Journal: Acta Neuropathologica

Article Title: IL-10-providing B cells govern pro-inflammatory activity of macrophages and microglia in CNS autoimmunity

doi: 10.1007/s00401-023-02552-6

Figure Lengend Snippet: Human B cells secrete relevant amounts of IL-10 and B cell depletion is associated with an enhanced activation of blood myeloid cells. a , b CD27 − and CD27 + B cells isolated from peripheral blood mononuclear cells (PBMC) of healthy donors ( n = 11) were cultured without stimulation (unstim), stimulated with CpG (4 µg/ml) or stimulated with CD40 ligand (CD40L; 1 µg/ml) and anti-IgM antibody (40 µg/ml) for 48 h. Concentrations of secreted a IL-10 and b IL-6 were determined using ELISA. Frequency of c CD19 + B cells ( n = 14), d CD14 + cells ( n = 14) and e mean fluorescence intensity (MFI) of MHC class II (MHCII) on CD14 + cells (data points were included if CD14 frequency > 1% in ( d ); n = 7) was determined in PBMC of patients with multiple sclerosis (MS) before (pre) and after (post) anti-CD20 antibody treatment. f CD14 + cells were isolated from PBMC of patients with MS ( n = 17) before (pre RTX) and 11–28 weeks after anti-CD20-mediated B cell depletion (post RTX) and stimulated with LPS for 18 h. Number of TNF-α producing CD14 + cells determined by ELISpot. The mean ± standard error of the mean is indicated in all graphs. Asterisks indicate significant difference calculated using paired two-tailed t -test (* P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

Article Snippet: 3.5 × 10 5 MACS-purified B cells of patients with MS and healthy controls, as well as CD27-positive and CD27-negative B cells from healthy individuals, were plated in 96-well u-bottom plates and stimulated with either 4 µg/ml CpG or with 40 µg/ml affiniPure F(ab')2 fragment rabbit anti-human IgM (Fc5 Fragment Specific, Jackson Immuno Research) and 1 µg/ml recombinant human CD40L (R&Dsystem).

Techniques: Activation Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Enzyme-linked Immunospot, Two Tailed Test

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Overview of the treatment scheme. i.t: intratumorally; i.v: intravenously; ICOSL: inducible co-stimulator ligand.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques:

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Viral DNA extraction from CsCl-purified virus particles using proteinase K. The AdV-D24-ICOSL-CD40L sequencing library was made using a Nextera XT kit (Illumina). The size of the library was assessed by high-resolution agarose gel electrophoresis. The presence of the 24-bp deletion in the E1A CR2 domain, the CMV-ICOSL-IRES-CD40L expression cassette inserted in place of the E3 region and the hybrid Ad5/3 fiber were confirmed. Analysis of sequence coverage did not detect the presence of a virus sub-population with a genome characterized by a major rearrangement such as a large insertion or deletion. The figure was generated by SnapGene ® .

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: DNA Extraction, Purification, Sequencing, Agarose Gel Electrophoresis, Expressing, Generated

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of ICOSL and CD40L expression in tested melanoma cells. ( A ) ICOSL concentration was detected on supernatants collected 24–48 h after treatment using ELISA kit. ( B ) CD40L was detected from supernatants collected 24–48 h after treatment using an ELISA kit according to the manufacturer’s instructions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (ns = not significant, p > 0.05). ( C ) Transgene expression. The expression of ICOSL and CD40L from AdV-D24-ICOSL-CD40L was assessed by infecting A549 cells with the virus, harvesting proteins 48 h after the infection and detecting ICOSL and CD40L by Western blot. 1–4: protein extracts from A549 cells infected with AdV-D24-ICOSL-CD40L clones #1–4. N: protein extract from non-infected A549 cells. M: PageRuler Prestained Protein Ladder (ThermoScientific # 26616). ICOSL MW: 70 kDa (due to heavy protein glycosylation); CD40L MW: 30 kDa.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Infection, Western Blot, Clone Assay

