Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 3. Effects of organic extracts of Violina pumpkin leaves on osteogenic markers. (A) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of hexane (Hex), acetone (Ac) and methanol (Me) extracts for 72 h. Representative optical photomicrographs of immunostaining are reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and are expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.01 vs. DMSO. (B) ALP activity was determined after 7 and 14 days of extract treatment in osteogenic medium. Data are presented as fold changes with respect to control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.05 vs. OM and Hex-treated groups. (C) Representative images of mineralized nodule formation detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in presence of extracts for 21 days. (D) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.05 vs. OM, ◦p < 0.01 vs. Hex and Me-treated groups. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Marker, Immunostaining, Software, Activity Assay, Control, Staining, Cell Culture, Extraction

Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 4. Effects of TLC subfractions of acetone raw extract on cell viability and osteogenic markers. (A) hOBs were treated with 5–250 µg/mL of TLC subfractions 2, 6 and 7 (Fr2, Fr6, Fr7) for 72 h. The viability was monitored with MTT assays. Data represent mean ± SD (n = 3). * p < 0.05 vs. control cells (CTR), ** p < 0.001 vs. CTR, ◦p < 0.001 vs. DMSO-treated group. (B) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of TLC subfractions or raw acetone extract for 72 h. Representative optical photomicrographs of immunostaining are reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and are expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.05 vs. DMSO, ** p < 0.001 vs. DMSO, ◦p < 0.001 vs. Ac, ◦◦p < 0.01 vs. Ac, # p < 0.001 vs. Fr2, ## p < 0.01 vs. Fr2. (C) ALP activity was determined after 7 and 14 days for TLC subfraction and raw acetone extract treatments in an osteogenic medium. Data are presented as fold changes with respect to the control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.01 vs. OM, ◦◦p < 0.05 vs. OM, # p < 0.05 vs. Fr7-treated group. (D) Representative images of the formation of mineralized nodules detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in the presence of TLC subfractions or raw acetone extract for 21 days. (E) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.01 vs. OM, ◦p< 0.01 vs. Ac-treated group, # p < 0.01 vs. Fr2-treated group. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Expressing, Marker, Immunostaining, Software, Activity Assay, Staining, Cell Culture, Extraction

Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 5. Effects of a pure compound (PU-13OH-FA) obtained from subfraction 2 on cell viability and osteogenic markers. (A) hOBs were treated with 5–250 µg/mL of compound PU-13OH-FA for 72 h. The viability was monitored with an MTT assay. Data represent mean ± SD (n = 3). * p < 0.05 vs. control cells (CTR), ** p < 0.001 vs. CTR, ◦p < 0.05 vs. DMSO-treated group, ◦◦p < 0.01 vs. DMSO-treated group. (B) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of Fr2 and PU-13OH-FA for 72 h. Representative optical photomicrographs of immunostaining is reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.001 vs. DMSO. (C) ALP activity was determined after 7 and 14 days of Fr2 and PU-13OH-FA treatment in osteogenic medium. Data are presented as fold changes with respect to the control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.01 vs. OM. (D) Representative images of mineralized nodules formation detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in the presence of Fr2 and PU-13OH-FA for 21 days. (E) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.001 vs. OM, ◦p < 0.05 vs. Fr2-treated group. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: MTT Assay, Control, Expressing, Marker, Immunostaining, Software, Activity Assay, Staining, Cell Culture, Extraction

