Journal: Biomedicines

Article Title: Stability of Myeloid Cell Phenotype and Function Across a Broad Age Range in Humans and Cynomolgus Monkeys, and a Dominant Contribution of Humoral Factors in the Control of Bacterial Infection

doi: 10.3390/biomedicines14010071

Figure Lengend Snippet: Confocal microscopy of HMGB1 and γH2AX localization. ( A ) Representative images of monocytes (MO) and granulocytes (GR). HMGB1 or γH2AX staining are highlighted in orange, the DAPI in blue, the overlay of fluorescent both markers is shown in white. ( B ) quantification of staining nucleus and cytoplasm between two age groups, n = 480 (30 cells of each type and each marker from each donor). Statistical difference analysis by Mann–Whitney U-test (ns—not significant).

Article Snippet: Finally, the cells were resuspended in 50 μL Perm/Wash buffer containing either anti-γH2AX antibodies (polyclonal, Cusabio, Wuhan, China; cat. #CSB- PA105411 , 1:100) or anti-HMGB1 antibodies (clone EPR3507, Abcam, Waltham, MA, USA; cat. #ab79823, 1:100).

Techniques: Confocal Microscopy, Staining, Marker, MANN-WHITNEY

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Effects of matrine on intestinal pathology, inflammatory function, and JAK2 expression in experimental colitis mice Dextran Sulfate Sodium (DSS)-induced murine model of colitis was established in mice; matrine and 5-ASA treatment was administered as described. A – C Body weight and colon length were measured. D , E Histopathological alterations in colon tissues were evaluated using H & E staining; DAI scores were assigned according to the staining. F , G The levels of IL-1β, TNF-α, IL-6, MPO, NO, and MDA in colon tissues were examined using commercial assay kits. H , I The protein levels of JAK2 in mice colon tissues were determined using Immunohistochemical staining (IHC staining) and Immunoblotting. Magnification = 100 × or 200 × for IHC staining. J The protein levels of p-JAK2, p-STAT3, and STAT3 were detected using Immunoblotting. **p < 0.01, vs. PBS group; #p < 0.05, ##p < 0.01 vs. DSS group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Expressing, Staining, Immunohistochemical staining, Immunohistochemistry, Western Blot

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Effects of JAK2 overexpression on intestinal epithelial cells upon inflammation MODE-K cells were transfected with a JAK2-overexpressing plasmid (JAK2 oe) or a control vector, with or without subsequent LPS stimulation. A The protein levels of JAK2 and p-JAK2 and the inflammatory factors IL-1β, TNF-α, and IL-6 were determined by Immunoblotting. B Cellular levels of MPO, NO, and MDA were measured using biochemical kits. C Intracellular ROS production was assessed by DCFH-DA staining and quantified with flow cytometry. D The protein levels of the bile acid metabolism-related factors FXR, MRP3 and MRP4 were examined by Immunoblotting. E The mRNA levels of MRP3, MRP4, OSTα, and OSTβ were measured by qRT-PCR. **p < 0.01 vs. Vector group; ##p < 0.01 vs. Vector group; &&p < 0.01 vs. LPS + Vector group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Western Blot, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Effects of matrine on intestinal epithelial cell function upon inflammation Mouse intestinal epithelial cell line, MODE-K, was stimulated with LPS with or without matrine treatment (1, 2, 3 mg/ml), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); The levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); The protein levels of p-JAK2, JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. PBS group; #p < 0.05, ##p < 0.01 vs. LPS group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Dynamic effects of matrine and JAK2 on intestinal epithelial cell function upon inflammation MODE-K cells were stimulated with LPS with or without matrine treatment (2 mg/ml), transfected with JAK2-overexpressing plasmid (JAK2 oe), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); the levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); the protein levels of JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. LPS group; #p < 0.05, ## p < 0.01 vs. LPS + matrine + vector group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, Evolutionary analysis of the ASCH domain reveals high conservation across species. b, A scheme showing functional domains of EOLA1 in Homo sapiens (top) and Mus musculus (bottom). The ASCH domain is shown in red. c, Schematic diagram of sgRNA targeting mouse Eola1 locus. d, Western blotting analysis confirming Eola1 knockout clones in B16-F10 and HL-1 cell lines with anti-EOLA1 antibody. GAPDH was used as a loading control. e, Sanger sequencing results of Eola1 KO cell lines with genetic mutations introduced with the CRISPR/Cas9 system.