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of cell viability by MTS assay (cell cytotoxicity assay). ( A ) Cell viability was evaluated 72 h post-infection with AdV-24-ICOSL-CD40L and AdV-24 at the concentration of 100 VP/cell and combination with anti-PD1 (pembrolizumab). Data are expressed as the percentage of viable cells according to MTS cell viability assay protocol (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega). Statistical analysis was carried out with a Mann–Whitney test to compare two groups ( p > 0.05). ( B , C ) Microscopic photographs visualizing the morphology and cytopathic effect (CPE) of the cells representing the investigated groups and assessed by MTS assay at 48 and 72 h post-infection (magnification 10×) (** = p ≤ 0.001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: MTS Assay, Cytotoxicity Assay, Infection, Concentration Assay, Viability Assay, Proliferation Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Immunogenic cell death assessment. ( A ) Evaluation of calreticulin (CRT) exposure by melanoma cell lines after treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured 48 h post-treatments with anti-calreticulin antibody staining AlexaFluor ® 488 and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( B ) Assessment of ATP release after the treatment. ATP concentration in a supernatant was evaluated 72 h after infection with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. ( C ) Evaluation of high-mobility group box 1 (HMGB-1) release after treatment with oncolytic adenoviruses and the combination with anti PD-1. HMGB-1 level was measured from the supernatant collected 72 h after infection with ELISA kit (MBL International, Woburn, MA, USA), according to manufacturer’s dispositions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (* = p ≤ 0.05; ** = p ≤ 0.001, *** = p ≤ 0.0001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Infection, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Antitumor efficacy of oncolytic vectors and the combination therapy with anti PD-1 antibody in murine melanoma B16V (1 × 10 6 cells/flank, one mouse had 2 tumors/6 tumors per group) xenograft immunocompetent C57BL/6 model. ( A ) Tumor volume (mm 3 ) measured through the study. The treatment was performed once per day on days 1–6. The mice were treated according to the scheme with viruses (i.t.) and anti PD-1 antibody (i.v.) ( B ) At the end of the study, mice were sacrificed and tumors harvested for weight assessment. ( C , D ) Tumor volume measurement on days 6 and 20. ( E ) Body weight measurements throughout the study. ( F ) Survival profile was calculated by Kaplan–Meier test. ( H ) Evaluation of CRT exposure after the treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured in the end of the study (after mice sacrifice) with anti-calreticulin antibody staining and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( G ) Assessment of ATP release after the treatment. ATP concentration from the tumors was evaluated in the end of the study (after mice sacrifice) with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. Error bars, mean ± SEM, * = p ≤ 0.05, *** = p ≤ 0.0001.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Cell Viability Assay

Journal: Journal of Virology

Article Title: Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

doi: 10.1128/jvi.01870-08

Figure Lengend Snippet: FIG. 1. Generation of CD40L-pseudotyped SIVs. (A) CD40L- dSIV was generated by transient cotranfection of pCD40L, pVSV-G, and pSIVENgfp into 293T cells. pSIVENgfp is a plasmid encod- ing the full-length SIVmac239 proviral DNA with deletions in the env and nef genes and an insertion of the GFP gene in the nef region. pVSV-G is the plasmid containing the VSV-G gene under a CMV promoter. pCD40L contains the CD40L gene under a CMV promoter. Deletions are indicated by shaded regions, the inserted GFP gene is shown as an open box, and pA refers to the polyadenylation signals from the expression plasmids. (B) 293T cells cotransfected with pCD40L, pVSV-G, and pSIVENgfp were able to express all three genes (CD40L, VSV-G, and GFP). Cells were fixed, permeabilized, and double stained with anti-CD40L conjugated with Alexa Fluor 555 and anti-VSV-G conjugated with Alexa Fluor 647. Protein expression was visualized by confocal microscopy.

Article Snippet: Cells were incubated with goat anti-human CD40L polyclonal antibody (R&D Systems) and rabbit anti-VSV-G polyclonal antibody.

Techniques: Generated, Plasmid Preparation, Expressing, Staining, Confocal Microscopy

Journal: Journal of Virology

Article Title: Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

doi: 10.1128/jvi.01870-08

Figure Lengend Snippet: FIG. 2. Verification of proteins present in CD40L-dSIV as deter- mined by Western blotting. (A) Purified CD40L-dSIV particles were analyzed by Western blotting. Lysates from cells transfected with ei- ther pCD40L or pVSV-G served as controls. VSV-G and Gag p27 were present in both dSIV and CD40L-dSIV particles, while CD40L was present only in the CD40L-dSIV particles. Blots were stained with anti-VSV-G (upper panels), anti-Gag (middle panels), or anti-CD40L (lower panels) antibodies. (B) CD40L-dSIV viral stock was run on either a continuous 20 to 60% sucrose gradient or a 6 to 18% iodixanol (OptiPrep) gradient, and 20 fractions were collected from the bottom of the tube. Each fraction was analyzed by ELISA for human CD40L, SIV Gag p27, and VSV-G. The highest concentrations of these three proteins in both gradients were found in the same fraction in two separate experiments (duplicates not shown).