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

Article Title: Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4 + T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

doi: 10.4049/jimmunol.2000137

Figure Lengend Snippet: FIGURE 3. Metformin + 2-DG treatment downregulates transcripts of genes involved in activation-induced glycolysis and polyamine metabolism, plus key transcriptional regulators. (A and B) RNA-Seq analyses showing expression of genes involved in (A) glycolytic and (B) polyamine metabolism in primary human CD4+ T cells activated for 24 h in the presence of metformin + 2-DG or vehicle alone. Each tile in the heatmaps represents average log transcripts per million (TPM) from triplicates of one of three human donors. (C) RNA-Seq analyses showing gene expression of metabolic transcription factors in primary human CD4+ T cells activated with anti-CD3/anti-CD28 in the presence of metformin (5 mM), 2-DG (1 mM), metformin + 2-DG, or vehicle alone. Each tile in the heatmap represents average log TPM from triplicates of one of three human donors. (D) Immunoblot of MYC and HIF-1A in primary human CD4+ T cells activated with anti-CD3/anti-CD28 in the presence of metformin (5 mM), 2-DG (1 mM), metformin + 2-DG, or vehicle alone.

Article Snippet: Abs against MYC (catalog no. 5605), HIF-1A (catalog no. 14179), TSC1 (catalog no. 6935), TSC2 (catalog no. 4308), phospho-S6 ribosomal protein (catalog no. 4857), S6 ribosomal protein (catalog no. 2317), phospho-p70 S6 kinase (catalog no. 9205), p70 S6 kinase (catalog no. 2708), phospho-4E-BP1 (catalog no. 2855), 4E-BP1 (catalog no. 9644), and b-tubulin (catalog no. 86298) were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, RNA Sequencing, Expressing, Gene Expression, Western Blot

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

Article Title: Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4 + T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

doi: 10.4049/jimmunol.2000137

Figure Lengend Snippet: FIGURE 5. Deletion of MYC in primary human T cells impairs proliferation and glutamine uptake. (A) Immunoblot confirming loss of MYC (left) and HIF-1A (right) protein in human MYC KO and HIF-1A KO CD4+ T cells activated for 24 h with anti-CD3/anti-CD28. (B) Cell proliferation measured by dilution of CTV dye. Representative CTV dilution plot and proliferative index of human MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+ T cells following 96 h of activation with anti-CD3/anti-CD28. (C and D) Flow cytometry analysis of (C) CD98 and (D) SLC7A5 surface expression on MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+ T cells activated for 24 h with anti-CD3/anti-CD28. Mean fluorescence intensity (MFI) shown are the mean 6 SEM (n = 3–6). *p , 0.05, **p , 0.01. (E) 14C-glutamine uptake allowed to proceed for 1 h in MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+

Article Snippet: Abs against MYC (catalog no. 5605), HIF-1A (catalog no. 14179), TSC1 (catalog no. 6935), TSC2 (catalog no. 4308), phospho-S6 ribosomal protein (catalog no. 4857), S6 ribosomal protein (catalog no. 2317), phospho-p70 S6 kinase (catalog no. 9205), p70 S6 kinase (catalog no. 2708), phospho-4E-BP1 (catalog no. 2855), 4E-BP1 (catalog no. 9644), and b-tubulin (catalog no. 86298) were from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Activation Assay, Flow Cytometry, Expressing

Journal: Frontiers in cell and developmental biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner.

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: FIGURE 2 | vMIA-mediated mitochondrial fragmentation is independent on the fission machinery proteins DLP1 and MFF. (A) Immunofluorescence analyses of MEFs MAVS-MITO cells: (a) control cells, (b) DLP1 silenced cells, (c) MFF silenced cells: (a–c) anti-TIM23; (d–f) overexpression of vMIA-Myc: (d) anti-TIM23, (e) anti-Myc, (f) merge image of d and e; (g–i) overexpression of vMIA-Myc in DLP1 silenced cells: (g) anti-TIM23, (h) anti-Myc, (i) merge image of g and h; (j–l) overexpression of vMIA- Myc in MFF silenced cells: (j) anti-TIM23, (k) anti-Myc, (l) merge image of j and k. Bars represent 10 µm. (B,C) Statistical analysis of mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS-MITO cells in the absence of DLP1 or MFF, respectively. Approximately 600 cells were analysed per condition. Data represents the means ± SEM of three independent experiments analysed using two-way ANOVA with Bonferroni’s multi comparations test (ns = non-significant, ****– p < 0.0001). Error bars represent SEM.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and αTubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Control, Over Expression

Journal: Frontiers in cell and developmental biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner.

