. The primary antibodies used for western blotting were the same as those used in immunofluorescence and were used at the same dilutions, with the additional antibodies: mouse anti-phosphorylated-histone-H3 at residue S10 (6G3, Cell Signaling Technology, MA), 1:2000; mouse anti-β-actin (A5441, Sigma-Aldrich), 1:3000; rabbit anti-GFP (598, MBL, Nagoya, Japan), 1:1000; mouse anti-GFP (11-814-460-001, Roche), 1:1000; goat anti-GFP (AB0020-200, SICGEN antibodies, Cantanhede, Portugal), 1:1000-2000; anti-PP1α (sc-6104, Santa Cruz Biotechnology), 1:2000; anti-PP1β (sc-6107, Santa Cruz Biotechnology), 1:2000; anti-PP1γ (sc-6109, Santa Cruz Biotechnology), 1:10,000; rabbit anti-NUP107 (A301-787A, Bethyl Laboratories, Montgomery, TX), 1:1000; rabbit anti-HP-1 (C7F11, Cell Signaling Technology), 1:500; and rabbit anti-histone-3 (ab1791, Abcam), 1:4000. .. The mAb414 antibody was used (MMS-120P, Covance) to detect NUP62 at a 1:3000 dilution.

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    Cell Signaling Technology Inc hif 1α
    AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, <t>HIF-1α,</t> and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01
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    AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Over Expression, In Vivo, Mouse Assay, Injection, Staining, Immunohistochemistry, Expressing, Imaging, Plasmid Preparation, Concentration Assay, Two Tailed Test

    Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Invasion Assay, Inhibition, Western Blot, Plasmid Preparation, Staining, Mouse Assay, Injection, Two Tailed Test

    Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Activation Assay, Inhibition

    AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Immunohistochemistry, Staining

    AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Plasmid Preparation, Expressing, Over Expression, Western Blot, Invasion Assay, Two Tailed Test

    AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Two Tailed Test

    AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 10 6 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. ** P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 10 5 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Over Expression, In Vivo, Mouse Assay, Injection, Staining, Immunohistochemistry, Expressing, Imaging, Plasmid Preparation, Concentration Assay, Two Tailed Test

    Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. ** P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Invasion Assay, Inhibition, Western Blot, Plasmid Preparation, Staining, Mouse Assay, Injection, Two Tailed Test

    Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: Upstream analysis of the AK4 metabolic gene signature predicted that HIF-1α is transcriptionally activated in lung adenocarcinomas. a Genes were ranked according to their corresponding Pearson correlation coefficient ( R ) to AK4 expression. Genes that are positively correlated with AK4 ( R ≥ 0.3) or negatively correlated with AK4 ( R ≤ − 0.3) were further filtered with enzyme or transporter annotations and defined as the AK4 metabolic gene signature. b KEGG pathway analysis of the AK4 metabolic gene signature. Bar chart represents top 20 significant metabolic pathways ranked according to – log enrichment P value. P values were calculated using Fisher exact test. c GSEA plots of lung cancer prognostic, hypoxic response, and glucose metabolism genes in the AK4 metabolic gene signature. d Left, the ingenuity upstream regulator analysis algorithm predicted significant activation or inhibition of upstream regulators in the AK4 metabolic gene signature. An activation z score of more than 2 or less than − 2 was considered to indicate significant activation or inhibition, respectively. Right, a heatmap illustrating both direct and indirect HIF-1α-regulated genes that are positively or negatively correlated with AK4 expression. e Left, overall survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature. Right, relapse-free survival analysis of patients stratified according to the AK4-HIF-1α gene expression signature

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Activation Assay, Inhibition

    AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 is positively correlated with HIF-1α expression in NSCLC patients. a Representative IHC images of AK4 and HIF-1α expression scores in serial sections of NSCLC tissues. b Spearman’s rho correlation analysis of the IHC staining results for AK4 and HIF-1α in 100 NSCLC patients. c Overall survival analysis of 100 lung cancer patients stratified according to HIF-1α alone or both AK4 and HIF-1α

