Journal: Advanced Science

Article Title: Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3‐Regulated Histone Deacetylation and Transcription

doi: 10.1002/advs.202307804

Figure Lengend Snippet: Both 65k Δ/Δ and ankrd11 Δ/Δ mutants exhibit enhanced levels of H3K9ac and H4K5ac in the head. A) The NMJ boutons are reduced in the two deletion strains, but less than the reduction in the U12‐deletion strain. Quantitation of the NMJ boutons is shown on the right. B) The larva locomotion is impaired in the two deletion strains. Quantitation of the relative mobility are shown on the right. C) H3K9ac and H4K5ac were enhanced in the head of the two deletion strains. D) Quantitative analyses of the changed levels of histone acetylations. E) Retained intron events in the head of the two deletion strains. Numbers in brackets are counting of minor introns. F) Validation of inhibited splicing of three minor introns in the two deletion mutants by RT‐PCR. Red lines, minor introns. G) Analyses of differentially expressed genes (DEGs) in the head of the two mutants. Numbers in brackets are counting of MIGs. H) Validation of three DEGs in the two deletion mutants by RT‐PCR. The length of PCR amplicons is indicated. Statistical data are shown as mean ± SD. p values were calculated using two‐sided unpaired t‐test; * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001; ns, no significance.

Article Snippet: After removing debris, the lysates were first pre‐cleared by 25 μL of Protein A magnetic beads (Thermo, 88 846) for overnight incubation, and then applied to FLAG magnetic beads (Sigma, M8823) or V5 agarose beads (GNI, 4510‐V5) or pre‐conjugated 65K antibody (Proteintech, 25820‐1‐AP) with protein A‐G magnetic beads (Beyotime, P2108) for IP and co‐IP; Protein A magnetic beads were used as negative controls.

Techniques: Quantitation Assay, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3‐Regulated Histone Deacetylation and Transcription

doi: 10.1002/advs.202307804

Figure Lengend Snippet: The 65K‐ANKRD11 interaction is conserved in human. A) Schematics for HsANKRD11‐truncated proteins that were expressed with V5‐tag in human 293T cells. Borders and domains are indicated. ANK, ankyrin repeat domain; RD, transcription repression domain; AD, transcription activation domain. B) Co‐IPs by the truncated HsANKRD11s indicated that T6, covers aa 1160 to 1470 in the long uncharacterized region, is sufficient for interaction with Hs65K. C) Hs65K was co‐IPed by an HDAC3 antibody, but not by an HDAC1 or HDAC2 antibody. D) Knockdown of HsANKRD11 reduced the Hs65K association with HDAC3. E) H3K9ac, H4K5ac, and H4K8ac were enhanced in both Hs65K‐KD and HsANKRD11‐KD cells. KDs were performed using shRNAs, and the empty vector was used as a control. F) Quantitation of the histone acetylation levels in Hs65K‐KD and HsANKRD11‐KD cells. Statistical data are shown as mean ± SD. p values were calculated using two‐sided unpaired t‐test; * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001; ns, no significance.

Article Snippet: After removing debris, the lysates were first pre‐cleared by 25 μL of Protein A magnetic beads (Thermo, 88 846) for overnight incubation, and then applied to FLAG magnetic beads (Sigma, M8823) or V5 agarose beads (GNI, 4510‐V5) or pre‐conjugated 65K antibody (Proteintech, 25820‐1‐AP) with protein A‐G magnetic beads (Beyotime, P2108) for IP and co‐IP; Protein A magnetic beads were used as negative controls.

Techniques: Activation Assay, Knockdown, Plasmid Preparation, Control, Quantitation Assay

Journal: Advanced Science

Article Title: Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3‐Regulated Histone Deacetylation and Transcription

