Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 1. MEP50 is expressed in the human epidermis and in cultured keratinocytes. A, foreskin tissue sections were stained with anti-MEP50, and binding was visualized with peroxidase-conjugated secondary antibody (top panels). The control is IgG. The arrows indicate MEP50 nuclear localization in suprabasal keratinocytes. Foreskin tissue sections (bottom panels) were fixed and stained with anti-MEP50, and antibody binding was visualized using a FITC-conjugated secondary antibody. The arrows indicate nuclear MEP50 accumulation. Scale bars 10 m. B, colocalization of endogenous and expressed MEP50. KERn were electroporated with 3 g of pcDNA3 or pcDNA3-FLAG-MEP50. After 48 h, protein lysates were tested by immunoblot using anti-FLAG and anti-MEP50. -actin was used as the loading control. After 48 h, the cells were fixed and costained with anti-FLAG (green) and anti-MEP50 (red). Similar results were observed in each of three experiments. The staining indicates MEP50 distribution in the nucleus and cytoplasm. Scale bars 10 m.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Cell Culture, Staining, Binding Assay, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 2. MEP50 and PRMT5 are required for keratinocyte proliferation. A, KERn were electroporated twice with control, MEP50, or PRMT5 siRNA, and 15,000 cells/well were plated. After overnight attachment, the cell number was determined (day 0) and at the indicated times thereafter. The values are mean S.E. (n 3). *, p 0.005. B, immunoblot detection of MEP50 and PRMT5. The immunoblot confirms a reduction in MEP50 in MEP50 siRNA-treated cultures. PRMT5 is also reduced.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 3. MEP50 regulation of p21Cip1 promoter activity: a role for p53. A, MEP50 suppresses p21Cip1 expression. KERn were electroporated with the indicatedplasmid,and,after24h,extractswerepreparedforquantitativeRT-PCRdetectionofp21Cip1mRNAandimmunoblotdetectionofp21Cip1andMEP50. EV, empty vector. B, opposing action of MEP50 and p53. A schematic of the human p21Cip1 promoter shows the Sp1 and p53 transcription factor response elements. The numbers indicate the distance in nucleotides relative to the transcription start site. KERn were transfected with 0.5 g of p21-2316, which encodes the full-length wild-type p21Cip1 promoter, or the promoter harboring mutations at the p53-1 or p53-2 sites, linked to luciferase. After 24 h, extracts were prepared for the luciferase activity assay. The values are mean S.E. (n 3). C–E, KERn were transfected with 0.5 g of PG13-Luc (the p53 gene promoter linked to luciferase) or p21-2326 and the indicated micrograms of pcDNA3 or pcDNA3-FLAG-MEP50. 48 h post-transfection, cell extracts were prepared and assayed for promoter activity. The values are mean S.E. (n 3). *, p 0.005.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Transfection, Luciferase

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 4. MEP50 knockdown reduces MEP50 and PRMT5 association at the p21Cip1 promoter. A and B, KERn were electroporated with 3 g of con- trol siRNA or MEP50 siRNA. After 48 h, extracts were prepared for ChIP analy- sis. DNA from 1 million cells was sheared, and 50,000 cell equivalents of DNA were used for immunoprecipitation. The primers span the p21Cip1 promoter region that includes the p53-2 site. The values are mean S.E. (n 3). *, p 0.005. C, extracts were prepared from the electroporated cells after 48 h to confirm MEP50 knockdown.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Knockdown, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 5. PKC and p38 regulate MEP50 and PRMT5 level and activity. A, KERn were infected with tAd5-EV, Ad5-PKC, or tAd5-HA-p38 (multiplicity of infection (MOI) 10), and, at 48 h, extracts were prepared for detection of PKC, MEP50, H3R8me2s, and H4R3me2s. Similar results were obtained in three different experiments. EV, empty vector. B and C, KERn were infected as above, and, at 48 h, extracts were prepared for ChIP using primers spanning the p21Cip1 promoter region that includes the p53-2 site. The values are mean S.E. (n 3). *, p 0.005).