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Immunohistochemistry, Staining

    AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 shifts metabolism toward aerobic glycolysis and increases oxidative stress. a Global GSEA statistics of differentially expressed genes in AK4-overexpressing CL1-0 cells compared with vector-expressing CL1-0 cells. b GSEA plots and KEGG metabolic pathways of Glycolysis_Gluconeogenesis and Glutathione_Metabolism pathways between CL1-0 AK4 cells and CL1-0 Vec cells. c Relative ATP levels, glucose consumption, and lactate production upon AK4 overexpression in CL1-0 cells. ** P ≤ 0.01 d Relative ADP/ATP ratio upon AK4 overexpression in CL1-0 cells under Nx or Hx. * P ≤ 0.05 e Intracellular ROS level of vector-expressing CL1-0 cells and AK4-expressing CL1-0 cells; the ROS level was normalized to vector-expressing CL1-0 cells. ** P ≤ 0.01. f WB analysis of HIF-1α in CL1-0 vector- and AK4-expressing cells treated with or without 10 mM NAC under Nx or Hx. g Invasion assay of CL1-0 vector- or AK4-expressing cells treated with 10 mM NAC under Nx or Hx. h Intracellular ROS level of shNS-expressing CL1-5 cells and shAK4-expressing CL1-5 cells. The ROS level was normalized to shNS-expressing CL1-5 cells. ** P ≤ 0.01. i Time course analysis of HIF-1α and AK4 protein expression in CL1-5 cells treated with 10 mM NAC for the indicated time. j Invasion assay of CL1-5 cells treated with or without 10 mM NAC. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. * P ≤ 0.05; ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Plasmid Preparation, Expressing, Over Expression, Western Blot, Invasion Assay, Two Tailed Test

    AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01

    Journal: Journal of Hematology & Oncology

    Article Title: Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

    doi: 10.1186/s13045-019-0698-5

    Figure Lengend Snippet: AK4 stabilizes and exaggerates HIF-1α protein expression to promote EMT. a Upper, endogenous AK4 and HIF-1α protein expression in human NSCLC cell lines. Bottom, correlation between AK4 and HIF-1α protein expression in NSCLC cell lines. b Left, WB analysis of AK4, HIF-1α, E-cadherin, and vimentin upon AK4 knockdown in CL1-5 cells. Right, WB analysis of AK4, AMPK, phospho-AMPK (Thr172), and E-cadherin upon AK4 knockdown in A549 cells treated with or without TGF-β (5 ng/mL) for 24 h. c WB analysis of HIF-1α, AK4, E-cadherin, vimentin, and Snail in CL1-0 and H1355 vector- or AK4-overexpressing cells exposed to hypoxia for the indicated time. d RT-PCR analysis of AK4 , HIF1A , CDH1 , HK2 , GLUT1 , VEGFA , and GAPDH in CL1-0 vector- or AK4-expressing cells under normoxic and hypoxic conditions. e WB analysis of HIF-1α from CL1-0 vector- or AK4-expressing cells treated with the proteasome inhibitor MG-132 under Nx and Hx conditions. f WB analysis of HIF-1α and AK4 from CL1-0 vector- or AK4-expressing cells treated with CHX for 20, 40, and 60 min. g WB analysis of HIF-1α and hydroxylated HIF-1α from CL1-0 vector or AK4-expressing cells treated with MG-132 under Nx. h Invasion assay of CL1-0 vector- or AK4-expressing cells under normoxia (Nx) or hypoxia (Hx). ** P ≤ 0.01. i Invasion assay of H1355 vector- or AK4-expressing cells under Nx or Hx. ** P ≤ 0.01. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. ** P ≤ 0.01

    Article Snippet: The following antibodies were used to detect AK4, HIF-1α, E-cadherin, and pimonidazole in tissues: AK4 (Genetex, 1:200), HIF-1α (Cell Signaling, 1:100), E-cadherin (Cell Signaling, 1:100), and pimonidazole (Hypoxyprobe, INC).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Two Tailed Test