Journal: Pharmaceutics

Article Title: Ceragenins in Combination with Ivacaftor Prevent the Formation of Biofilm by Bacteria That Cause Rhinosinusitis

doi: 10.3390/pharmaceutics18010001

Figure Lengend Snippet: Changes in P. aeruginosa Xen 5 luminescence upon addition of CSA-13 ( A ), CSA-44 ( B ), CSA-131 ( C ), colistin (COL) ( D ), and ivacaftor (IVA) ( E ) tested at concentrations of 5, 10, 20, and 40 μg/mL in comparison to untreated control. Data represent mean ± SE, n = 3. * indicates statistical significance compared to untreated bacteria cells ( p ≤ 0.05).

Article Snippet: CSA-13, CSA-44, and CSA-131 exhibit antibacterial activity against S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), and M. catarrhalis (ATCC 49616), reducing biofilm formation in a concentration-dependent manner ( ).

Techniques: Comparison, Control, Bacteria

Journal: Pharmaceutics

Article Title: Ceragenins in Combination with Ivacaftor Prevent the Formation of Biofilm by Bacteria That Cause Rhinosinusitis

doi: 10.3390/pharmaceutics18010001

Figure Lengend Snippet: Changes in S. aureus Xen 30 luminescence upon addition of CSA-13 ( A ), CSA-44 ( B ), CSA-131 ( C ), vancomycin (VAN) ( D ), and ivacaftor (IVA) ( E ) tested at concentrations of 5, 10, 20, and 40 μg/mL in comparison to untreated control. Data represent mean ± SE, n = 3. * indicates statistical significance compared to untreated bacteria cells ( p ≤ 0.05).

Article Snippet: CSA-13, CSA-44, and CSA-131 exhibit antibacterial activity against S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), and M. catarrhalis (ATCC 49616), reducing biofilm formation in a concentration-dependent manner ( ).

Techniques: Comparison, Control, Bacteria

Journal: Pharmaceutics

Article Title: Ceragenins in Combination with Ivacaftor Prevent the Formation of Biofilm by Bacteria That Cause Rhinosinusitis

doi: 10.3390/pharmaceutics18010001

Figure Lengend Snippet: The formation of S. aureus (ATCC 29213) ( A ), P. aeruginosa (ATCC 27853) ( B ), and M. catarrhalis (ATCC 25238) ( C ) biofilm in the presence of ceragenins CSA-13, CSA-44, CSA-131, vancomycin (VAN), colistin (COL), and meropenem (MEM) alone (green bars), in the presence of ivacaftor (yellow bars) (all at doses ranging from 0.1 to 20 µg/mL) and when combined with IVA (5 µg/mL) (orange bars). Biofilm formation in treated samples was normalized to the untreated control (0 µg/mL; 100% viability; indicated as black horizontal line). Results are presented as a mean ± SD from 3 technical replicates. * indicates statistical significance ( p < 0.05) when compared to single treatments.

Article Snippet: CSA-13, CSA-44, and CSA-131 exhibit antibacterial activity against S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), and M. catarrhalis (ATCC 49616), reducing biofilm formation in a concentration-dependent manner ( ).

Techniques: Control

Journal: Pharmaceutics

Article Title: Ceragenins in Combination with Ivacaftor Prevent the Formation of Biofilm by Bacteria That Cause Rhinosinusitis

doi: 10.3390/pharmaceutics18010001

Figure Lengend Snippet: Storage modulus G’ of P. aeruginosa biofilms upon exposure to ceragenins CSA-13, CSA-44, and CSA-131 (at doses of 10 and 50 µg/mL) alone or when combined with ivacaftor (IVA, at doses of 10 and 20 µg/mL). Panel ( A ) demonstrates the storage modulus as a function of time, panels ( B – D ) demonstrate the average values of the storage modulus recorded for each sample during a 60 s analysis. Data are presented as mean ± SE. * indicates statistical significance when compared to single treatments.

