Journal: Bioorganic & Medicinal Chemistry Letters

Article Title: Antiproliferative activity of 2,3-disubstituted indoles toward apoptosis-resistant cancers cells

doi: 10.1016/j.bmcl.2013.03.110

Figure Lengend Snippet: Figure 1. Cellular imaging of B10 against melanoma SKMEL-28 and NSCLC A549 cells illustrating noncytotoxic, but cytostatic, antiproliferative mechanism at MTT colorimetric assay-related GI50 concentrations after 72 h of cell culture with the drug.

Article Snippet: Yet, C-3 ether and thioether indoles B appear to the most potent, with ether indole B6 N NHR1 NH NH2R1 O H N O R2 R2 O R2 C N SR1 O S S NR4R5 NR4R5 S R2 E HCl, EtOH (b) (d) N NH2R1 R2 HCl, EtOH R3 R3 2. cer cells Table 1 (continued) # Structure GI50 in vitro valuesa (lM) # Structure GI50 in vitro valuesa (lM) U373 Hs683 A549 SKMEL B16 U373 Hs683 A549 SKMEL B16 B10 N H Cl Cl S 28 31 27 34 28 E5 N Me S Br N S CO2Et >100 85 72 78 50 C1 N H Cl NH O O O 66 45 37 55 27 E6 N H S Br N S Br 9 22 22 29 33 C2 N H NH O O N Cl F 24 15 9 13 22 E7 N H S N S Br 36 26 31 28 40 C3 N H NH O 38 69 38 67 44 E8 N H S Br N S OH 32 24 24 28 29 C4 N H NH O BnS 47 33 30 30 35 E9 N H S Br N S CO2Et 35 36 34 36 27 a U373 (ECACC code 89081403) cell line was cultured in MEM medium supplemented with 5% heat inactivated fetal bovine serum; Hs683 (ATCC code HTB-138), SKMEL-28 (ATCC code HTB-72), A549 (DSMZ code ACC107) and B16F10 (ATCC code CRL-6475) cell lines were cultured in RPMI medium supplemented with 10% heat inactivated fetal bovine serum; MEM and RPMI cell culture media were supplemented with 4 mM glutamine, 100 lg/mL gentamicin and penicillin–streptomycin (200 U/mL and 200 lg/mL). exhibiting single-digit micromolar GI50 values.

Techniques: Imaging, Colorimetric Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Expressing, Isolation, Derivative Assay, Reverse Transcription, Positive Control, Sequencing, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 is internalized by human B cells. (A) Western blot results analyzing the uptake of C3 by Raji B cells. Cells were treated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met, 10% hiFBS,) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37°C. C3 uptake was investigated by Western blot using the goat polyclonal anti-C3 antibody (Quidel) under non reducing conditions. Data shown are representative of four independent experiments. (B) Western blot results confirming the presence of internalized C3 in the cytoplasm. Cells were incubated with distinct sources of C3 (20% NHS, 200 μg/ml C3, 200 μg/ml C3met) in DGVB++ or EDTA-GVB buffer for 1 h at 37°C. After lysis, cytoplasmic and membrane fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody (Quidel) under non reducing conditions. The purity of distinct cellular fractions was verified using antibodies against B-tubulin (cytoplasmic marker) and Na/K ATPase (membrane marker) under reducing conditions. Results shown are representative of two independent analyzes.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Western Blot, Incubation, Lysis, Membrane, Marker

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a bind to distinct types of DNA. (A–C) Gel shift assays presenting interaction between C3(a) and genomic DNA isolated from Raji cells. Isolated genomic DNA was incubated either with pre-titrated concentrations of C3 (A) or C3a (B) or C3b (C) and separated by agarose gel electrophoresis. Formation of high molecular weight DNA-protein complexes is indicated by slower DNA migration (DNA shift) compared to migration of the free nucleic acid. The presence of C3 and its cleavage fragments in the pre-formed protein-DNA complexes was confirmed with anti-C3a and anti-C3d antibodies caused supershift. One representative experiment out of five performed is shown. (D) Gel shift assays showing that C3(a)—DNA interaction is not ionic in nature. C3 or C3a were incubated with genomic DNA of Raji cells in the presence of increasing NaCl concentration (ranging from 150 to 1,200 mM). Binding of C3 and C3a to DNA was observed at 8X higher salt concentration than the physiological 150 mM. (E) Interaction of C3 and C3a with DNA is not restricted to genomic DNA. Linearized pCEP4 vector was incubated with either C3 or C3a and DNA-protein complex formation was investigated by gel shift assay. Results illustrated are one representative out of three independent experiments. (F,G) ELISA results confirming interaction between C3(a) and DNA. ELISA microplate surfaces were coated either with DNA (F) or C3, C3a, C3b (G) . After incubation with distinct concentrations of C3 proteins (F) or Raji genomic DNA (G) , protein-DNA interactions were detected using anti-C3d, anti-C3a (anti-C3 ELISA, F ), or anti-dsDNA antibody (anti-DNA ELISA, G ). Results shown are mean absorbance values measured at 490 nm with SD of four independent experiments. (H) ELISA results showing presence of C3-DNA complexes in formaldehyde cross-linked chromatin fraction of Raji B cells. Cells were incubated in the presence or absence of C3, C3met, fixed with 1% formaldehyde to cross-link protein-DNA complexes and chromatin isolated using commercial kit. C3-DNA complexes were investigated by ELISA on anti-C3 or anti-DNA coated plates. Data are shown as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to non-treated cells (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01).

