Journal: Cell Death & Disease

Article Title: Enhancing cell death in B-cell malignancies through targeted inhibition of Bcl-3

doi: 10.1038/s41419-024-07067-w

Figure Lengend Snippet: A Lysates were prepared from RS4;11, SUP-B15, Karpas-422, SU-DHL-8, Rec-1, RL, Granta-519, and WSU-NHL cells and subjected to Western Blot analysis toward Bcl-3 and Actin. B Lysates from a large panel of B-cell leukemia and B-cell lymphoma cells including Karpas-422, RS4;11, SU-DHL-8, WSU-NHL, JVM2, SP53, Granta-519, Mino, UPN2, Ramos, HBL2, RL, and Rec-1 were subjected to Western Blot analysis toward Bcl-3 and Actin. C mRNA from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, RL, Rec-1, and Granta-519 cells were prepared and subjected to real-time quantitative (qPCR) analysis of Bcl-3 and housekeeping gene GAPDH. All reactions were performed in triplicates. Data are represented as relative mRNA levels mean ± SEM. D Left. Lysates from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, RL, Rec-1, and Granta-519 cells were subjected to Western Blot analysis toward p105/p50 and Actin Right. mRNA from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, RL, Rec-1, and Granta-519 cells were prepared and subjected to real-time quantitative (qPCR) analysis of p50 and housekeeping gene GAPDH. All reactions were performed in triplicates. Data are represented as relative mRNA levels mean ± SEM. E Left: Lysates from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, RL, Rec-1, and Granta-519 cells were subjected to Western Blot analysis toward p100/p52 and Actin Right: mRNA from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, RL, Rec-1, and Granta-519 cells were prepared and subjected to real-time quantitative (qPCR) analysis of p52 and housekeeping gene GAPDH. All reactions were performed in triplicates. Data are represented as relative mRNA levels mean ± SEM. F Left: mRNA from Rec-1, and Granta-519 cells were prepared and subjected to real-time quantitative (qPCR) analysis of cyclin D1 and housekeeping gene GAPDH. All reactions were performed in triplicates. Data are represented as relative mRNA levels mean ± SEM. Right: mRNA from Karpas-422, RS4;11, SU-DHL-8, SUP-B15, WSU-NHL, and RL cells were prepared and subjected to real-time quantitative (qPCR) analysis of cyclin D1 and housekeeping gene GAPDH. All reactions were performed in triplicates. Data are represented as relative mRNA levels mean ± SEM.

Article Snippet: Following blocking, membranes were probed with antibodies towards Bcl-3 (SC-185, Santa Cruz), p50 (SC-8414, Santa Cruz), p52 (SC-7386, Santa Cruz), β-Actin (#ab6276, Abcam), cIAP1 (SC-271419, Santa Cruz), or cleaved caspase 3 (Asp175, #9660, Cell Signaling).

Techniques: Western Blot

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 1. p65/p52 nuclear colocalization. a Immunofluorescence analysis of p65 and p52 proteins in M2 macrophages, MφTAMMDA-MB−231 TCM, and MφTAMMCF-7 TCM

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: Immunofluorescence

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 2. p65/p52 putative target genes (PTGs). a Gene expression analysis of PTGs was evaluated in untreated and treated macrophages by qRT-PCR. Error bars represent ±SEM. One-way ANOVA and Tukey correction were used to evaluate the differences between means; * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4 for each experimental group. Immunofluorescence analysis of p65 and p52 proteins in b MφTAMMDA-MB−231 TCM and c MφTAMMCF-7 TCM treated with QNZ, compared to control DMSO. p65 was stained in red and p52 in green. Nuclei were counterstained with DAPI. Scale bars represent 5 μm. Gene expression analysis of NF-κB PTGs by qRT-PCR in d MφTAMMDA-MB−231 TCM and e MφTAMMCF-7 TCM treated with QNZ, compared to control DMSO. Error bars represent ±SEM. Student's t-test was used to analyze the efficacy of QNZ treatment for each gene, compared to the control treatment; * p < 0.05, ** p < 0.01, *** p < 0.001. n = 3 for each experimental group.