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Activity Assay, Infection, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE7.MEP50andPRMT5regulatedifferentiationandproliferationin an epidermal equivalent model. KERn were electroporated twice with con- trol or MEP50 or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days of exposure at the air-liquid interface, the equivalents were har- vestedandsectioned.AandB,MEP50andPRMT5arerequiredforappropriate skin equivalent formation. KERn were electroporated twice with control, MEP50, or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days of exposure at the air-liquid interface, the equivalents were harvested and stained with hematoxylin and eosin. The nylon support membrane is indicated by an asterisk, and the extent of the epidermis is indicated (E). Sim- ilarresultswereobservedinthreeseparateexperiments.Thegraphcompares epidermal equivalent thickness among control, MEP50, and PRMT5 siRNA cultures. The values are mean S.E. (n 3). *, p 0.005. Scale bars 100 m. C and D, MEP50 and PRMT5 are required for cell proliferation. KERn were electroporated twice with control, MEP50, or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days, the equivalents were stained with Ki67 antibody and Hoechst. The membrane (asterisk) and extent of epidermis (E) are shown. The graph quantitates the number of Ki67-positive cells in the control, MEP50, and PRMT5 siRNA rafts. Similar results were observed in three separate experiments. Significant differences were determined using Student’s t test (*, p 0.005). Note that the blue-green spots are Ki67 staining compared with the larger stained circular structures, which are nonspecific staining to the nylon membrane.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Control, Staining, Membrane

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE 6. TPA regulates MEP50 and PRMT5 levels and activity. A, KERn were treated with 50 ng/ml TPA for 48 h, and extracts were prepared for detection of MEP50, PRMT5, H3R8me2s, and H4R3me2s. Similar results were obtained in three different experiments. B and C, KERn were treated with 50 ng TPA/ml, and, after 48 h, mRNA extracts were isolated for ChIP analysis and detection of MEP50 and PRMT5 interaction and H3R8-me2s and H4R3me2s formation at the p21Cip1 promoter. Similar results were obtained in three different experiments. D, cells were treated for 48 h with TPA, and p21Cip1 levels were monitored.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Activity Assay, Isolation

Journal: Journal of Biological Chemistry

Article Title: Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

doi: 10.1074/jbc.m115.642868

Figure Lengend Snippet: FIGURE8.Proposedregulatorymodel:abalancebetweentranscriptional and epigenetic regulation. Arrows indicate a stimulus, and flat-headed bars inhibition. TPA stimulation or PKC overexpression activates MEKK1, MEK3, p38, and p53 to increase p21Cip1 expression, which leads to reduced kerati- nocyte proliferation. PRMT5/MEP50 arginine dimethylates proteins in the p38 complex to inhibit p38 (20), and arginine dimethylates histones H3R8me2s and H4R3me2s in the p21Cip1 promoter to reduce p21Cip1 expres- sion, leading to increased cell proliferation. We propose that the PKC signal- ing is dominant in differentiated/non-proliferative cells and that PRMT5- MEP50 is dominant in non-differentiated/proliferative cells. The dashed line indicates that PKC/p38 signaling can reduce PRMT5 and MEP50 expression to suppress pro-proliferation signaling.

Article Snippet: Antibodies and Reagents—Rabbit polyclonal antibodies for MEP50 (catalog no. 2823), PRMT5 (catalog no. 2252), and p21Cip1 (catalog no. 2947) were obtained from Cell Signaling Technology (Danvers, MA), and mouse monoclonal anti- -actin (catalog no. A-5441), anti-FLAG antibody (catalog no. 8592), and anti-FLAG M2-FITC (catalog no. F4049) were purchased from Sigma-Aldrich.

Techniques: Inhibition, Over Expression, Expressing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

doi: 10.1128/AAC.00179-13

Figure Lengend Snippet: Synergistic bacteriostatic activities of different antimicrobial combinations against 10 colistin-resistant KPC-producing Klebsiella pneumoniae isolates