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: FIGURE 5 | vMIA inhibits MAVS oligomerization at peroxisomes and mitochondria. MFF is essential for the vMIA-mediated inhibition of MAVS oligomerization at peroxisomes but not at mitochondria. (A,B) HEK293T cells infected with SeV in the presence or absence of vMIA. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (A) Peroxisome-enriched fraction, (B) Mitochondria-enriched fraction. (C,D) HEK293T cells infected with SeV in the presence or absence of vMIA and in the absence of MFF. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (C) Peroxisome-enriched fraction, (D) Mitochondria-enriched fraction. (A–D) Immunoblots were performed with antibodies against MAVS, Myc-tag, COXIV, PEX14 and PMP70. (E) Whole cell lysates were resolved by SDS-PAGE. SeV infection, and consequential activation of MAVS downstream signalling, was confirmed using anti-p-STAT1. vMIA-Myc overexpression and MFF silencing were also confirmed using anti-Myc and anti-MFF, respectively. Antibody against Actin was used as loading control.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and αTubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Inhibition, Infection, Isolation, Western Blot, SDS Page, Activation Assay, Over Expression, Control

Journal: International journal of molecular sciences

Article Title: Trehalose Attenuates In Vitro Neurotoxicity of 6-Hydroxydopamine by Reducing Oxidative Stress and Activation of MAPK/AMPK Signaling Pathways.

doi: 10.3390/ijms251910659

Figure Lengend Snippet: Figure 7. Trehalose inhibits 6-OHDA-induced activation of JNK, p38, and AMPK. (a–f) SH-SY5Y cells were incubated with or without trehalose (100 mM) and then treated or not with 6-OHDA (60 µM) for another 2 or 6 h. The levels of phosphorylated and total forms of JNK (a), p38 MAPK (b), ERK (c), AMPK (d), AKT (e), and 4EBP1 (f), together with loading controls (actin or tubulin), were assessed by immunoblotting, and the representative immunoblots are shown. Densitometry data are mean ± SD values from three independent experiments (* p < 0.05 vs. no treatment; # p < 0.05 vs. 6-OHDA).

Article Snippet: 2024, 25, 10659 19 of 23 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MAO-A (Abcam, Cambridge, UK), sequestosome 1/p62, β-actin (both from Novus Biologicals, Littleton, CO, USA), glyceraldehyde 3 phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, Waltham, MA, USA), or primary mouse anti-human antibody against β-tubulin (Proteintech, Rosemont, IL, USA).

Techniques: Activation Assay, Incubation, Western Blot

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 5. Consequences of pharmacological CatS inhibition on the maturation of proCatC and proNSPs in HL-60 cells. HL-60 cells were cultured for 7 days in the presence of 10 µM of IcatSRO-11 or IcatS#54 or after adding DMSO containing medium alone. A) Western blot analysis of cell lysates using antiCatC antibody directed against the 23-kDa heavy chain of CatC demonstrating the accumulation of ~ 35-kDa fragment composed of truncated propeptide, the heavy chain and the light chain in presence of IcatSRO-11 or IcatS#54. Western blot analysis of cell lysates using antiCatS antibody as protein control showing not impact of inhibitors on proCatS processing. The percentage of proteolytic activities of CatC, PR3 and CatG was determined using fluorogenic substrates GF-AMC, Abz-VAD(nor)VADYQ- EDDnp, and Abz-TPFSGQ-EDDnp, respectively. Similar results were obtained in three independent experiments. B) Western blot analysis of cell lysates incubated with or without human α1PI using antiPR3 or antiCatG antibody. The de novo formation of irreversible α1PI-NSP complexes of about 75-kDa revealing activation of PR3 and CatG in presence of IcatS#54.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Incubation, Activation Assay