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Functional Assay, Western Blot, Knock-Out, Clone Assay, Control, Sequencing, CRISPR

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a , Schematic diagram of screening strategy. Cell populations with sgRNA library were cultured in glucose medium or galactose medium (galactose was used to replace glucose) for 48 hours. Dead cells from galactose medium or living cells from glucose medium were collected to carry out next-generation sequencing for evaluating the abundance of sgRNAs. b , Genes essential for mitochondrial oxidative phosphorylation (OXPHOS) -linked energy metabolism were identified in the screening. A rank-ordered plot shows the RRA (Robust Rank Aggregation) scores and corresponding p -values from the CRISPR screening results. c , Fold change (FC) distribution of sgRNAs targeting Eola1, Sp1, Stat1, and Samhd1 as indicated in red (upregulated) and blue (downregulated) lines, overlaid on gray gradient depicting the overall distribution. d , e , Growth curves of wild-type (WT) and EOLA1 KO cells cultured in the medium of glucose ( d ) or galactose ( e ). f , Eola1 depletion dramatically induced cell apoptosis when cancer cells cultured in galactose medium. Representative flow cytometry images (left) and quantification analysis (right) of the apoptotic B16-F10 melanoma cells and HL-1 cells cultured in glucose medium or galactose medium for 48 hours. FITC: fluorescein isothiocyanate; PI: propidium iodide. Note: d , e , One-way ANOVA with Tukey’s test. f , two-tailed unpaired Student’s t-tests. * P <0.05, **** P <0.0001, ns: not significant. d , e , f , Data were presented as mean ± SD (n=3).

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Cell Culture, Next-Generation Sequencing, Phospho-proteomics, CRISPR, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, Evolutionary conservation of EOLA1’s N-terminal domain. Sequence alignment across diverse species reveals key conserved residues (red). b, Fluorescence imaging analysis of EOLA1 subcellular localization after N-terminal mitochondrial targeting signal (MTS) deletion (red). Mitochondria were labeled with TFAM (green). Exogenously expressed MTS-deficient EOLA1 (delMTS-EOLA1) carried a C-terminal FLAG-HA tag.

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Sequencing, Fluorescence, Imaging, Labeling

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, Mitochondrial proteins predicted by TargetP 2.0 in human and mouse (left), and their overlap with MitoCarta 3.0 annotations (right). b, Fluorescence analysis of the subcellular location of human EOLA1 protein (red), and HSP60 (green) was used as a marker for mitochondria. The exogenously expressed EOLA1 was tagged with a C-terminal FLAG and HA tandem epitope. c, Western blotting analysis of EOLA1 in the subcellular fractions of PLC/PRF/5 ( Homo sapiens , left) and B16-F10 melanoma cells ( Mus musculus , right). Tubulin was used as a cytoplasmic marker, Histone H1.2 as a nuclear marker, and HSP60 as a mitochondrial marker. d, Determination of EOLA1 sub-mitochondrial localization by Proteinase K digestion assay using purified mitochondria from HEK293T cells. PK, Protease K; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. e, f, Seahorse analysis of WT and Eola1 KO in B16-F10 melanoma cells ( e ) and HL-1 cells ( f ). Quantification of the basal respiration, maximal respiration, and spare respiratory capacity was performed for the indicated cells. g-i, Electron microscopy images ( g ) of WT cells (left) and Eola1 knockout cells (right). Higher-magnification views are presented in panel h . The relative circularity ratio of mitochondria was quantified ( i ). Mito: Mitochondria, N: nucleus. Note: e , f , One-way ANOVA with Tukey’s test. i , two-tailed unpaired Student’s t-tests. ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. e , f , Data were presented as mean ± SD (n=6).

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Fluorescence, Marker, Western Blot, Purification, Membrane, Electron Microscopy, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, Pie chart representing the proportion of mitochondrial proteins identified in the EOLA1 interactome. b, EOLA1-interacting mitochondrial RNAs were identified via UV-RIP-seq. c, Protein levels of MRPS15 (mt-SSU marker) and MRPL11 (mt-LSU marker) were analyzed by Western blotting across fractions.