Article Snippet: Cells were incubated with goat anti-human CD40L polyclonal antibody (R&D Systems) and rabbit anti-VSV-G polyclonal antibody.

Techniques: Western Blot, Transfection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

doi: 10.1128/jvi.01870-08

Figure Lengend Snippet: FIG. 3. Expression of viral proteins in transduced DCs. Viral pro- tein expression was visualized by confocal microscopy. Virus particle- transduced DCs were fixed, permeabilized, and stained with anti-Gag p27 conjugated with Alexa Fluor 647. Upper panels show DCs trans- duced with dSIV, and lower panels show DCs transduced with CD40L-dSIV.

Article Snippet: Cells were incubated with goat anti-human CD40L polyclonal antibody (R&D Systems) and rabbit anti-VSV-G polyclonal antibody.

Techniques: Expressing, Confocal Microscopy, Virus, Staining, Transduction

Journal: Journal of Virology

Article Title: Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

doi: 10.1128/jvi.01870-08

Figure Lengend Snippet: FIG. 6. CD40L-dSIV-transduced DCs activated naive allogeneic CD8 T cells more efficiently in a MLR assay. T cells were incubated with virus particle-transduced or PBS control DCs at a ratio of 1:10 and cultured for 5 days. BrdU was added 18 h before harvesting, and T cells were restimulated to boost cytokine secretion 6 h before har- vesting. (A) T cells were analyzed by flow cytometry and gated using forward scatter, side scatter, and CD8. BrdU incorporation was used to evaluate T-cell proliferation. The percentage of CD8 BrdU T cells in the CD40L-dSIV group was up to twice that of CD8 T cells in the PBS and dSIV groups. The percentage of CD8 BrdU T cells secret- ing IFN- in the CD40L-dSIV group was also significantly higher than those in the PBS and dSIV groups. Mean data from all of the exper- iments are shown in the percentages of CD8 BrdU T cells and CD8 BrdU IFN- T cells. Bars represent means standard er- rors. Data were analyzed with two-tail paired t tests: *, P 0.03, compared individually with results for the dSIV and PBS groups (n 10). (B) Representative data from 1 set of MLR experiments out of 10.

Article Snippet: Cells were incubated with goat anti-human CD40L polyclonal antibody (R&D Systems) and rabbit anti-VSV-G polyclonal antibody.

Techniques: Mlr Assay, Incubation, Virus, Control, Cell Culture, Cytometry, BrdU Incorporation Assay

Journal: Journal of Virology

Article Title: Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

doi: 10.1128/jvi.01870-08

Figure Lengend Snippet: FIG. 7. CD40L-dSIVs induced stronger humoral and CMI responses in mice. (A) Sera collected from animals were analyzed by ELISA for SIV Gag-specific antibodies and by neutralization assay for anti-VSV antibodies. Sera were pooled for the 2-week samples in each group. “Week” refers to weeks postvaccination. (B) CD4-cell-depleted splenocytes from each mouse set were seeded in triplicate into 96-well plates precoated with anti-IFN- monoclonal antibody. Cells were pulsed with a Gag peptide pool (1 g/ml) for 24 h at 37°C. IFN--secreting cells were counted as SFC and normalized as SFC/106 cells. Bars represent means standard errors. A two-tailed paired t test was used for data analysis: *, P 0.05 compared to results with PBS; **, P 0.01 compared to results with PBS and dSIV individually (n 5 for groups at week one; n 10 for groups at week four).

Article Snippet: Cells were incubated with goat anti-human CD40L polyclonal antibody (R&D Systems) and rabbit anti-VSV-G polyclonal antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Two Tailed Test

Journal: Journal of Histochemistry & Cytochemistry

Article Title: Pathogenic Role of NF-κB Activation in Tubulointerstitial Inflammatory Lesions in Human Lupus Nephritis

doi: 10.1369/jhc.7a7368.2008

Figure Lengend Snippet: Figure 4 Microphotographs of immunostaining for CD40, CD40L, TNF-a, and IL-1b in controls and patients with LN. (A–D) Normal controls. Weak immunoreactivity with CD40 (A), CD40L (B), and TNF-a (C) was detected in renal tubules of normal kidneys, whereas IL-1b expression was absent in the normal kidney tissue (D). (E–H) MCD. Expression of CD40 (E) and CD40L (F) was barely observed in the tubulointerstitium of patients with MCD, whereas TNF-a expression (G) was localized to the apical surface of proximal tubules, and expression of IL-1b (H) was present in the peritubular capillaries of a patient with MCD. (I–L) LN. Pronounced overexpression of CD40 (I), CD40L (J), TNF-a (K), and IL-1b (L) was observed in damaged tubules and interstitial infiltrates in patients with class IV-G LN. Bar (A–D; E–H, K,L; I,J) 5 50 mm.