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 6. Processing of proCatC, proCatS and proNSPs in HL-60 cells cultured in the presence of compound 1 supplemented with IcatCXPZ-01. HL-60 cells were cultured for 14 days in the presence of compound 1 or after adding DMSO containing buffer alone. IcatCXPZ-01 was added at day 7. Processing of proCatC, proCatS and proNSPs in cell lysates were analyzed by western blotting. A) Western blot analysis of cell lysates using anti-CatC antibody directed against the 23-kDa heavy chain of CatC demonstrating the accumulation of ~ 35-kDa fragment in presence of compound 1. Proteolytic activities of NSPs are inhibited in presence of compound 1 supplemented with IcatCXPZ-01. The percentage of proteolytic activities of CatC, PR3 and CatG was determined as in Fig. 5. Results obtained in three independent experiments were shown. B) Western blot analysis of cell lysates using anti-CatS antibody showing alteration of proCatS processing with accumulation of inter mediate precursor in presence of compound 1 supplemented with IcatCXPZ-01. Similar results were obtained in three independent experiments. Incubation of cells only with compound 1 (data not shown) or IcatCXPZ-01 for 14 days did not alter the processing of proCatS (see Fig. 7C). C) Western blot analysis of cell lysates incubated with or without α1PI using anti-PR3 antibody. (Left) PR3 stability in cell lysates incubated without α1PI. (Right) PR3 in same cell lysates incubated with or without α1PI. The decrease of ~ 75-kDa irreversible α1PI-PR3 complexes formation and the detection of free PR3 in presence of compound 1 supplemented with IcatCXPZ-01 revealing unprocessing of PR3 zymogens. Similar results were obtained in three independent experiments. D) Diagram summary of proNSPs activation pathways in HL-60 cells. ProNSPs are activated by CatC (in blue) and NSPs-AAP-1 (in dark yellow) dependent pathways in HL-60 cells. CatC dependent activation pathway is initiated by proteolytic processing and maturation of proCatS. Combination of compound 1 with IcatCXPZ-01 partially inhibits the maturation of proCatS. The nitrile dipeptidyl CatC inhibitor IcatCXPZ-01 inhibits both CatC and NSPs-AAP-1 dependent maturation of proNSPs in HL-60 cells.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Cell Culture, Western Blot, Incubation, Activation Assay

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 10. Activation of CatC and NSPs in Ctss−/−C57BL/6 mice. A) Western blot analysis of WT and Ctss−/−bone marrow (BM) cell lysates using anti-CatS antibodies. B) Western blot analysis of bone marrow cells and blood neutrophils lysates using anti-CatC antibodies. Presence of mature immunoreactive CatC as revealed by the ~ 20-kDa heavy chain in Ctss−/−mice indicates that CatS is not required in proCatC maturation. C) Western blot analysis of bone marrow cells and blood neutrophils lysates incubated with or without human α1PI using anti-NE antibodies. The de novo formation of irreversible α1PI-scNE complexes of about 80-kDa reveals that NE is proteolytically active despite the absence of CatS. NE produced by murine neutrophils may be present in a cleaved (two-chains (tc)) and uncleaved (single chain (sc)) [64]. Similar results were obtained in three independent experiments.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Western Blot, Incubation, Produced

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 11. Activation of NSPs in Ctsc−/− C57BL/6 mice. A) Western blot analysis of bone marrow cell lysates using antiCatC, antiNE and antiCatG antibodies. Significantly reduced level of immunoreactive NE and CatG in Ctsc−/−mice bone marrow lysates compared with WT shows the elimination of NSP zymogens in absence of CatC (n = 4 animals). sc: single chain/tc: two chains (upper and lower bands). Myeloperoxidase (MPO) served as control for protein content. B) Residual activity of CatC, NE, PR3 and CatG in Ctsc−/−mice bone marrow lysates. Proteolytic activities were measured using fluorogenic substrates. Monitored selective NE, PR3 and CatG activities indicates the activation of NSPs in absence of CatC. The histograms show the mean ± SD of CatC (n = 5 animals) and NSP activities (n = 5 animals), each done in duplicates. Statistical analysis of the data was performed using a nonparametric test (Mann Whitney test), **p < 0.01. C) Consequences of CatC depletion in Ctsc−/−mice on the fate of proNSPs. Activation and degradation pathways of proNSPs identified in PLS patients are conserved in CatC defi cient mice.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Western Blot, Control, Activity Assay, MANN-WHITNEY