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Marker, Western Blot

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, b, Identification of EOLA1 interacting partners. EOLA1 complex was purified from EOLA1–FH stable cells using immunoaffinity purification. Eluted proteins were separated by SDS-PAGE and analyzed by silver staining ( a ). Western blotting analysis verified the mitochondrial Tu translation elongation factor (TUFM) as an EOLA1-binding partner ( b ). c-e, RNA immunoprecipitation (RIP) followed by qPCR demonstrates specific binding between EOLA1 and 12S mt-rRNA. Exogenous RIP-qPCR with ( c ) or without ( d ) UV crosslinking confirms direct RNA-protein interactions. Endogenous RIP-qPCR further validates this physiological association ( e ). The identification primers are provided in . f, g, EOLA1 affects mitochondrial mRNA translation. Sucrose gradient fractionation profiles of mitochondrial ribosomal subunits assessed by RT-PCR of 12S rRNA (mt-SSU) and 16S rRNA (mt-LSU) ( f , top). Distribution of mitochondrial-encoded mRNAs (ATP6, ND5, and COX1) measured by RT-PCR ( f , bottom), along with their corresponding protein levels measured by Western blotting. β-actin served as a loading control ( g ). The identification primers are provided in . h, Mitochondrial translation activity was assessed using the Mito-Click-iT assay. i, Mitochondrial-encoded mRNAs (mt-mRNAs) and ribosomal RNA levels (12S and 16S mt-rRNA) were quantified in control and EOLA1-knockout cells by RT-qPCR. The identification primers are provided in . Note: c , d , e , two-tailed unpaired Student’s t-tests. i , One-way ANOVA with Tukey’s test. *** P <0.001, **** P <0.0001. c , d , e , i , Data were presented as mean ± SD (n=3).

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Purification, Immunoaffinity Purification, SDS Page, Silver Staining, Western Blot, Binding Assay, RNA Immunoprecipitation, Fractionation, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Knock-Out, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, EOLA1 protein deficiency in knockout mice was validated by Western blotting analysis. GAPDH was used as a loading control. b, Western blotting analysis revealed significantly lower levels of mitochondrial protein in Eola1 -/- hearts compared to Eola1 +/+ . GAPDH was used as a loading control. c, Typical ventricular borders at end-diastole were identified by B-mode echocardiography. d, e, Compared to Eola1 +/+ mice, both male ( d ) and female ( e ) Eola1 -/- mice demonstrated significant enlargement in ventricular long-axis length ( i ), short-axis length ( ii ), and cross-sectional area ( iii ). f, Representative images of left ventricular motion patterns identified by M-mode echocardiography. g, Compared to Eola1 +/+ mice, male Eola1 -/- mice demonstrated a significant decrease in ejection fraction ( i ), shortening fraction ( ii ), and stroke volume ( iv ) of left ventricle. Cardiac output ( iii ) of left ventricle also showed a decreasing trend. h, Compared to Eola1 +/+ mice, female Eola1 -/- mice demonstrated a significant decrease in ejection fraction ( i ), shortening fraction ( ii ), cardiac output ( iii ) and stroke volume ( iv ) of left ventricle. i, Eola1 knockout leads to thickening of the anterior wall ( i ) and thinning of the posterior wall ( ii ) of the left ventricle in male mice. j, Eola1 deficiency causes thinning of the posterior wall ( ii ) and a trend of thickening of the anterior wall ( i ) of the left ventricle in female mice. k, l, Eola1 deficiency increases heart rate of both male ( k ) and female ( l ) mice. Note: d , e , g-l , two-tailed unpaired Student’s t-tests. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. Data were presented as mean ± SEM.

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Knock-Out, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, b, Schematic diagram of sgRNA targeting mouse Eola1 locus ( a ) and validation of Eola1 knockout in mice by PCR ( b ). The genotyping primers are provided in . c, Representative pictures of the Eola1 +/+ and Eola1 -/- mice. d, Comparison of body weight in female (left) and male (right) Eola1 +/+ and Eola1 -/- mice. e, Relative mRNA levels of mitochondrial-encoded transcripts were quantified by RT-qPCR in Eola1 +/+ and Eola1 -/- hearts. Note: e , two-tailed unpaired Student’s t-tests. **** P <0.0001, ns: not significant. Data were presented as mean ± SD (n=6).