Article Snippet: CSA-13, CSA-44, and CSA-131 exhibit antibacterial activity against S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), and M. catarrhalis (ATCC 49616), reducing biofilm formation in a concentration-dependent manner ( ).

Techniques:

Journal: Pharmaceutics

Article Title: Ceragenins in Combination with Ivacaftor Prevent the Formation of Biofilm by Bacteria That Cause Rhinosinusitis

doi: 10.3390/pharmaceutics18010001

Figure Lengend Snippet: Hemo- and cytocompatibility of ceragenins CSA-13, CSA-44, and CSA-131 (green bars), ivacaftor (yellow bars), and ceragenins (all at a concentration range from 1 to 40 µg/mL) combined with ivacaftor (5 µg/mL) (orange bars). The release of hemoglobin from isolated erythrocytes and the viability of NIH/3T3 cells exposed to the tested agents are presented in panels ( A , B ), respectively. Results are presented as mean ± SD from three replicates. * indicates statistical significance when compared to single treatments.

Article Snippet: CSA-13, CSA-44, and CSA-131 exhibit antibacterial activity against S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), and M. catarrhalis (ATCC 49616), reducing biofilm formation in a concentration-dependent manner ( ).

Techniques: Concentration Assay, Isolation

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 1. SIRT1 protein expression in unligated carotid arteries and at 7, 14, and 28 days after ligation. Protein extracted from pooled samples of mouse LCCA were used for Western blotting. Bottom, Representative Western blot for mouse Sirt1 (mSirt1, anti- body from Millipore) and -actin in unligated and ligated vessels. Top, Quantification of the densitometric analysis. Values are meansSEM (n3 with 3 mice in each pool). ***P0.001 versus unligated control (con indicates unligated control).

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Expressing, Ligation, Western Blot, Control

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 3. SIRT1 overexpression inhibits neointima formation following ligation in vivo. A, Left, Representative cross- sections of hematoxylin/eosin-stained carotid arteries without ligation (control) or ligated for 14 or 28 days. Scale bar: 50 m. Right, Quantitative analysis of I/M ratio in histological sections from WT or SMC-SIRT1 Tg mouse arteries. **P0.01 for SMC-SIRT1 Tg versus WT mice (n4 mice per group on control and day 14, n10 mice per group on day 28). B, Left, Representative sections of injured mouse carotid arteries at 28 days after ligation that were immunostained for markers of proliferation (PCNA to identify cells in S phase, brown). Scale bar: 25 m. Right, Quantification of PCNA-positive cells in the neointima of carotid arteries. The number of PCNA-positive cells was defined as the percentage of the total cells within a given area that was positive for PCNA staining. **P0.01 for SMC- SIRT1 Tg versus WT mice (n6 mice per group). The internal elastic lamina is indi- cated by black arrows.

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Over Expression, Ligation, In Vivo, Staining, Control

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 4. SIRT1 inhibits VSMC proliferation and migration in vitro. A, DNA synthesis in Ad-GFP- or Ad-SIRT1-infected (MOI100) VSMCs following serum starvation for 48 hours and then serum induction for 24 hours. Data represent the meansSEM (n4). ***P0.001 versus each Ad-GFP group. B, FACS analysis to measure the DNA content in Ad-GFP– or Ad-SIRT1–infected VSMCs following serum starvation for 24 hours and then serum stimulation at the indicated time points. Data are expressed as the percentage of the total cells and rep- resent the meansSEM (n4). *P0.05 versus Ad-GFP at the indicated time points. C through F, Migration assay for adenovirus-infected VSMCs following TNF- (30 ng/mL) induc- tion for 24 hours (C and D) or 8 hours (E and F). C and D, Scratch wound assay. Quantification of the average VSMC migration distance. E and F, Modified Boyden chamber assay. Data represent the meansSEM of 3 independent experiments. *P0.05, **P0.01, ***P0.001 versus each Ad-GFP or Ad-U6 group.