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 1. CIP2A is a cell-cycle regulated protein. A, HeLa cells were synchronized by either a double thymidine block or nocodazole block, released into fresh medium at indicated time points, and then analyzed by immunoblotting with antibodies against the indicated proteins. Synchronization and progression through the cell cycle was confirmed by fluorescence-activated cell sorting analysis. B, HeLa cells were stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (DNA, blue). C, an enlarged single-cell image from HeLa cells stained with anti-CIP2A antibody (green) and DAPI (blue). D, H1299 cells were transfected with the PTEN-expressing construct or empty vector. E, Hs68 cells were transfected with either control (Ctrl) siRNA or Plk1 siRNA. D and E, after transfection for 48 hours, PTEN-induced G1 arrest or Plk1 depletion–induced G2–M arrest was analyzed by immunoblotting with antibodies against the indicated proteins or by fluorescence-activated cell sorting analysis, respectively. F, Hs68 cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) or anti-cyclin B1 antibody (red) with DAPI (blue). Data shown represent typical results from at least four independent experiments. Scale bars, 10 mm.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Blocking Assay, Western Blot, FACS, Staining, Transfection, Expressing, Construct, Plasmid Preparation, Control

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 2. CIP2A depletion blocks nocodazole-induced mitotic arrest. A, HeLa cells were transfected with the indicated siRNAs (top) or transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv (bottom) for 48 hours and then analyzed by immunoblotting with anti-CIP2A antibody. B, C, F, and G, HeLa cells were transfected with the indicated siRNAs or Flag-CHFR vector for 36 hours and then incubated with 100 ng/mL nocodazole for 16 hours. MAD2 siRNA or the Flag-CHFR vector was used for a positive control of spindle checkpoint regulation or premitotic regulation, respectively. Cells were analyzed by light microscopy (B, left), fluorescence-activated cell sorting analysis (B, right), or immunoblotted with antibodies against the indicated proteins (C). D and E, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Nocodazole-treated cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) with DAPI (blue; D). E, the mitotic index was determined by the percentage of phospho-H3–positive cells (bottom) or by FACS (top) and was quantified using CellProfiler software (300 cells for each data point, n ¼ 3; , P < 0.001). F, nocodazole-treated cells were fixed with DAPI (blue) and the percentage of nonmitotic cells with multilobed or interphase nuclei was quantified (300 cells for each data point, n ¼ 3; , P < 0.01). G, representative confocal images of the cells that indicate the different nuclear morphologies, including mitotic arrest, checkpoint bypass, or premitotic arrest. Arrows, multilobed nuclei. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-H3–positive, phospho-H3–positive.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Incubation, Positive Control, Light Microscopy, FACS, Staining, Software

