Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal: bioRxiv

Article Title: Capturing Translation in Action with Protein Synthesis Profiling

doi: 10.1101/2025.11.17.688896

Figure Lengend Snippet: a, Structural model of APEX2-eEF2 bound to the ribosome. The large ribosomal subunit is shown in cyan, the small subunit in green, APEX2-eEF2 in magenta, and mRNA as a black line with the red circles indicating the alkyne modification. The blue sphere marks the ∼25 nm labeling radius of APEX2. b, Expression constructs for APEX2 and APEX2-eEF2 under the TDH3 promoter. c, Western blot detection of APEX2 and APEX2-eEF2 expression. Lanes: control (empty vector), APEX2 (27 kDa), and APEX2-eEF2 (118 kDa). Tubulin (Tub) was probed as a loading control. d, Schematic of the RNA tagging workflow. Yeast cells were incubated with alkyne-phenol (30 min), followed by H₂O₂ (5 min). After quenching, total RNA was extracted, conjugated to biotin-azide via click chemistry, and enriched with streptavidin beads. Both total and enriched RNA were used for preparing Illumina sequencing libraries. e, Agarose gel analysis of total RNA from control, APEX2, and APEX2-eEF2 cells. The presence of intact 25S and 18S rRNA bands indicates high RNA quality. M, molecular weight ladder. f, Detection of alkyne-labeled RNAs by conjugation with fluorescein-azide. Total RNA from control, APEX2, and APEX2-eEF2 cells was subjected to click chemistry and analyzed by agarose gel electrophoresis. Fluorescence was detected using a Typhoon imager. g, The same gel as in F, stained with SafeStain to verify equal RNA loading. h, Quantification of fluorescein-labeled RNA signal. The bar graph shows fluorescence intensity normalized to total RNA, averaged across two independent experiments. i, Gel-shift assay of biotin-labeled RNAs incubated with anti-biotin-AF488 antibody. RNAs from control, APEX2, and APEX2-eEF2 cells were conjugated with biotin-azide, bound by antibody, and resolved on an agarose gel. Antibody-RNA complexes are indicated by the black bar. Lanes: M, molecular weight ladder; control, RNA from control cells; APEX2, RNA from APEX2-expressing cells; APEX2-eEF2, RNA from APEX2-eEF2-expressing cells; Ab, antibody only.

Article Snippet: After incubation, RNA was purified using the Monarch RNA Cleanup Kit (New England BioLabs, Cat# T2040) according to the manufacturer’s instructions.

Techniques: Modification, Labeling, Expressing, Construct, Western Blot, Control, Plasmid Preparation, Incubation, Illumina Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Conjugation Assay, Fluorescence, Staining, Gel Shift

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A, B): m6A methylation of transcripts was detected by m6A qRT-PCR in DDX5-knockdown or DDX5 overexpressed MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48hand infected with VSV for 6h (A), and MEFs were transfected with Myc-DDX5 expressed vector (DDX5) or Myc tag control vector (Con) for 24h and infected with VSV for 6h (B). After extracting total RNA, purifying mRNA, and removing ribosomal RNA, purified mRNA was fragmented and incubated with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4h. RNA was isolated from the solution with phenol-chloroform, and cDNA was subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (C, D): The interaction between METTL3 and transcripts was detected through METTL3 RIP qRT-PCR in knockdown-DDX5 (C) or DDX5-expressing (D) MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48 h and infected with VSV for 6 h (C), and transfected with DDX5 expression plasmid (DDX5) or control vector (Con) for 24 h, infected with VSV for 6h, and subjected to METTL3 RIP qRT-PCR to detect GAPDH, TBK1, DHX58, IKKγ, and p65. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (E, F): Nuclear transcript retention increased in DDX5-knockdown MEFs. MEFs were transfected with DDX5 siRNA (siNC), infected with VSV for 8h, and lysed to extract nuclear to cytoplasmic RNA fractions. Then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (E) with RNU6 and GAPDH as the nuclear and cytoplasmic controls, respectively. The quantitative distribution of m6A modified DHX58, IKKγ, and p65 mRNAs in DDX5-knockdown MEFs were detected by m6A qRT-PCR (F). (G, H): Nuclear transcript export was increased in DDX5-expressing MEFs. MEFs were transfected with DDX5 expression plasmid (control vector), infected with VSV for 8h, and lysed to extract nuclear or cytoplasmic RNA; then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (G), and the quantitative distribution of these mRNAs was detected by m6AqRT-PCR (H). (I, J) : Immunoblot analysis of DHX58, IKKγ, and p65 in DDX5-knockdownMEFs (I) or DDX5-expressing MEFs (J) after infection with VSV at 0, 4, and 6 h. All data are mean ± SEM of biologically independent samples. Data are representative of three independent experiments. ns, no significant difference. * p <0.05, ** p <0.01, and *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Purification, Incubation, Isolation, Expressing, Western Blot, Modification