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Migration, In Vitro, DNA Synthesis, Infection, Scratch Wound Assay Assay, Boyden Chamber Assay

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 5. Regulation of cyclin D1 and MMP-9 expression/activity by SIRT1 in VSMCs. The levels of cyclin D1 or MMP-9 were quantified by Western blotting and normalized to that of -actin (top panel). A through D, Representative Western blots of Ad-SIRT1 or Ad-SIRT1 RNAi-infection in VSMCs (bottom panel). Values represent the meansSEM (n3). *P0.05, **P0.01, ***P0.001 versus each Ad-GFP or Ad-U6 group. E, Gelatin zymography assay to determine the enzymatic activities of MMP-9 and MMP-2. The gelatino- lytic activity of MMP-9 was normalized to that of MMP2 (top panel). Data represent the meansSEM (n3). *P0.05 versus Ad-GFP– infected VSMCs treated with TNF-. F, Representative Western blot showing VSMCs with or without TNF- (30 ng/mL) induction for 8 hours that were pretreated with or without the SIRT1 inhibitor Sirtinol (20 mol/L or 40 mol/L) for 16 hours. Data represent the meansSEM from 3 independent experiments. *P0.05, **P0.01 versus the control without both TNF- and Sirtinol treatment, #P0.05 versus the control by treatment with TNF- (30 ng/mL) for 8 hours.

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Expressing, Activity Assay, Western Blot, Infection, Zymography Assay, Control

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 7. Transcriptional regulation of cyclin D1 and MMP-9 by SIRT1 in VSMCs. A, Representa- tive semiquantitative RT-PCR results for Ad-GFP- or Ad-SIRT1-infected VSMCs (bottom panel). Quantitative analyses of PCR (normalized to that of -actin, top panel). Values represent the meansSEM (n3). **P0.01 versus Ad-GFP– infected VSMCs treated with 10% FBS, ***P0.001 versus each Ad-GFP group. B, Regula- tion of the promoter activities of cyclin D1 and MMP-9 by SIRT1 in A10 (cyclin D1-luc) or A7r5 (MMP-9-luc) cells, respectively. Data represent the meansSEM (n3). *P0.05, ***P0.001 versus each pcDNA3.1 group. C, Modulation of the repressive effect of SIRT1 on the promoter activi- ties of cyclin D1 and MMP-9 by the indicated cyclin D1 and MMP-9 promoter deletions or muta- tions, respectively. The relative cyclin D1 or MMP-9 luciferase activities are presented as the ratio of the indicated pcDNA3.1-SIRT1–modified to the pcDNA3.1-modified cyclin D1 or MMP-9 pro- moter activities. Data represent the meansSEM (n3). *P0.01, **P0.001 versus the fold change in cyclin D1-Luc and MMP-9-Luc, respectively.

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Luciferase

Journal: Circulation Research

Article Title: SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

doi: 10.1161/circresaha.110.237875

Figure Lengend Snippet: Figure 8. The repression of AP-1 activity by SIRT1 leads to decreased cyclin D1 and MMP-9 expression by SIRT1. A through D, ChIP of cyclin D1 (A and C) and MMP-9 (B and D) promoter complexes. Primary VSMCs (A and B) and primary VSMCs with Ad-U6 or Ad-SIRT1 RNAi infection (C and D) were used for immunoprecipitation with antimSirt1 (antibody from Millipore), anti–c-Fos, anti–c-Jun antibody, or control IgG. Immunoprecipitated chromatin fragments were applied to routine PCR (bottom panel of A and B) and quanti- fied by realtime-PCR (top panel of A and B, C and D) using specific primers sets. The images and data are representative of 3 inde- pendent experiments.