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 3. CIP2A depletion results in delay of mitotic entry and mitotic abnormalities. A, schematic of cell synchronization protocol by double thymidine block and for transfection with siRNA (top). A and B, HeLa cells were transfected with control (Ctrl) siRNA or CIP2A.1 siRNA, synchronized at the G1–S phase by a double thymidine block and released from the secondary thymidine block into medium with or without 100 ng/mL nocodazole (treated 6 hours after release). A, the mitotic index of control- or CIP2A-depleted cells was expressed as the percentage of phospho- H3–positive cells (500 cells at each time point; bottom). B, cells were analyzed by fluorescence-activated cell sorting (FACS) analysis (top) or immunoblotted with the indicated antibodies (bottom). C and D, control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes were synchronized by double thymidine block and released with fresh medium. C, the time of mitotic entry was determined by observing mitotic cell rounding with signs of DNA condensation and was monitored by time-lapse microscopy (left). Stable knockdown of CIP2A was determined by immunoblotting with anti-CIP2A antibody (right). D, representative time-lapse images of control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes during cell-cycle progression. E–H, HeLa cells were transfected with either control (Ctrl) siRNA or CIP2A.1 siRNA for 72 hours. E, CIP2Arv was cotransfected with CIP2A.1 siRNA for the rescue of CIP2A. The percentage of normal and aberrant nuclei was quantified using fluorescence microscopy (500 cells for each data point). F, representative images of CIP2A-depleted cells stained with anti-CIP2A antibody (green) and DAPI (blue). Arrows indicate aberrant nuclei. G, CIP2A-depleted cells were scored for abnormal mitosis (200 mitotic cells for each data point, n ¼ 4, error bars, SD). H, representative images of a normal mitotic cells (i) and CIP2A-depleted mitotic cells (ii–vi) stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (blue). The data shown represent typical results from at least three independent experiments. Scale bars, 10 mm. , P < 0.001. p-H3–positive, phospho-H3–positive; Noc, nocodazole.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Blocking Assay, Transfection, Control, FACS, shRNA, Knockdown, Expressing, Time-lapse Microscopy, Western Blot, Microscopy, Staining

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 4. CIP2A binds to Plk1 during mitosis. A and B, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and Flag-Plk1 vector for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Cells were analyzed by light microscopy (A) or immunoblotted with antibodies against the indicated proteins (B). C, lysates of HeLa cells were immunoprecipitated with anti-CIP2A antibody, anti-Plk1 antibody, or their respective control immunoglobulin G (IgG) antibodies and immunoblotted with anti-CIP2A or anti-Plk1 antibody. D, lysates of HeLa cells released from thymidine block for the indicated times were immunoprecipitated with anti-CIP2A antibody and immunoblotted with anti-Plk1 or anti-CIP2A antibody. E, lysates of 293T cells expressing Flag, Flag-tagged Plk1 (WT), polo-box–mutant (FAA) Plk1, N-terminal (N) Plk1, and C-terminal (C) Plk1 with Strep-CIP2A were pulled down with Strep-Tactin beads. The Flag-Plk1 protein associated with Strep-CIP2A was detected by immunoblotting with anti-Flag antibody. F–I, HeLa cells were fixed and incubated with mouse anti-CIP2A antibody together with rabbit anti-phospho-Plk1 (Thr210) antibody (top) or rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody (bottom), followed by in situ PLA analysis. Arrows indicate mitotic cells (F). G and I, dots per cell were counted using CellProfiler (100 cells for each data point; G) or 20 single cells in each respective cell-cycle phase (I); error bars, SD, , P < 0.001). The variation in the number of signal dots between G and I was due to the size difference between multiple- and single-cell images. H, representative confocal images of cells with PLA-positive signals in each respective cell-cycle phase. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-MPM2, phospho-MPM2.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Control, Plasmid Preparation, Incubation, Light Microscopy, Immunoprecipitation, Blocking Assay, Expressing, Mutagenesis, Western Blot, In Situ