doi: 10.1002/advs.202307804

Figure Lengend Snippet: Hs65K and HDAC3 synergistically promote each other's chromatin binding. A) Analyses of chromatin‐binding sites of Hs65K and HDAC3 in 293T cells using CUT&Tag data. The majority of Hs65K‐binding sites overlap with HDAC3‐binding sites from the view of both peaks (left) and genes with peaks (right). B) Density distribution of Hs65K‐ and HDAC3‐binding signals on gene bodies. Peak‐containing genes are grouped as common‐, all‐, and only‐binding. TSS, transcription start site; TES, transcription end site. C) The chromatin‐bindings near the TSS region of Hs65K and HDAC3 were significantly decreased when HsANKRD11 was knocked down by shRNA. 11‐KD , HsANKRD11‐KD ; ctrl , control . D) Peak signals of Hs65K and HDAC3 at the common‐binding sites were more seriously decreased than at the only‐binding sites in the HsANKRD11‐KD cells. p values were calculated using the Wilcoxon test; *** , p value < = 0.001. E) A large portion of the 65K‐ and HDAC3‐signals at the common‐binding sites were simultaneously decreased in the HsANKRD11‐KD cells. F–I) Experimental validation of the CUT&Tag analyses for chromatin‐binding of Hs65K and HDAC3 by individual PCRs. Four genes were selected for testing, including two common‐binding genes F) TRIM59 and G) CREB3L4 , H) an Hs65K‐only binding gene RP1‐29K1.6 , and I) an HDAC3‐only binding gene ARID1A . The location of protein‐binding peaks and PCR amplification regions for peaks and non‐peaks are indicated.

Article Snippet: After removing debris, the lysates were first pre‐cleared by 25 μL of Protein A magnetic beads (Thermo, 88 846) for overnight incubation, and then applied to FLAG magnetic beads (Sigma, M8823) or V5 agarose beads (GNI, 4510‐V5) or pre‐conjugated 65K antibody (Proteintech, 25820‐1‐AP) with protein A‐G magnetic beads (Beyotime, P2108) for IP and co‐IP; Protein A magnetic beads were used as negative controls.

Techniques: Binding Assay, shRNA, Control, Biomarker Discovery, Protein Binding, Amplification

Journal: Advanced Science

Article Title: Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3‐Regulated Histone Deacetylation and Transcription

doi: 10.1002/advs.202307804

Figure Lengend Snippet: The 65K‐ANKRD11 interaction modulates H3K9 acetylation and gene expression. A) Statistics of H3K9ac ChIP‐peaks and peak‐containing genes in three human cells. The overlapping between the three cells is analyzed. 11‐KD , HsANKRD11‐KD ; ctrl , control . B) Density distribution of H3K9ac‐signals surrounding TSSs in human cells. All genes are divided into four groups according to their binding with Hs65K and HDAC3: Hs65K‐only binding, HDAC3‐only binding, common‐binding, and non‐binding. C) Fold changes of H3K9ac‐signals after knockdown of Hs65K (left) and HsANKRD11 (right). D) Composition of the changed H3K9ac‐signals in the two KD cells. The H3K9ac‐signals are analyzed in four groups, each of them containing four parts according to the location relationship between the H3K9ac peaks and Hs65K, HDAC3 peaks. The numbers of decreased and increased H3K9ac‐signals at the chromatin domains with colocalized triple‐peaks are shown in red and blue, respectively. E) Overlapping of the changed H3K9ac‐signals at chromatin domains with the colocalized triple‐peaks between the two knockdown cells. The significantly increased H3K9ac‐signals show a larger overlapping between the two KD cells than the decreased H3K9ac‐signals. F) Most of the chromatin domains with colocalized triple‐peaks and increased H3K9ac‐signals in both two KD cells exhibit simultaneously decreased binding of Hs65K and HDAC3 in the HsANKRD11‐KD cells. G) Forty‐four genes are up‐regulated in both knockdown cells due to the decreased binding of Hs65K and HDAC3 and reduced deacetylation of H3K9 at their chromatin domains with the colocalized triple‐peaks. Three genes for further experimental validation are indicated. H,I) Compared with both down‐regulated 70 genes, both up‐regulated 44 genes have significantly more reduced deacetylation of H3K9 in both the Hs65K‐KD and HsANKRD11‐KD cells (H). p values were calculated using the Wilcoxon test; * , p value <0.05, *** p value <0.001. The paired boxplot of increased H3K9ac sites and expression changes (I).

Article Snippet: After removing debris, the lysates were first pre‐cleared by 25 μL of Protein A magnetic beads (Thermo, 88 846) for overnight incubation, and then applied to FLAG magnetic beads (Sigma, M8823) or V5 agarose beads (GNI, 4510‐V5) or pre‐conjugated 65K antibody (Proteintech, 25820‐1‐AP) with protein A‐G magnetic beads (Beyotime, P2108) for IP and co‐IP; Protein A magnetic beads were used as negative controls.