Article Snippet: Synergistic bactericidal activity (∑FBC ≤ 0.5) was documented in 8/13 CST-R KPC-Kp isolates with CST+RIF, 3/13 strains with CST+TIG, 2/13 strains with MER+GEN, IMI+GEN, and CST+GEN, and 1/13 strains with IMI+GEN, CST+MER, and GEN+TIG, while no synergistic bactericidal activity was observed for MER+TIG or IMI+TIG ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Isolate ST ΣFBC a of b : CST-RIF MER-GEN CST-TIG CST-GEN CST-IMI GEN-TIG IMI-GEN CST-MER MER-TIG IMI-TIG 2018/12 ST512 0.16 0.50 NA 2.00 0.19 NA 0.31 1.25 NA NA 2604/12 ST512 0.52 0.38 NA 0.28 0.63 0.31 2.00 0.75 NA NA 2550/12 ST512 2.00 0.63 0.02 2.00 2.00 2.00 0.53 0.52 NA 0.53 2762/12 ST512 2.00 0.75 2.00 2.00 2.00 2.00 2.00 NA NA NA 3031/12 ST512 0.14 2.00 0.31 2.00 NA NA NA NA NA NA 3177/12 ST512 0.31 2.00 2.00 2.00 NA NA 2.00 1.00 NA 2.00 3325/12 ST512 2.00 2.00 2.00 2.00 2.00 2.00 NA NA 0.56 NA 3342/12 ST512 0.31 2.00 2.00 2.00 NA 2.00 NA NA NA 2.00 3434/12 ST512 2.00 1.00 2.00 0.75 NA NA NA NA NA NA 3515/12 ST512 0.09 0.75 2.00 0.50 2.00 2.00 0.75 2.00 NA NA 2/12 ST101 0.37 NA 2.00 2.00 NA NA NA NA NA NA 4306/11 ST101 0.37 2.00 0.25 0.75 0.5 NA 2 0.5 NA NA 101/11 ST101 0.07 NA NA 2.00 NA NA NA NA NA NA No. (% c ) with synergism 8 (61.5) 2 (15.4) 3 (23.1) 2 (15.4) 2 (15.3) 1 (7.7) 1 (7.6) 1 (7.6) 0 (0) 0 (0) Open in a separate window a ∑FBC = FBC-A + FBC-B (FBC, fractional bactericidal concentration; FBC-A, MBC of agent A in the presence of agent B divided by MBC of agent A alone; FBC-B, MBC of agent B in the presence of agent A divided by MBC of agent B alone; MBC, minimum bactericidal concentration). b Shading indicates synergism.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

doi: 10.1128/AAC.00179-13

Figure Lengend Snippet: Synergistic bactericidal activities of different antimicrobial combinations against 10 colistin-resistant KPC-producing Klebsiella pneumoniae isolates

Article Snippet: Synergistic bactericidal activity (∑FBC ≤ 0.5) was documented in 8/13 CST-R KPC-Kp isolates with CST+RIF, 3/13 strains with CST+TIG, 2/13 strains with MER+GEN, IMI+GEN, and CST+GEN, and 1/13 strains with IMI+GEN, CST+MER, and GEN+TIG, while no synergistic bactericidal activity was observed for MER+TIG or IMI+TIG ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Isolate ST ΣFBC a of b : CST-RIF MER-GEN CST-TIG CST-GEN CST-IMI GEN-TIG IMI-GEN CST-MER MER-TIG IMI-TIG 2018/12 ST512 0.16 0.50 NA 2.00 0.19 NA 0.31 1.25 NA NA 2604/12 ST512 0.52 0.38 NA 0.28 0.63 0.31 2.00 0.75 NA NA 2550/12 ST512 2.00 0.63 0.02 2.00 2.00 2.00 0.53 0.52 NA 0.53 2762/12 ST512 2.00 0.75 2.00 2.00 2.00 2.00 2.00 NA NA NA 3031/12 ST512 0.14 2.00 0.31 2.00 NA NA NA NA NA NA 3177/12 ST512 0.31 2.00 2.00 2.00 NA NA 2.00 1.00 NA 2.00 3325/12 ST512 2.00 2.00 2.00 2.00 2.00 2.00 NA NA 0.56 NA 3342/12 ST512 0.31 2.00 2.00 2.00 NA 2.00 NA NA NA 2.00 3434/12 ST512 2.00 1.00 2.00 0.75 NA NA NA NA NA NA 3515/12 ST512 0.09 0.75 2.00 0.50 2.00 2.00 0.75 2.00 NA NA 2/12 ST101 0.37 NA 2.00 2.00 NA NA NA NA NA NA 4306/11 ST101 0.37 2.00 0.25 0.75 0.5 NA 2 0.5 NA NA 101/11 ST101 0.07 NA NA 2.00 NA NA NA NA NA NA No. (% c ) with synergism 8 (61.5) 2 (15.4) 3 (23.1) 2 (15.4) 2 (15.3) 1 (7.7) 1 (7.6) 1 (7.6) 0 (0) 0 (0) Open in a separate window a ∑FBC = FBC-A + FBC-B (FBC, fractional bactericidal concentration; FBC-A, MBC of agent A in the presence of agent B divided by MBC of agent A alone; FBC-B, MBC of agent B in the presence of agent A divided by MBC of agent B alone; MBC, minimum bactericidal concentration). b Shading indicates synergism.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

doi: 10.1128/AAC.00179-13

Figure Lengend Snippet: Synergistic bacteriostatic activities of different antimicrobial combinations against 10 colistin-resistant KPC-producing Klebsiella pneumoniae isolates