Article Snippet: After blocking with Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies against SIRT1 (anti-mSirt1, 1:2000, cat. no. 07-131, Millipore; anti-hSIRT1, 1:1000, cat. no. sc-15404, Santa Cruz Biotechnology Inc.), MMP-9 (1:1000, cat. no. sc-6840, Santa Cruz Biotechnology Inc.) , cyclin D1 (1:1000, cat. no. sc-8396, Santa Cruz Biotechnology Inc.), MMP-2 (1:1000, cat. no. sc-13595, Santa Cruz Biotechnology Inc.), cyclin E (1:1000, cat. no. sc-481, Santa Cruz Biotechnology Inc.), CDK2 (1:1000, cat. no. sc-163, Santa Cruz Biotechnology Inc.), CIRCRESAHA/2010/237875 (OLD #204008) /R2 5 CDK4 (1:1000, cat. no. sc-260, Santa Cruz Biotechnology Inc.), p27 (1:1000, cat. no. sc-1641, Santa Cruz Biotechnology Inc.), survivin (1:1000, cat. no. sc-10811, Santa Cruz Biotechnology Inc.) and AT1R (1:1000, cat. no. sc-1173, Santa Cruz Biotechnology Inc.) at 4°C overnight.

Techniques: Activity Assay, Expressing, Infection, Immunoprecipitation, Control

Journal: EBioMedicine

Article Title: Age-Specific Profiles of Antibody Responses against Respiratory Syncytial Virus Infection

doi: 10.1016/j.ebiom.2017.01.014

Figure Lengend Snippet: Evaluation of serum IgG to recognize antigenic sites I, II, and IV in F protein. Sera were subjected to competitive ELISA by using monoclonal antibody specific to antigenic sites I, II, or IV. The amounts of serum IgG specific for each antigenic epitopes are represented by IC50 resulting in 50% reduction of biding RSV F with competitive monoclonal antibody. The limitation of the IC50 detection was defined as 5. The concentration of serum IgG specific to antigenic site II was calculated based on the result of palivizumab on competitive ELISA. The mean ± 95% CI was estimated by the linear model, and were shown in the graphs. IC50 of antigenic site I-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 26; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 32; adults, n = 23. IC50 of antigenic site I-specific IgG in convalescent phase: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 32; adults, n = 23. GMRs C/A of antigenic site I-specific IgG: 0–3 mo, n = 21; 4–6 mo, n = 25; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. IC50 of antigenic site II-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 28; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 33; adults, n = 23. IC50 of antigenic site II-specific IgG in convalescent phase: 0–3 mo, n = 27; 4–6 mo, n = 29; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 33; adults, n = 23. GMRs C/A of antigenic site II-specific IgG: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 32; adults, n = 23. IC50 of antigenic site IV-specific IgG in acute phase: 0–3 mo, n = 29; 4–6 mo, n = 28; 7–12 mo, n = 28; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. IC50 of antigenic site IV-specific IgG in convalescent phase: 0–3 mo, n = 27; 4–6 mo, n = 29; 7–12 mo, n = 32; 13–18 mo, n = 35; 19–36 mo, n = 33; adults, n = 23. GMRs C/A of antigenic site IV-specific IgG: 0–3 mo, n = 24; 4–6 mo, n = 27; 7–12 mo, n = 26; 13–18 mo, n = 34; 19–36 mo, n = 31; adults, n = 23. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Palivizumab (Synagis®) was purchased from AbbVIE Inc. A neutralization mouse monoclonal antibody against antigenic site I (131-2A) was purchased from Millipore, whereas that against antigenic site IV (6H6-2B4) was obtained by selecting hybridoma clones produced from rats immunized with the recombinant F protein.