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 5. CIP2A regulates the stability and activity of Plk1 during mitosis. A, HeLa cells were transfected with the indicated siRNAs, synchronized, and treated with cycloheximide (CHX), which was added 7 hours after release from G1–S. At the indicated times after the addition of cycloheximide, cells were analyzed by immunoblotting with the indicated antibodies. B, the levels of Plk1 were quantified using ImageJ software (n ¼ 3; error bars, SD, , P < 0.01). C, at 7 hours after release from G1–S, cells were treated with MG132 for 3 hours. D, cells cotransfected with vectors for HA-ubiquitin (Ub) and empty or CIP2A-Myc were released from G1–S for 1 hour (interphase) or 7 hours (mitosis) and MG132 was added for 3 hours. Lysates of the cells were immunoprecipitated with anti- Plk1 antibody and immunoblotted with anti-HA antibody. E, HeLa cells transfected with the indicated siRNAs targeting CIP2A, Cdh1, or Cdc20 were synchronized at the G1–S phase, released for 10 hours and then analyzed by immunoblotting with the indicated antibodies. F, HeLa cells transfected with control (Ctrl) siRNA or CIP2A.1 siRNA were synchronized by a double thymidine block and released into fresh medium. Cells were analyzed by immunoblotting with the indicated antibodies. G, purified Strep-CIP2A protein (1mg) incubated with or without recombinant His-Plk1 (0.1 mg) was used as thecontrol to exclude the possibility that purified Strep-CIP2A protein was associated with kinases that can phosphorylate casein (left). Recombinant His-Plk1 (0.1 mg) was incubated with 50 mmol/L BI2536 alone or with serial concentrations of purified Strep-CIP2A proteins (0.25, 0.5, or 1 mg) for 30 minutes (right). Plk1 activity was determined by in vitro kinase assay using casein as a substrate. Data shown represent typical results from at least three independent experiments.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Transfection, Western Blot, Software, Ubiquitin Proteomics, Immunoprecipitation, Control, Blocking Assay, Incubation, Recombinant, In Vitro, Kinase Assay

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 6. Association between CIP2A and Plk1 in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. A, quantification of CIP2A and Plk1 staining intensities in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. B and C, representative microscopic images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts stained with anti-CIP2A antibody (B) or anti-Plk1 antibody (C). Scale bars, 200 mm. D, representative high-magnification images of gastric cancer tissues stained with the indicated antibodies. Scale bars, 100 mm. E and F, tissue sections were incubated with rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody followed by in situ PLA analysis. Representative confocal images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts (E, left and F). Scale bars, 20 mm. Four different areas were obtained for each sample and 200 to 500 cells were quantified per area (mm2) using CellProfiler (E, right). Nuclei were stained with Hoechst (blue). The green signal represents autofluorescence. N, normal lung tissue. C, cancer tissue. A and E (left), data are presented as box-and-whisker plots. , P < 0.001 compared with their normal tissue counterparts.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Staining, Incubation, In Situ, Whisker Assay

Journal: FEBS letters

Article Title: Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence.

doi: 10.1016/j.febslet.2012.05.052

Figure Lengend Snippet: Fig. 3. Knockdown of 6PGD induces cellular senescence and p53 upregulation. H1975 cells were treated with 2 lg/ml doxycycline for 5 days. Cells were either stained for b-GAL activity (A–C) or lysed and used in a chromogenic b-GAL assay for quantification (D) according to a published procedure with slight modifications (21). p53 expression was also determined by western blots (E). Experiments were done in triplicate and were repeated at least three times. Representative results are shown. Error bars represent standard deviation. Anova (p = 0.0002) with Turkey post hoc test with confidence interval at 95%. ⁄Indicates the change is statistically significant.

Article Snippet: A rabbit polyclonal anti p53 antibody raised against human p53 was purchased from Cell Signaling (9282S).

Techniques: Knockdown, Staining, Activity Assay, Expressing, Western Blot, Standard Deviation