Article Snippet: Antagonism was not observed with any antibiotic combination ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Isolate ST ΣFIC a of b : CST-RIF MER-GEN CST-GEN CST-MER CST-IMI CST-TIG IMI-GEN GEN-TIG MER-TIG IMI-TIG 2018/12 ST512 0.09 0.08 0.07 0.13 0.26 2.00 0.07 2.00 2.00 2.00 2604/12 ST512 0.38 0.38 0.07 1.13 0.25 2.00 0.38 0.56 2.00 2.00 2550/12 ST512 0.08 0.63 0.07 0.16 0.38 0.13 0.13 0.63 2.00 0.56 2762/12 ST512 0.09 1.00 0.75 2.00 1.13 0.75 0.56 0.56 2.00 2.00 3031/12 ST512 0.16 0.25 0.25 2.00 2.00 0.38 2.00 1.00 2.00 2.00 3177/12 ST512 0.09 0.50 0.53 0.50 2.00 1.00 0.75 2.00 2.00 1.00 3325/12 ST512 0.28 0.16 0.19 2.00 0.63 0.31 4.00 0.63 1.00 2.00 3342/12 ST512 0.09 0.63 1.00 2.00 2.00 0.63 2.00 1.00 2.00 1.00 3434/12 ST512 0.19 0.75 0.56 1.00 3.00 0.75 2.00 2.00 2.00 2.00 3515/12 ST512 0.06 0.75 0.52 0.50 0.19 0.26 0.56 0.75 2.00 2.00 2/12 ST101 0.08 2.00 0.56 2.00 2.00 0.50 2.00 2.00 2.00 2.00 4306/11 ST101 0.12 0.75 0.53 0.50 0.50 0.56 0.56 2.00 2.00 2.00 101/11 ST101 0.09 2.00 0.75 2.00 2.00 2.00 2.00 2.00 2.00 2.00 No. (% c ) with synergism 13 (100) 5 (38.5) 5 (38.5) 5 (38.5) 5 (38.5) 5 (38.5) 3 (23.1) 0 (0) 0 (0) 0 (0) Open in a separate window a ∑FIC = FIC-A + FIC-B (FIC, fractional inhibitory concentration; FIC-A, MIC of agent A in the presence of agent B divided by MIC of agent A alone; FIC-B, MIC of agent B in the presence of agent A divided by MIC of agent B alone). b Shading indicates synergism.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

doi: 10.1128/AAC.00179-13

Figure Lengend Snippet: Synergistic bactericidal activities of different antimicrobial combinations against 10 colistin-resistant KPC-producing Klebsiella pneumoniae isolates

Article Snippet: Antagonism was not observed with any antibiotic combination ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Isolate ST ΣFIC a of b : CST-RIF MER-GEN CST-GEN CST-MER CST-IMI CST-TIG IMI-GEN GEN-TIG MER-TIG IMI-TIG 2018/12 ST512 0.09 0.08 0.07 0.13 0.26 2.00 0.07 2.00 2.00 2.00 2604/12 ST512 0.38 0.38 0.07 1.13 0.25 2.00 0.38 0.56 2.00 2.00 2550/12 ST512 0.08 0.63 0.07 0.16 0.38 0.13 0.13 0.63 2.00 0.56 2762/12 ST512 0.09 1.00 0.75 2.00 1.13 0.75 0.56 0.56 2.00 2.00 3031/12 ST512 0.16 0.25 0.25 2.00 2.00 0.38 2.00 1.00 2.00 2.00 3177/12 ST512 0.09 0.50 0.53 0.50 2.00 1.00 0.75 2.00 2.00 1.00 3325/12 ST512 0.28 0.16 0.19 2.00 0.63 0.31 4.00 0.63 1.00 2.00 3342/12 ST512 0.09 0.63 1.00 2.00 2.00 0.63 2.00 1.00 2.00 1.00 3434/12 ST512 0.19 0.75 0.56 1.00 3.00 0.75 2.00 2.00 2.00 2.00 3515/12 ST512 0.06 0.75 0.52 0.50 0.19 0.26 0.56 0.75 2.00 2.00 2/12 ST101 0.08 2.00 0.56 2.00 2.00 0.50 2.00 2.00 2.00 2.00 4306/11 ST101 0.12 0.75 0.53 0.50 0.50 0.56 0.56 2.00 2.00 2.00 101/11 ST101 0.09 2.00 0.75 2.00 2.00 2.00 2.00 2.00 2.00 2.00 No. (% c ) with synergism 13 (100) 5 (38.5) 5 (38.5) 5 (38.5) 5 (38.5) 5 (38.5) 3 (23.1) 0 (0) 0 (0) 0 (0) Open in a separate window a ∑FIC = FIC-A + FIC-B (FIC, fractional inhibitory concentration; FIC-A, MIC of agent A in the presence of agent B divided by MIC of agent A alone; FIC-B, MIC of agent B in the presence of agent A divided by MIC of agent B alone). b Shading indicates synergism.