Techniques: Competitive ELISA, Concentration Assay

Journal: The Journal of Immunology Author Choice

Article Title: c-Abl–Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes

doi: 10.4049/jimmunol.1801616

Figure Lengend Snippet: LPS stimulation induces c-Abl’s nuclear transportation. (A) LPS stimulation induces nuclear import of c-Abl. RAW 264.7 cells were challenged with LPS for various lengths of time. Cell lysates from nuclear and cytoplasmic fractions were subjected to Western blotting to determine the subcellular distribution of c-Abl. (B) Immune-fluorescence staining verifies LPS-induced nuclear import of c-Abl. RAW 264.7 cells were mock treated or LPS exposed (±STI571) for 1 h, and then cells were fixed and permeabilized and incubated with anti–c-Abl rabbit polyclonal Ab and TRITC-conjugated secondary Ab. The nuclei of the cells were stained with DAPI. Similar results were obtained from at least three independent experiments. Original magnification ×180.

Article Snippet: Mouse mAb against PARP1 (1:2000, B-10, sc-74470) and rabbit polyclonal Ab against c-Abl (1:3000, K-12, sc-131) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Fluorescence, Staining, Incubation

Journal: Nucleic Acids Research

Article Title: SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

doi: 10.1093/nar/gkz1127

Figure Lengend Snippet: Inhibition of SIRT1/2 with cambinol during MO-to-MAC differentiation results in an aberrant pro-inflammatory phenotype. (A) A representative western blotting analysis showing SIRT1 and acetylated lysine 16 of histone H4 (H4K16ac) protein expression of peripheral blood CD14+ MOs stimulated with 25 ng/mL of M-CSF for 0, 1, 3 and 5 days. Relative band intensities of SIRT1 and H4K16ac were normalised against β-Actin and H4 respectively (right panel) from two independent experiments. (B) Western blot analysis of protein expression of SIRT1 and SIRT2 in MO exposed to M-CSF for 0, 1, 3 and 5 days and activated by the addition of LPS for 18 hrs. RNA expressions of SIRT1 and SIRT2 were normalised against RPL38 . (C) Secreted levels of IL-10 and IL-1β were analysed by ELISA in MACs differentiated in the presence of DMSO or treated with 50 nM of cambinol. (D) FACS analysis of surface markers CD163 (FITC), CD86 (APC), CD80 (PE) and CD83 (APC) in MOs and M-CSF MACs before and after treatment with LPS for 18 hrs in the presence and absence of cambinol (50 nM). (E) Gene expression analysis of IL1B , IL1A , TNF and CD163 in MOs, cambinol- or DMSO-treated M-CSF MACs differentiated for 5 days and MACs treated with LPS for an additional 18 hrs. (A-E) Statistical significance of at least three independent experiments was calculated by paired student t-test (* p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001). (F) Heatmap representation of z-scores obtained from log2 expression of genes that undergo significant changes (FDR < 0.05) in cambinol-treated (M-CSF + Camb) compared to DMSO-treated (M-CSF) MACs, in which 533 and 303 genes become downregulated (log2FC < 0 and FDR < 0.05) and upregulated (log2FC > 0 and FDR < 0.05) respectively. (G) GO analyses of cambinol-downregulated (left) and -upregulated (right panel) genes using the DAVID tool (https://david.ncifcrf.gov). (H) Bubble plot representation of HOMER TF motif enrichment analysis. A window of -2000 bp upstream of TSS was used for all genes analysed.

Article Snippet: Membranes were then incubated with primary antibodies against SIRT1 (07-131, Millipore), SIRT2 (ab51023, Abcam), NF-κB p65 (sc-372, Santa Cruz Biotechnologies, TX, USA), p65K310ac (ab19840, Abcam, Cambridge, UK), DNMT1 (NB100-56519, Novus), DNMT3A (3598, Cell Signaling), DNMT3B (sc-20704, Santa Cruz) and β-Actin (ab6276; Abcam).