Article Snippet: Other primary antibodies used were polyclonal rabbit anti-IKK-a, monoclonal mouse anti-IkB-a and -phosphorylated IkB-a (serine 32), and polyclonal goat anti-CD40L antibodies (Santa Cruz Biotechnology); polyclonal rabbit anti-GM-CSF antibody (Rockland Immunochemicals); monoclonal mouse anti-CD40 antibody (Serotect; Oxford, UK); and monoclonal mouse anti-CD68KP1 antibody (Dako; Carpinteria, CA).

Techniques: Immunostaining, Expressing, Over Expression

Journal: Journal of Histochemistry & Cytochemistry

Article Title: Pathogenic Role of NF-κB Activation in Tubulointerstitial Inflammatory Lesions in Human Lupus Nephritis

doi: 10.1369/jhc.7a7368.2008

Figure Lengend Snippet: Figure 5 Semiquantitative analysis of tubular expression CD40, CD40L, TNF-a, IL-1b, ICAM-1, IL-6, and GM- CSF. *p,0.05 vs NC; ap,0.05 vs MCD; #no marker expression is detectable. NC, normal control; MCD, minimal change disease; LN, lupus nephritis.

Article Snippet: Other primary antibodies used were polyclonal rabbit anti-IKK-a, monoclonal mouse anti-IkB-a and -phosphorylated IkB-a (serine 32), and polyclonal goat anti-CD40L antibodies (Santa Cruz Biotechnology); polyclonal rabbit anti-GM-CSF antibody (Rockland Immunochemicals); monoclonal mouse anti-CD40 antibody (Serotect; Oxford, UK); and monoclonal mouse anti-CD68KP1 antibody (Dako; Carpinteria, CA).

Techniques: Expressing, Marker, Control

Journal: PLoS ONE

Article Title: Functional characterization of T-cells from palatine tonsils in patients with chronic tonsillitis

doi: 10.1371/journal.pone.0183214

Figure Lengend Snippet: CD4(+)/CD8(+) T-cells were isolated from tonsils of the different disease scenarios and their activation status was determined via flow cytometry analysis of the mixed CD4(+)CD8(+) preparations. (A) CD69, CD25 and CD154 activation marker surface expression determined by flow cytometry and plotted as percentage of all T-cells. (B) Double immune-staining for PD-1 and CD69 in all T-cells. Only statistically significant groups are labelled (* p<0.05). CT = chronic tonsillitis; PTA = peritonsillar abscess; HY = tonsillar hyperplasia; ton = tonsil; abs = abscess.

Article Snippet: Antibodies for flow cytometry analysis: anti-human CD3-FITC (clone MEM-57), anti-human CD4-PE (clone MEM-241), anti-human CD8-APC (clone MEM-31), anti-human CD45RA-FITC (clone HI100) and anti-human CD62L-APC (clone LT-TD180) from ImmunoTools (Friesoythe, Germany); Anti-human CD152 (CTLA-4)-PE (clone 14D3) and anti-human CD279 (PD-1)-FITC (clone MIH4) from eBioscience Inc. (San Diego, USA); CD25-APC human (clone 4E3), CD69-APC human (clone FN50), CD154-APC human (clone 5C8), anti-FoxP3-PE human (clone 3G3) and anti-IL-2-PE human (clone N7.48A) were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Isolation, Activation Assay, Flow Cytometry, Marker, Expressing, Staining

Journal: PLoS ONE

Article Title: Functional characterization of T-cells from palatine tonsils in patients with chronic tonsillitis

doi: 10.1371/journal.pone.0183214

Figure Lengend Snippet: CD4/CD8 T-cells isolated from tonsils were challenged with the indicated formulations of anti CD3 and/or CD28 Abs or with a mix of phorbol ester and Ionomycine (TPA/Iono) and subjected to flow cytometric determination of activation marker CD69 (A) , CD25 (B) and CD154 (C) expression, always plotted as percentage of all T-cells. All measurements were carried out on the mixed CD4(+)CD8(+) preparations. Representative fluorescence profiles for stimulated (black line) versus non-stimulated samples (grey curves) are shown on the left side of each panel. Beads: anti-CD3 and anti-CD28 Abs immobilized on beads. CT = chronic tonsillitis; PTA = peritonsillar abscess; HY = tonsillar hyperplasia; ton = tonsil; abs = abscess.