Techniques:

Journal: PLoS ONE

Article Title: Increased Precursor Cell Proliferation after Deep Brain Stimulation for Parkinson's Disease: A Human Study

doi: 10.1371/journal.pone.0088770

Figure Lengend Snippet: The figures illustrate Sox 2-positive labeling in the ependymal layer, adjacent to the hypothalamus, of ( a ) DBS and ( b ) normal brains (in all figures red = autofluorescent blood cells, green = Sox2 or δ-GFAP). The graph (e) shows the mean numbers of Sox2-positive cells in the ependymal layer in normal (negative), and DBS (positive) brain tissue samples. Overall, the DBS-positive cases show a statistically significant difference in the mean numbers of Sox2-positive cells. Scale bar is equivalent for figures a–d = 100 µm. ( c ) The figures illustrate δ-GFAP positive labeling in the ependymal layer, adjacent to the hypothalamus of DBS and ( d ) normal brains. ( e–g ) Boxplots of the cell count data by group. The bold horizontal lines indicate the group medians. The top and bottom of the boxes represent the 75 th and 25 th percentiles, respectively. An asterisk indicates an outlying data point (a point farther above or below the box than 1.5× the box height). The graph ( e ) shows the mean number of Sox2-positive cells in the ependymal layer in normal and PD-DBS brain tissue samples. The graph ( f ) shows the mean number of δ-GFAP-positive cells in the ependymal layer in normal and PD-DBS brain tissue samples. The graph ( g ) shows the mean number of MCM2-positive cells in the peri-lead region in normal and PD-DBS brain tissue samples.

Article Snippet: For immunohistochemistry, the following primary antibodies were used: Sox2 (mouse, R&D, 1∶500, MAB 2018), MCM2 (rabbit, Cell Signaling, 1: 500, D7G11), and δ-GFAP (rabbit, a generous gift from Dr. Elly Hol, Netherlands Institute for Neuroscience, 1∶1500).

Techniques: Labeling, Cell Counting

Journal: PLoS ONE

Article Title: Increased Precursor Cell Proliferation after Deep Brain Stimulation for Parkinson's Disease: A Human Study

doi: 10.1371/journal.pone.0088770

Figure Lengend Snippet: Boxplots of gene expression relative to 18S for Sox2: control and PD-DBS groups. The permutation test p-value shows that the expression level is significantly higher in the PD-DBS group at the 0.05 level. Boxplots of expression relative to 18S for MCM2: control and PD-DBS groups. The permutation test p-value shows that the expression level is significantly higher in the PD-DBS group at the 0.05 level. The relative gene expression level was normalized on the basis of the expression of a reference gene (18S) and was also normalized on the basis of the expression of a reference sample (calibrator). Final results are expressed in arbitrary units in gene expression relative to the expression of 18S gene and calibrator sample. The relative expression was calculated by 2 −ΔΔCT , where CT = fluorescence threshold value; ΔCT = CT of the target gene – CT of the reference gene (18S) ; ΔΔCT = ΔCT of the treated sample – ΔCT of the reference sample. A pool of five normal tissue samples served as the reference sample. The increased levels of gene expression of Sox2 and MCM2 correlate with the immunolabelling and cell count data.

Article Snippet: For immunohistochemistry, the following primary antibodies were used: Sox2 (mouse, R&D, 1∶500, MAB 2018), MCM2 (rabbit, Cell Signaling, 1: 500, D7G11), and δ-GFAP (rabbit, a generous gift from Dr. Elly Hol, Netherlands Institute for Neuroscience, 1∶1500).

Techniques: Expressing, Fluorescence, Cell Counting