Techniques: Inhibition, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Nucleic Acids Research

Article Title: SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

doi: 10.1093/nar/gkz1127

Figure Lengend Snippet: Inhibition of SIRT1/2 inhibition with cambinol affects DNA hypermethylation during MO-to-MAC differentiation. (A) Heatmap representation of significant (FDR < 0.05) DNA methylation changes during MO-to-MAC differentiation comparing DMSO-treated MACs (M-CSF) with MOs. (B) Violin plots depicting the distribution and probability density of z-scores of hypo- (upper) and hypermethylated (lower panel) CpGs. (C) Principal component analysis (PCA) of hypo- and hypermethylated CpGs of the three cell populations (Mo, M-CSF and M-CSF + Camb). (D) GO analysis of hypermethylated CpGs, in which CpGs were mapped to genes using GREAT online tool (http://great.stanford.edu/public/html) by applying the basal plus extension settings, using EPIC array as background. (E) Representation of the beta values of selected hypermethylated CpGs that map to relevant genes. (F) HOMER TF motif enrichment analysis of hypermethylated CpGs were carried out using a window of ±250 bp centring around the CpGs. CpGs annotated in the EPIC 850K array were used as background. (G) Histone marks enrichment analysis of hypermethylated CpGs were carried out by crossing DNase-seq and ChIP-seq data of H3K9me3, H3K9/14ac, H3K4me3, H3K4me1, H3K36me3, H3K27me3 and H3K27ac of MOs and MACs downloaded from the Blueprint database (refer to Materials and Methods for data processing).

Article Snippet: Membranes were then incubated with primary antibodies against SIRT1 (07-131, Millipore), SIRT2 (ab51023, Abcam), NF-κB p65 (sc-372, Santa Cruz Biotechnologies, TX, USA), p65K310ac (ab19840, Abcam, Cambridge, UK), DNMT1 (NB100-56519, Novus), DNMT3A (3598, Cell Signaling), DNMT3B (sc-20704, Santa Cruz) and β-Actin (ab6276; Abcam).

Techniques: Inhibition, DNA Methylation Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

doi: 10.1093/nar/gkz1127

Figure Lengend Snippet: MO-to-MAC DNA methylation gains occur concomitantly with a loss of DNase I hypersensitivity and activating histone marks, and SIRT1/2 inhibition upregulates activating histone marks. ( A ) DNase-seq and ChIP-seq data of H3K27ac, H3K4me1 and H3K4me3 of MOs and MACs were downloaded from the Blueprint database (refer to Materials and Methods for details). Odds ratios were calculated for bins of 10 bp up to ±2500 bp centering around hypo- (left panel) and hypermethylated (right panel) CpGs in which CpGs annotated in the EPIC 850K array were used as background. ( B ) Schematic representation of DNase I hypersensitivity, H3K27ac, H3K4me3 and H3K4me1 marks near hypermethylated CpGs in ADORA2A , RUNX3 , IL2RA and JAK3 . MOs are represented in red and MACs are represented in blue. ( C ) Chromatin immunoprecipitation (ChIP) of H4K16ac (yellow), H3K4me1 (green), H3K4me3 (light blue) and H3K27ac(dark blue) MOs, MACs and LPS-activated MACs in the presence and absence of 50 μM cambinol. ChIP amplicon positions in relation to hypermethylated CpGs and genes ADORA2A , RUNX3 , IL2RA and JAK3 are depicted in ( B ). Three independent experiments were performed and statistical significance was calculated using paired Student's t -tests (* P -value < 0.05, ** P -value < 0.01 and *** P -value < 0.001).

Article Snippet: Membranes were then incubated with primary antibodies against SIRT1 (07-131, Millipore), SIRT2 (ab51023, Abcam), NF-κB p65 (sc-372, Santa Cruz Biotechnologies, TX, USA), p65K310ac (ab19840, Abcam, Cambridge, UK), DNMT1 (NB100-56519, Novus), DNMT3A (3598, Cell Signaling), DNMT3B (sc-20704, Santa Cruz) and β-Actin (ab6276; Abcam).

