Journal: Life Science Alliance

Article Title: The noncoding RNA GRAS1 interacts with NKAP and protects against DNA damage and cell death

doi: 10.26508/lsa.202403105

Figure Lengend Snippet: (A) RNA antisense purification–mass spectrometry experimental outline. (B) RT-qPCR analysis of GRAS1, U1 snRNA, and GAPDH mRNA in nuclear and cytoplasmic fractionations of A549 cells (left panel). Immunoblot of Lamin A/C as a marker for nuclear fraction, and GAPDH as a marker for cytoplasmic fraction (right panel); N = 3. (C) Validation of GRAS1 RNA capture and recovery by biotinylated antisense oligonucleotide probes against GRAS1. RT-qPCR analysis of GRAS1 RNA and TUBB control mRNA in three fractions, including input with probes added, unbound sample flowthrough, and elution; N = 3. (D) Total proteins identified in RAP-MS of GRAS1 RNA, U1 snRNA positive control, and luciferase mRNA negative control elution samples. (E) GRAS1 interactome proteins identified by RAP-MS, with at least two unique peptides, after excluding proteins that were also found in U1 and luciferase control captures (left panel). The top 10 GRAS1-interacting proteins and their molecular weights (right panel). (F) GRAS1 RNA enrichment measured by RT-qPCR after immunoprecipitation of NKAP in A549 cells, compared with immunoprecipitation with IgG control antibody; N = 3. (G) Immunoblot of NKAP and p65 in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2. (H) Quantification of immunoblot (G). NKAP and p65 expression were calculated after normalization to the GAPDH loading control for each sample; N = 3. (I) RT-qPCR analysis of GCLC, ANGPT1, BDKRB1, and NR3C1 expression levels in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, normalized to GAPDH control mRNA; N = 3. (J) RT-qPCR analysis of NKAP mRNA expression in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, normalized to GAPDH control mRNA; N = 3. (K) Immunoblot of NKAP and p65 in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, with 5 μM MG-132 treatment. (L) Quantification of immunoblot (K). NKAP and p65 expression were calculated after normalization to GAPDH loading control for each sample; N = 6. Error bars represent SD in all panels; (ns) P > 0.05; (***) P < 0.001 by two-tailed t test.

Article Snippet: The following primary antibodies were used: NKAP polyclonal antibody (ab229096; Abcam, 1:1,000 dilution), phospho-histone (γ) H2A.X Ser139 20E3 (60566SF; Cell Signaling Technology, 1:1,000 dilution), p53 (10442-1-AP; Proteintech, 1:1,000 dilution), GAPDH (60004-1-Ig; Proteintech, 1:3,000 dilution), Lamin A/C (10298-1-AP; Proteintech, 1:1,000 dilution), NF-κB p65 (10745-1-AP; Proteintech, 1:1,000 dilution), and TIGA1 (MBS9610109; MyBioSource, 1:1,000 dilution).

Techniques: Purification, Mass Spectrometry, Quantitative RT-PCR, Analysis, Western Blot, Marker, Biomarker Discovery, Control, Positive Control, Luciferase, Negative Control, Immunoprecipitation, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells decreases neutrophil infiltration and NETs formation through NF‐κB p65/IL‐8 signaling. (A) Bubble plot of the enriched KEGG pathways showing that the NF‐κB signaling pathway was enriched in HCT116 cells (marked with a red border). (B) GSEA revealed that MIIP knockdown in HCT116 cells significantly activated the NF‐κB signaling pathway. (C) Western blotting analysis of key proteins involved in the NF‐κB p65 signaling pathway in human CRC cells with different levels of MIIP expression. (D) Representative images of H&E and IHC staining for MIIP, p‐p65, IL‐8, and Ly6G in tumors from the cecum of an orthotopic CRC mouse model between the MIIP‐overexpressing group and the control group (n = 5 per group). Scale bar, 50 µm. (E) Statistical analysis of Ly6G + neutrophils and the expression of p‐p65 and IL‐8 in different groups. (F) Representative images and statistical quantification of IF staining for MPO (red), Cit‐H3 (green), and DAPI (blue) in tumor tissue from the cecum and comparisons between the two groups ( n = 5). Scale bar, 25 µm. (G) Western blotting of NF‐κB p65 and NF‐κB p‐p65 in CRC cells treated with or not treated with an NF‐κB inhibitor (Bay 11–7082, 10 µM). (H) RT‐qPCR of CXCL8 mRNA in CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (I) ELISA of IL‐8 in CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (J) Quantification of neutrophils that migrated to the CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (K–M) ELISA of MPO‐DNA and representative images and statistical quantification of IF staining for NETs formation after neutrophils were cultured with CM from SW480 and HCT116 cells treated or not treated with an NF‐κB inhibitor ( n = 3–5). Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and the Mann‐Whitney U test in (E); the Mann‐Whitney U test in (F); and two‐way ANOVA in (H, I, J, K, and M).