Techniques: Gene Expression, Control, Binding Assay, Knockdown, Biomarker Discovery, Expressing

Journal: Advanced Science

Article Title: Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3‐Regulated Histone Deacetylation and Transcription

doi: 10.1002/advs.202307804

Figure Lengend Snippet: Minor spliceosomal component 65K interacts with ANKRD11 and facilitates HDAC3‐regulated histone deacetylation and gene expression. (Left) In the WT condition, HDAC3 deacetylates the histone proteins H3 and H4, which is facilitated by its cofactor ANKRD11. The interaction between ANKRD11 and the minor spliceosome‐specific protein 65K/RNPC3 enhances the chromatin‐binding of HDAC3 to further facilitate the deacetylation of histones, especially on H3K9 and H4K5, and results in regulated gene expression. (Right) Caused by the deficiency of 65K or ANKRD11, the defective 65K‐ANKRD11 interaction weakens the chromatin‐binding of HDAC3 and decreases the deacetylation activity, resulting in changed gene expression. This regulatory mechanism is conserved in both Drosophila and human cells.

Article Snippet: After removing debris, the lysates were first pre‐cleared by 25 μL of Protein A magnetic beads (Thermo, 88 846) for overnight incubation, and then applied to FLAG magnetic beads (Sigma, M8823) or V5 agarose beads (GNI, 4510‐V5) or pre‐conjugated 65K antibody (Proteintech, 25820‐1‐AP) with protein A‐G magnetic beads (Beyotime, P2108) for IP and co‐IP; Protein A magnetic beads were used as negative controls.

Techniques: Gene Expression, Binding Assay, Activity Assay

Journal: Microbiology Spectrum

Article Title: Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

doi: 10.1128/spectrum.00341-25

Figure Lengend Snippet: Western blot analysis of RVFV mAb library. Anti-RVFV mAbs 1RV1, 1RV2, 1RV3, 1RV4, and 1RV5 (250 ng/mL) were used in lanes 1–5, respectively. Reactivity to probe non-reduced RVFV rNP ( A ) and reduced RVFV rNP ( B ) is demonstrated. HRP-conjugated goat anti-mouse IgG was used as the detection marker.

Article Snippet: Subsequent immunizations were performed through intraperitoneal injections of RVFV rNP (BEI Resources) emulsified with Freund’s incomplete adjuvant (Millipore Sigma).

Techniques: Western Blot, Marker

Journal: Microbiology Spectrum

Article Title: Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

doi: 10.1128/spectrum.00341-25

Figure Lengend Snippet: Sensitivity and specificity testing of the RVFV LFI prototype. RVFV rNP suspended in 1× DPBS ( A ) or NHS ( B ) was applied to the sample pad of the LFI prototype at varying concentrations. Presence or absence of a test line, as interpreted by the eye, is depicted as (+) or (−) to indicate the visual result. Gamma-irradiated RVFV lysate suspended in NHS (1:200) was assayed on the RVFV LFI prototype ( C ). Antigen biomarkers from other pathogens were assayed for cross-reactivity (100 ng/mL) in NHS ( D ).

Article Snippet: Subsequent immunizations were performed through intraperitoneal injections of RVFV rNP (BEI Resources) emulsified with Freund’s incomplete adjuvant (Millipore Sigma).

Techniques: Irradiation

Journal: Microbiology Spectrum

Article Title: Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

doi: 10.1128/spectrum.00341-25

Figure Lengend Snippet: Spot size, density of cAb, and signal intensity reported as functions of the volume of 1RV4 immobilized; VFI membranes were run with 2.5 ng/mL of RVFV rNP in 5 mL of buffer ( A and B ). Image of 1RV4 cAb optimization membrane after assay ( C ). Optimization of dAb concentration involved measuring signal intensity with varying levels of AuNP-1RV5 ( D ).

Article Snippet: Subsequent immunizations were performed through intraperitoneal injections of RVFV rNP (BEI Resources) emulsified with Freund’s incomplete adjuvant (Millipore Sigma).

Techniques: Membrane, Concentration Assay

Journal: Microbiology Spectrum

Article Title: Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

doi: 10.1128/spectrum.00341-25

Figure Lengend Snippet: Calibration curve for detection of RVFV rNP in spiked buffer samples ( A ). LOD study for the detection of RVFV rNP in spiked 10% NHS samples ( B ). Background (mean negative + 3σ) signal is shown by red dotted line for control samples, and the LOD is indicated as “*” in each graph.

Article Snippet: Subsequent immunizations were performed through intraperitoneal injections of RVFV rNP (BEI Resources) emulsified with Freund’s incomplete adjuvant (Millipore Sigma).

Techniques: Control