Techniques: DNA Methylation Assay, Inhibition, ChIP-sequencing, Chromatin Immunoprecipitation, Amplification

Journal: Nucleic Acids Research

Article Title: SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

doi: 10.1093/nar/gkz1127

Figure Lengend Snippet: SIRT1 and 2 interact with DNMTs to drive DNA hypermethylation at inflammatory loci. (A) Co-immunoprecipitation assays were performed in MOs differentiated to MACs in the presence of M-CSF for 5 days. Protein extracts were immunoprecipitated utilising anti-DNMT1, -DNMT3A, -DNMT3B, -SIRT1 and –SIRT2 antibodies, in which IgG was used as a negative control and total protein extract was used as input. (B) Co-immunoprecipitation of MACs differentiated in the presence of DMSO or 50 µM cambinol for 5 days. (C) Chromatin immunoprecipitation of two independent experiments of SIRT1 (pink), SIRT2 (red) and PU.1 (blue) was performed in isolated monocytes and MACs differentiated in the presence of M-CSF for 5 days in the presence and absence of cambinol, before and after LPS stimulation for 18 hrs. (D) MOs were transfected with 100nM of corresponding siRNA and differentiated to MACs in the presence of M-CSF for 5 days, as described in Materials and Methods. Annotated numbers indicate relative band intensities compared to non-targeting control (NT). (E) Pyrosequencing of hypermethylated CpGs in MOs and MACs transfected with NT, siSIRT1 and siSIRT2 prior to LPS activation. (F) Gene expression of SCL1A2 , TNFAIP3 , JAK3 , RUNX3 and ADORA2A in MOs and LPS-activated MACs transfected with NT, siSIRT1 and siSIRT2, as normalised against RPL38 . (D-F) Statistical significance of at least three independent experiments was calculated using paired student t-tests (* p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001).

Article Snippet: Membranes were then incubated with primary antibodies against SIRT1 (07-131, Millipore), SIRT2 (ab51023, Abcam), NF-κB p65 (sc-372, Santa Cruz Biotechnologies, TX, USA), p65K310ac (ab19840, Abcam, Cambridge, UK), DNMT1 (NB100-56519, Novus), DNMT3A (3598, Cell Signaling), DNMT3B (sc-20704, Santa Cruz) and β-Actin (ab6276; Abcam).

Techniques: Immunoprecipitation, Negative Control, Chromatin Immunoprecipitation, Isolation, Transfection, Control, Activation Assay, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells

doi: 10.3390/ijms26115024

Figure Lengend Snippet: Phenotypic features of CliniMACS Prodigy-manufactured RevCAR T cells. RevCAR T cells were stained with mAbs against ( A ) CD4, CD8, ( B ) CD62L, CD45RO, ( C ) CD69, ( D , E ) PD-1, Tim-3, and Lag-3 for flow cytometric analysis. Data are shown for three individual donors. ( B ) T N /T SCM —naive/stem cell memory (CD62L + CD45RO − ); T CM —central memory (CD62L + CD45RO + ); T EM —effector memory (CD62L − CD45RO + ); T TE —terminal effector (CD62L − CD45RO − ).

Article Snippet: For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells

doi: 10.3390/ijms26115024

Figure Lengend Snippet: Phenotypic features of CliniMACS Prodigy-manufactured Dual-RevCAR T cells. Dual-RevCAR T cells were incubated with mAbs against ( A ) CD4, CD8, ( B ) CD62L, CD45RO, ( C ) CD69, ( D , E ) PD-1, Tim-3, and Lag-3 for 15 min at 4 °C in the dark and analyzed via flow cytometry. Data of two individual donors are presented. ( B ) T N /T SCM —naive/stem cell memory (CD62L + CD45RO − ); T CM —central memory (CD62L + CD45RO + ); T EM —effector memory (CD62L − CD45RO + ); T TE —terminal effector (CD62L − CD45RO − ).

Article Snippet: For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark.

Techniques: Incubation, Flow Cytometry