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A): m6A methylation of transcripts was detected in DDX5 +/+ or DDX5 +/- primary mouse macrophages infected for 8 h with VSV (MOI = 10). After extracting total RNA and purifying mRNA, mRNA was used to perform m6A qRT-PCR by incubating with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4 h. RNA was isolated and subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained in the control group, and the expression of DDX5 was analyzed bywestern blotting. (B): Immunoblot analysis of DDX5, DHX58, p65, and IKKγ in lysates of DDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (C, D): ELISA of IFN-β (C) and IL-6 (D) in cell supernatants ofDDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (E, F): ELISA of IFN-β (E) and IL-6 (F) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or VSV (5×10 8 plaque-forming units/g body weight) for 8h (n = 6). (G, H): ELISA of IFN-β (G) and IL-6 (H) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or SeV (1×10 8 plaque-forming units/g body weight) for 8h (n = 6). (I, J): The TCID 50 dose of VSV (I) or SeV (J) was measured in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice. (K): Pathological lesions in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice observed by hematoxylin-eosin staining with intraperitoneal injection of PBS, VSV (5×10 8 plaque-forming units/g body weight) or SeV (1×10 8 plaque-forming units/g body weight) for 12h. Scale bars, 100 μm. All data are presented as mean ± SEM of biologically independent samples. n = number of biological replicates. Data are representative of three independent experiments. NS, no significant difference. ** p <0.01, *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Infection, Quantitative RT-PCR, Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 1. BMPER expression, localization, and regulation by FoxO3a in endothelial cells. A, Expression of BMPER in human vascular endothelial cells of different origin. Cells were lysed and subjected to Western blot analysis with the indicated antibodies. B and C, Localization of BMPER was characterized by immuno- cytochemistry in C166 mouse yolk sac endothelial cells. Corre- sponding serum was used as negative control. Nuclei were stained with DAPI. Scale bar100 m. D and E, Silencing of FoxO3a in HUVECs with 2 different siRNAs compared to scram- bled siRNA control resulted in enhanced BMPER expression shown by RT-PCR (D) and Western blot analysis (E). Seventy- two hours after transfection, mRNA expression was analyzed by using specific primers for FoxO3a, BMPER, and human RNA polymerase II. Western blot analysis was performed with the indicated antibodies. -Tubulin served as loading control. Rep- resentative Western blots are shown, along with densitometric analysis of the time course of BMPER expression. F, BMPER mRNA expression at 72 and 96 hours after transfection with FoxO3awt or the constitutively active mutant FoxO3aA3 com- pared to empty vector. BMPER mRNA was quantified by real- time (quantitative) PCR using specific primers for BMPER and hRP as internal control. BMPER mRNA expression was calcu- lated using the CT method. MeansSD. *P0.05 vs control.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Western Blot, Immunocytochemistry, Negative Control, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 2. Specific silencing of BMPER by siRNA in HUVECs. A, BMPER mRNA expression after 24 and 48 hours posttrans- fection with the siRNA BMPER I and II, respectively, compared to scrambled siRNA control. BMPER mRNA was quantified by real-time (quantitative) PCR using specific primers for BMPER and human RNA poly- merase II as internal control. Knockdown efficiency was calcu- lated using CT method. MeansSD; n4. *P0.001 vs con- trol. B, Representative semiquantitative RT-PCR analysis 24 hours posttransfection is shown. C, Western blot analysis was performed with the indicated antibodies 48 hours posttransfection.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Circular maps show fold enrichment profiles of EcTopoI (ChIP-Seq, light orange; Topo-Seq, cyan and dark orange for two separate DNA strands), RNAP (ChIP-Seq, green), and DNA gyrase (Topo-Seq, dark-red). Additionally, GC-content (%, purple) and mean expression levels (FPKM, RNA-Seq, gray) for annotated TUs (inner blue segments) are also shown. Blue asterisks indicate positions of rRNA operons on the innermost orange ring representing E. coli DY330 genome. The numbers on the outside of the orange ring indicate genome coordinates in megabase pairs (Mbs). Three gaps around ~0.3, ~0.8, and ~1.2 Mb correspond to deletions in the E. coli DY330 genome relative to the E. coli W3110 reference genome. Insets provide a zoom-in view of representative regions with high EcTopoI signals. Coordinates in kb are indicated on top of each inset. For ChIP-Seq, fold enrichment is given relative to the input sample in all figures. The maps were constructed with the Circos tool , and the insets were prepared using IGV .