Article Snippet: CRC cells were treated with an NF‐κB inhibitor (10 μM, Bay 11–7082, MCE, USA).

Techniques: Knockdown, Western Blot, Analysis, Expressing, Immunohistochemistry, Control, Staining, Tissue, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP modulates the NF‐κB p65/IL‐8 signaling pathway ‐mediated neutrophil infiltration and NETs formation through interactions with PKR. (A) IP–MS revealed PKR as a MIIP‐interacting protein. (B) The binding structure of MIIP and PKR was predicted using HDOCK molecular docking software. (C) Endogenous interaction between MIIP and PKR were detected via Co‐IP in HCT116 cells. (D) 293T cells were cotransfected with MIIP‐Flag and PKR‐HA, and reverse and forward Co‐IP analyses were performed. (E) Co‐IP assays were used to determine the PKR‐binding region of MIIP. (F) Co‐IP assays revealed that the PKR binding site for MIIP encompasses its IKKβ‐binding domain. (G) The expression of PKR was determined by Western blotting in CRC cells with different levels of MIIP expression. (H) Co‐IP was performed to examine the effect of MIIP expression on the interaction between PKR and IKKβ. (I–M) Double‐stable transgenic CRC cell lines were used in a rescue assay to demonstrate that the regulatory effects of MIIP on the NF‐κB p65‐mediated IL‐8 signaling pathway (I–K) and NETs formation (L, M) were dependent on PKR ( n = 3–5). All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using two‐way ANOVA in (J–M).

Article Snippet: CRC cells were treated with an NF‐κB inhibitor (10 μM, Bay 11–7082, MCE, USA).

Techniques: Protein-Protein interactions, Binding Assay, Docking Assay, Software, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transgenic Assay, Rescue Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Targeting Microglial HDAC5 Suppresses NLRP3 Inflammation via NF ‐ κB Deacetylation to Alleviate Ischemic Brain Injury

doi: 10.1002/cns.71161

Figure Lengend Snippet: HDAC5 deacetylates NFκB p65 and indirectly regulates NLRP3 expression. (A) BV2 cells were transfected with lentivirus for 48 h and then harvested for Co‐IP assay. (B) Molecular docking analysis predicting the interaction between HDAC5 and NF‐κB p65. The enlarged inset shows the predicted potential binding site. (C) Co‐IP assay showing the interaction between HDAC5 and NF‐κB p65 in BV2 cells. (D) Western blot assay to detect the expression of acetylated proteins after Hdac5 KD. (E) Hdac5 KD in BV2 cells and subsequent Co‐IP assay. (F) Western blot assay to detect NFκB p65 protein expression in cytoplasm and nucleus after Hdac5 KD. (G, H) Quantitative analysis of NFκB p65 protein form (D) ( n = 4). (I) Schematic of the predicted NFκB p65 (Rela) binding site in the promoter of NLRP3. All quantitative data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 by one‐way ANOVA.

Article Snippet: Equal amounts of protein were incubated overnight at 4°C with anti‐HDAC5 antibody (Santa Cruz), anti‐acetylated‐lysine antibody (Cell Signaling Technology), anti‐NF‐κB p65 antibody (Santa Cruz), or the corresponding species‐matched IgG as a negative control.

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Docking Assay, Analysis, Binding Assay, Western Blot