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α selectively modulates ANP, BNP, and NPR-1 expression. BEAS-2B cells were treated with 10, 20, and 40 ng/mL TNF- α for 24 h. ((a), (b)) ANP, ((c), (d)) BNP, and ((e), (f)) NPR-1 gene expression at mRNA ((a), (c), and (e)) and protein level ((b), (d), and (f)). Western blots were obtained by using the specific rabbit polyclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each one tested in quadruplicate and expressed as percentage of control (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 versus untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α modulation of NPs and NPR-1 may require activation of NF- κ B pathway. BEAS-2B cells were treated with 10, 20, and 40 ng/mL TNF- α alone or in combination with NF- κ B inhibitor BAY 11-7082 (1 μ M), for 24 h. ((a), (b)) ANP, ((c), (d)) BNP, ((e), (f)) NPR-1 gene expression at mRNA ((a), (c), and (e)) and protein level ((b), (d), (f)), and (g) Phospho-I κ B- α protein level. Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each of one tested in quadruplicate and expressed as percentage of control (* P < 0.05, ** P < 0.01, **** P < 0.0001 versus Controls). (° P < 0.05, °° P < 0.01, and °°°° P < 0.0001 versus BAY). C: Controls (untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Activation Assay, Gene Expression, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α modulation of protein levels of BNP and NPR-1 may require activation of NF- κ B pathway. BEAS-2B cells were treated 10 ng/mL TNF- α alone or in combination with the inhibitor QNZ (10 μ M), for 24 h. ((a), (b)) BNP, ((c), (d)) NPR-1 gene expression at mRNA ((a), (c)) and protein ((b), (d)) level, and (e) Phospho-I κ B- α protein level. Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each one tested in quadruplicate and expressed as percentage of control (** P < 0.01, and **** P < 0.0001 versus Controls). (°° P < 0.01 versus QNZ). C: Controls (untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Activation Assay, Gene Expression, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α modulation of BNP and NPR-1 expression may require activation of MAPKs pathway. BEAS-2B cells were treated with 10 ng/mL TNF- α alone or in combination with 10 μ M SB 203580 (p38 MAPK inhibitor), SP 600125 (JNK 1/2 inhibitor), or U-0126 (ERK 1/2 inhibitor), for 24 h. Panels show: ((a), (b)) BNP, ((c), (d)) NPR-1 gene expression at mRNA ((a), (c)) and protein ((b), (d)) level, and (e) Phospho-p38 MAPK protein level. Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each one tested in quadruplicate and expressed as percentage of control. (* P < 0.05, ** P < 0.01, and *** P < 0.001 versus Controls) ( •• P < 0.01 versus SB) ( § P < 0.05, §§ P < 0.01 versus SP) (°° P < 0.01, °°° P < 0.001 versus U-0126). C: Controls (untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Expressing, Activation Assay, Gene Expression, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α decreases AQP1 and AQP5 expression in BEAS 2B cells. BEAS-2B cells were treated with 10, 20, and 40 ng/mL TNF- α for 24 h. Gene expression of AQP1 ((a), (b)) and AQP5 ((c), (d)) at mRNA ((a), (c)) and protein ((b), (d)) level. Western blots were obtained by using the specific rabbit polyclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each one tested in quadruplicate and expressed as percentage of control (* P < 0.01, ** P < 0.001, and *** P < 0.0001 versus untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: TNF- α Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B

doi: 10.1155/2013/159349

Figure Lengend Snippet: TNF- α downregulation of AQP1 and AQP5 expression may require activation of NF- κ B pathway.BEAS-2B cells were treated with 10, 20, and 40 ng/mL TNF- α alone or in combination with NF- κ B inhibitor BAY 11-7082 (1 μ M), for 24 h. Gene expression of AQP1 ((a), (b)) and AQP5 ((c), (d)) at mRNA ((a), (c)) and protein ((b), (d)) level. Western blots were obtained by using the specific rabbit polyclonal Abs. The blots were stripped of the bound Ab and reprobed with mouse anti- β -actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three different cultures each one tested in quadruplicate and expressed as percentage of control (* P < 0.01, ** P < 0.001, *** P < 0.0001 versus Controls; and °°° P < 0.0001 versus BAY). C: Controls (untreated cells).

Article Snippet: Rabbit polyclonal antibodies (Abs) against ANP, NPR-1, AQP1, and AQP5 as well as the mouse monoclonal Ab against β -actin and the appropriate HRP-conjugated secondary Abs, were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Techniques: Expressing, Activation Assay, Gene Expression, Western Blot, Control