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Construct

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Representative regions of the E. coli chromosome with EcTopoI ChIP-Seq peaks matching the EcTopoI Topo-Seq TCSs ( dps , potF ) and a region lacking EcTopoI-binding and activity ( nuoN ). EcTopoI cleavage activity is shown strand-specifically. A control, non-induced culture, is shown. Positions of regions used for ChIP-qPCR and affinity measurements are indicated by gray rectangles. b Affinity of purified EcTopoI to three amplified genomic regions from the panel a measured by EMSA. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected. c Metagene plot of EcTopoI ChIP-Seq enrichment (untreated condition, black curve) and EcTopoI Topo-Seq cleavage signal (blue and red curves for coding and template strands, respectively). Confidence bands around the mean metagene signal are represented by ±SEM. Analysis was performed for the HETU set. Regions used for further quantification of enrichment in panel d are shown by colored areas on the plot. d Mean EcTopoI cleavage signal in different regions relative to HETUs. Means were compared by a two-sided Welch t -test. P values <4e-3 are indicated by asterisks and Bonferroni correction for multiple testing was applied. Bars represent mean values ±SEM, n = 200 TUs. e Metagene plot of EcTopoI Topo-Seq cleavage activity for all TUs, LETU, and HETU sets. Cleavage is shown strand-specifically. f Logo of EcTopoI-binding motif identified in sequences under the ChIP-Seq peaks. A motif is shown in both orientations. g EcTopoI cleavage motif identified by alignment of TCSs. The cleavage site between nucleotides −1 and 1 is indicated by a dashed line. The cleavage signal (N3E) is plotted below. h Affinity of purified EcTopoI to oligonucleotides measured with EMSA. The binding of forward (left) and reverse-complement (right) oligonucleotides is shown. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected (Supplementary Table ). i Affinity of purified EcTopoI to oligonucleotides measured with MST. Data were represented as mean values ± SEM, minimum of three independent MST experiments were performed. For source data, see Table in the Source Data file. j Cleavage of oligonucleotides by purified EcTopoI. A control with EcTopoI inactivated by a high temperature is indicated with a boxed + sign. Cleavage products are marked with a blue arrow. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Binding Assay, Activity Assay, Control, ChIP-qPCR, Affinity Purification, Amplification, Concentration Assay, Gel Shift, Purification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Growth curves for E. coli DY330 topA -SPA harboring pCA24 GFP (i), pCA24 14 kDa CTD (ii), or pCA24 topA (iii) plasmids. Data for induced (+IPTG 1 mM) and non-induced (−IPTG) cultures are shown. Shade represents a 0.95 confidential interval of the mean of three biological replicates. Gray lines mark aliquots collection for plasmid extraction. b Quantification of cell length in CTD or GFP producing cultures (left). Cells >10 µm are collected into an overflowing bin. The number of cells is indicated in parentheses. Representative fields are shown on the right. c Graphical representation of truncated versions of a topA gene constructed by recombineering in E. coli BW25113. d Growth curves of E. coli BW25113 strains with truncated versions of topA and the wild-type control (left). Shade represents 0.95 confidential intervals of the