Article Snippet: Antibodies for flow cytometry analysis: anti-human CD3-FITC (clone MEM-57), anti-human CD4-PE (clone MEM-241), anti-human CD8-APC (clone MEM-31), anti-human CD45RA-FITC (clone HI100) and anti-human CD62L-APC (clone LT-TD180) from ImmunoTools (Friesoythe, Germany); Anti-human CD152 (CTLA-4)-PE (clone 14D3) and anti-human CD279 (PD-1)-FITC (clone MIH4) from eBioscience Inc. (San Diego, USA); CD25-APC human (clone 4E3), CD69-APC human (clone FN50), CD154-APC human (clone 5C8), anti-FoxP3-PE human (clone 3G3) and anti-IL-2-PE human (clone N7.48A) were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Isolation, Activation Assay, Marker, Expressing, Fluorescence

Journal: Cell Death & Disease

Article Title: CD40L mediated alternative NFκB-signaling induces resistance to BCR-inhibitors in patients with mantle cell lymphoma

doi: 10.1038/s41419-017-0157-6

Figure Lengend Snippet: a REC-1 and MAVER-1 cells were stimulated with CD40L (100 ng/ml) or H 2 O as a control for 18 h, and cytoplasmic (Cy) and nuclear (Nu) protein fractions were analyzed by Western blot. α-Tubulin and lamin B served as controls for the purity of the individual cell compartment fractions. b MCL cell lines were stimulated with CD40L (100 ng/ml) or H 2 O as a control overnight followed by TPCA-1 treatment at the indicated concentration. After an additional 24 h, viability was determined by MTT assay (* p < 0.001; ** p < 0.0001; *** p < 0.00001)

Article Snippet: Primary antibodies for PARP (#9532), NIK (#4994), β-actin (#4970), CD40L (#15094), Bcl-xL (#54H6) and secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG-HRP antibody (#7074) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Control, Western Blot, Concentration Assay, MTT Assay

Journal: Cell Death & Disease

Article Title: CD40L mediated alternative NFκB-signaling induces resistance to BCR-inhibitors in patients with mantle cell lymphoma

doi: 10.1038/s41419-017-0157-6

Figure Lengend Snippet: The MCL cell lines REC-1 a and MAVER-1 b were preincubated with CD40L (100 ng/ml) or H 2 O for 18 h and subsequently treated with either sotrastaurin (STN, 3 μM), ibrutinib (Ibru, 400 nM) or DMSO as a control for an additional 24 h. Cytoplasmic and nuclear protein extracts were analyzed by Western blot for the indicated proteins; “sh.” stands for a short and “l.” for a long exposure time for RelB detection. c L-929 cells were transfected with a CD40L expression plasmid or a control plasmid and co-cultured with REC-1 and MAVER-1 cells for 18 h. Subsequently, the cells were treated with sotrastaurin (3 µM), ibrutinib (400 nM) or DMSO for an additional 24 h, and whole cell lysates were analyzed by Western blot

Article Snippet: Primary antibodies for PARP (#9532), NIK (#4994), β-actin (#4970), CD40L (#15094), Bcl-xL (#54H6) and secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG-HRP antibody (#7074) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Control, Western Blot, Transfection, Expressing, Plasmid Preparation, Cell Culture

Journal: Cell Death & Disease

Article Title: CD40L mediated alternative NFκB-signaling induces resistance to BCR-inhibitors in patients with mantle cell lymphoma

doi: 10.1038/s41419-017-0157-6

Figure Lengend Snippet: a , b Viability assays were performed after prestimulation of REC-1 and MAVER-1 cells with CD40L (100 ng/ml, 18 h) or H 2 O as a control and additional treatment with sotrastaurin ( a , 3 µM) or ibrutinib ( b , 400 nM) for 48 h. Viability was determined with the MTT assay. c REC-1 cells were treated with various drugs (Akti-1/2, 1 µM; NVP-BEZ 235, 25 nM; AZD8330, 500 nM; ERK inhibitor II, 10 µM; 2-Bromoaldisine, 1 µM) or DMSO for 20 min before stimulation with CD40L (100 ng/ml, 18 h) or H 2 O as a control and subsequent treatment with sotrastaurin (3 µM), ibrutinib (400 nM) or DMSO for additional 48 h. Viability was determined with the MTT assay (* p < 0.001; ** p < 0.0001; *** p < 0.00001)

Article Snippet: Primary antibodies for PARP (#9532), NIK (#4994), β-actin (#4970), CD40L (#15094), Bcl-xL (#54H6) and secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG-HRP antibody (#7074) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Control, MTT Assay