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Gel Shift, Isolation, Incubation, Agarose Gel Electrophoresis, High Molecular Weight, Migration, Concentration Assay, Binding Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Confirmation of C3-DNA interaction by ChIP sequencing. (A) ChIP-seq identified peaks from anti-C3 immunoprecipitated chromatin samples were classified based on the location (UCSC annotation data) and are shown in the following genome regions: intergenic, introns, downstream 5 Kbp, upstream 5 Kbp, and exons. The interacting DNA elements localized mainly the intergenic and intronic regions. (B) ChIP-seq statistics of identified peaks including peak numbers and genome rate (%). C3 binding to genomic DNA is strongly increased upon stimulation of B cells with the polyclonal activator, CpG oligonucleotide. (C) ChIP-PCR was run on ChIP DNA of CpG-activated samples using primers specific for the ChIP-seq identified regions in IL-7 and L-selectin genes. Enrichment of the specific DNA loci was calculated with the percent input method and illustrated as % input or as fold changes over samples without C3 treatment (two-way ANOVA with Sidak's multiple comparison, * p < 0.05, ** p < 0.01). The values represent the mean ± SEM of four independent experiments.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: ChIP-sequencing, Immunoprecipitation, Binding Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: Although this product was recognized by several different antibodies against C3 (Quidel #A304 , Complement Technology #A213, Sigma #C7761, DAKO #A0062 and #A0063), it is probably unspecific since it can be detected in the Crispr/Cas9 edited, C3 knock out A549 cells and mock transfected HEK293 cells ( ).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Comparison, Labeling

Journal: Nature Communications

Article Title: Boosting NAD + with a small molecule that activates NAMPT

doi: 10.1038/s41467-019-11078-z

Figure Lengend Snippet: Effects of SBI-797812 on NAMPT activity in cultured cells. A549 cells treated with SBI-797812 for 4 h. NMN ( a ), NAD + ( b ), and NADP ( c ) were measured by LC-MS/MS. Data shows means ± s.d.; * p < 0.0001 vs. DMSO. NMN: n = 35, 8, 12, 20 for vehicle, 0.4, 2, 10 μM SBI-797812, respectively; NAD + : n = 32, 8, 12, 20 for vehicle, 0.4, 2, 10 μM SBI-797812; NADP: n = 36, 12, 20 for vehicle, 2, 10 μM SBI-797812. One-way ANOVA with Tukey’s multiple comparisons test. NADH ( d ) or NADPH ( e ) were not increased in A549 cells treated with SBI-797812 (10 μM) for 4 h. NADH and NADPH were assayed by LC-MS/MS. Data shows means ± s.d. n = 16 except for NADPH (SBI-797812), n = 15. f NAD + biosynthesis in A549 cells treated for 4 h with vehicle, SBI-797812 (10 μM) or FK-866 (1 μM), and 13 C/ 15 N-NAM (20 μM). NAM(M + 4), MeNAM(M + 4), NMN(M + 4), NAD + (M + 4), and NADP(M + 4) were assayed by LC-MS/MS. Data shows means ± s.d.; n = 5. * p < 0.001 vs. Control. One-way ANOVA with Dunnett’s multiple comparisons test. g , h SBI-797812 increased NMN and NAD + in human primary myotubes. Cells treated with SBI-797812 (0.4, 2, 10 μM) for 4 h. NMN and NAD + quantified by LC-MS/MS and normalized to cell protein. Data shows means ± s.d.; n = 24. * p < 0.05, ** p < 0.01, # p = 0.0001 vs. Control. One-way ANOVA with Dunnett’s multiple comparisons test. e . f For Fig. 4a–h, source data are provided as a Source Data file

Article Snippet: In a related protocol, A549 cells were exposed to media also containing 20 μM NAM ( 13 C 3 , 15 N) (Cambridge Isotope Laboratories) along with vehicle or SBI-797812 for 4 h. Cells were harvested and processed as described above.

Techniques: Activity Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Control