mean. Quantification of doubling time for exponential regions of growth curves (right). e Quantification of cell length for E. coli BW25113 strains with truncated versions of topA and the wild-type (left). Vertical dashed line marks 2*mean cell length for wild-type. Representative fields are shown on the right. f Mutations in gyrase genes ( gyrA , gyrB ) found in E. coli BW25113 topAΔ30 clones. An asterisk indicates amplification of a chromosomal region containing TopoIV genes; clones lacking compensatory mutations are highlighted in green. g Supercoiling of pCA24 GFP (i), pCA24 topA (ii), and pCA24 14 kDa CTD (iii) plasmids extracted from exponentially growing E. coli DY330 topA -SPA. Time-points correspond to panel a . Supercoiling of pCA24 GFP (iv) plasmid extracted from exponentially growing E. coli BW25113 topAΔ 30 (time-course, on the left) or E. coli BW25113 wt (two rightmost lanes). (v) Supercoiling level of the pCA24 GFP plasmid extracted from overnight cultures of different clones of E. coli BW25113 topA mutants and from the wild-type control. Clone numbers correspond to panel f . Nic - nicked plasmid, L - linear plasmid, −sc - negatively supercoiled plasmid, HCF - hypercompacted plasmid. h Metagene plots of normalized strand-specific read coverage depth obtained in DRIP-Seq experiments for E. coli DY330 topA -SPA for HETU (upper panel, rRNA operons were excluded) and LETU (lower panel) sets. Schematic TUs are shown below. Data for CTD-/Rif- condition are shown with a dashed line, coverage depths for the coding and template strands are indicated by dark-red and dark-blue fillings, respectively. Data for CTD+/Rif− condition are shown with a solid line, coverage depths for the coding, and template strands are indicated by light-red and light-blue fillings, respectively. i DRIP-Seq data for pCA24 14 kDa CTD for CTD+/Rif− and CTD+/Rif+ conditions and corresponding RNase HI-treated controls. Coverage depths for “−” and “+” strands are shown in light-blue and light-red, respectively. A linearized map of the plasmid is shown below. Source data are provided as a Source Data file.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: Plasmid Preparation, Extraction, Construct, Control, Clone Assay, Amplification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Average normalized enrichment of TopoI, DNA gyrase, and RNAP over transcription units of E. coli ( a , “open” model) and Mycobacterium ( b , “closed” model). Graphical representations of twin-domain sub-models are shown below. ChIP-Seq data for M. tuberculosis MtbRNAP, MtbGyrase, and M. smegmatis MsmTopoI was taken from publicly available datasets , , . c Other “semi-open” hypothetical variations of the twin-domain model, based on the interaction of key topoisomerases (TopoI, DNA gyrase) with RNAP and their activity within a complex. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Activity Assay

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: Direct-zol RNA Miniprep Kit , Zymo Research , R2054.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Expressing, Software, Microscopy

Journal: Molecular cell

Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF

doi: 10.1016/j.molcel.2019.07.039

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA Polymerase I, Large (Klenow) Fragment , New England Biolabs , Cat. # M0210.

Techniques: In Vitro, RNA Binding Assay, Recombinant, Staining, Flow Cytometry, Sample Prep, Expressing, Imaging, Knock-In, Plasmid Preparation, Software