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: Gene Expression, Quantitative RT-PCR, Immunofluorescence, Control, Staining

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 4. p65 and p52 downregulation in TAMs. Western blot analysis of p65 and p52 protein was performed in a MφTAMMDA-MB−231 TCM and b MφTAMMCF-7 TCM

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: Western Blot

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 5. HSPG2 regulatory elements. a In silico analysis of HSPG2 on the UCSC Genome Browser showed four regulatory regions with RELA (p65) and NFKB2 (p52) binding sites, as shown in the lower panel (JASPAR 2022 TFBS and ReMap density tracks). b The luciferase assay of the HSPG2 regulatory regions was performed in M2 macrophages and TAMs. Data represents firefly/renilla luminescence. Error bars represent ±SEM. Student's t-test: *p < 0.05, **p < 0.01, ***p < 0.001. n = 4 for each experimental group. ChIP-qPCR analysis of c p65 and d p52 was performed in M2 macrophages and TAMs on HSPG2_R3 and HSPG2_R4 regulatory regions. Quantitative PCR data is presented as a percentage of the input chromatin control, compared to control IgG. Student's t-test was performed to compare the antibody enrichment to IgG. ns = not significant enrichment of antibody compared to control IgG; *p < 0.05 were none reported. Student's t-test was also performed to compare the antibody enrichment between the two regulatory regions: *p < 0.05, **p < 0.01, ***p < 0.001. n = 4 for each experimental group.

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: In Silico, Binding Assay, Luciferase, ChIP-qPCR, Real-time Polymerase Chain Reaction, Control

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 6. CSF-1 regulatory elements. a In silico analysis of CSF-1 on the UCSC Genome Browser showed two regulatory regions with overlapped RELA (p65) and NFKB2 (p52) binding sites, as shown in the lower panel (JASPAR 2022 TFBS and ReMap density tracks). b The luciferase assay of the two CSF-1 regulatory regions was performed in M2 macrophages and TAMs. Data represents firefly/renilla luminescence. Error bars represent ±SEM. Student's t-test: *p < 0.05. n = 4 for each experimental group. ChIP-qPCR analysis of c p65 and d p52 was performed in M2 macrophages and TAMs on CSF-1_R1 and CSF-1_R2 regulatory regions. Quan titative PCR data is presented as a percentage of the input chromatin control, compared to control IgG. Student's t-test was performed to compare the antibody enrichment to IgG. ns = not significant enrichment of antibody compared to control IgG; *p < 0.05 were none reported. Student's t-test was also performed to compare the antibody enrichment on the two regulatory regions: *p < 0.05, **p < 0.01. n = 4 for each experimental group.

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: In Silico, Binding Assay, Luciferase, ChIP-qPCR, Control

Journal: Life sciences

Article Title: Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs).

doi: 10.1016/j.lfs.2024.123059

Figure Lengend Snippet: Fig. 7. TGFB1 and LILRB1 regulatory elements. ChIP-qPCR analysis of a p65 and b p52 was performed in M2 macrophages and TAMs on TGFB1_R1 and TGFB1_R2 regulatory regions. Quantitative PCR data is presented as a percentage of the input chromatin control, compared to control IgG. n = 3 for each experimental group. ChIP-qPCR analysis of c p65 and d p52 was performed in M2 macrophages and TAMs on the LILRB1 p65/p52 binding site. Quantitative PCR data is presented as a percentage of the input chromatin control, compared to control IgG. n = 3 for each experimental group. Student's t-test was performed to compare the antibody enrichment to IgG. ns = not significant enrichment of antibody compared to control IgG; *p < 0.05 were none reported. Student's t-test was also performed to compare the antibody enrichment on the regulatory region: *p < 0.05, **p < 0.01.

Article Snippet: 1 % of the total volume was conserved as Input DNA, while collected samples were equally split and incubated on a rotating platform at 4 ◦C overnight with the following primary antibodies: anti-p65 (D14E12; Cell Signaling Technology), anti-p52 (sc7386; Santa Cruz Biotechnology) and normal IgGs (12–370; MilliporeSigma) at a concentration of 1 μg per 1 × 106 cells.

Techniques: ChIP-qPCR, Real-time Polymerase Chain Reaction, Control, Binding Assay