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Biomarker Discovery, Knock-Out, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: EOLA1, a mitochondrial matrix protein identified by CRISPR screening, interacts with TUFM/12S mt-rRNA to promote the protein synthesis of OXPHOS subunits. Its loss impairs mt-mRNA translation and causes heart failure in mice, revealing a mitochondrial translation-cardiac function axis pivotal for cardiovascular homeostasis.

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: CRISPR

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Schematic representation of the B. fragilis NCTC9343 (hereafter referred to as NCTC9343) T6SS locus. Predicted gene products are shown above the indicated genes. Core T6SS structural components are shaded in gray; five hcp variants are colored in red. b Experimental design for antibiotic treatment and competitive colonization in C57BL/6 J mice. Numbers indicate days before (−) and after (+) strains gavage. c In vivo competition assays between killer strains (wild-type NCTC9343, T6SS-deficient mutants Δ tssC or Δ hcp1 – hcp5 ) and a Bte1-sensitive prey strain (Δ tssC ΔV1-ErmR) in antibiotic-treated C57BL/6 J mice (5 mice per group). Fecal samples collected at specified time points were homogenized in PBS, diluted, and plated on selective BHI agar (200 μg/mL gentamycin + 15 μg/mL erythromycin) for c.f.u. counting. The black dashed line indicates the limit of detection (L.O.D.). Data are presented as the arithmetic mean ± SEM. Unpaired two-tailed Student’s t-tests were used for comparing means between two groups. Source data are provided as a Source Data file. d In vitro co-culture assays between the indicated killer and prey strains. Wild-type NCTC9343 (WT) and its isogenic deletion mutants (Δ tssC or Δ hcp1 – hcp5 ) serve as killers. Bte1 sensitive mutant (NCTC9343 ΔV1-CamR) serves as prey. Prey survival was determined by serial dilution and plating on chloramphenicol-supplemented BHI agar. e Immunoblot analysis of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT) and its isogenic deletion mutants (Δ tssC or Δ hcp1 – hcp5 ). DnaK serves as a cytoplasmic loading control. f In vitro co-culture assays using NCTC9343 (WT), Δ tssC , Δ hcp2 , Δ hcp3 , Δ hcp2 - hcp3 ( Δ hcp23) or the indicated plasmid-based complementary strains (Δ hcp2::phcp2 , Δ hcp3::phcp3 , Δ hcp2 - hcp3::phcp2-hcp3 ) as killers and Bte1 sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was determined by serial dilution and plating on chloramphenicol-supplemented BHI agar. g Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), its isogenic deletion mutants (Δ tssC , Δ hcp2 , Δ hcp3 or Δ hcp2 - hcp3 ) and the indicated plasmid-based complementary strains (Δ hcp2::phcp2 , Δ hcp3::phcp3 or Δ hcp2 - hcp3::phcp2-hcp3 ). For ( d – g ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: In Vivo, Two Tailed Test, In Vitro, Co-Culture Assay, Mutagenesis, Serial Dilution, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a In vivo pull-down analysis to detect the interactions of Hcp1, Hcp2, Hcp3 and Bte1 in NCTC9343, hcp3 deletion strain or hcp2 deletion strain. Whole cell lysates were used as input for in vivo pull-down. The 6 × His-tagged Bte1 was enriched using Ni-affinity resin. The coprecipitated proteins (output) were detected by western immunoblots with antibodies specific to the indicated proteins. b In vitro pull-down analysis to detect the interactions of Hcp2, Hcp3 and Bte1. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. c In vitro pull-down analysis to detect the interactions of Hcp1 and Bte1. The strep-tagged Hcp1 was enriched using Strep-affinity resin. No interaction between Hcp1 and Bte1 was detected. d Top view 2D class averages of the in vitro purified Hcp2-Hcp3 heterohexamers obtained from cryoSPARC. The unique α-helix in Hcp2 (red dashed box) was used to determine subunit stoichiometry. Both 3:3 and 4:2 (Hcp2:Hcp3) assemblies were observed in vitro. e Sequence alignment of Hcp2 NCTC9343 and Hcp3 NCTC9343 . The key amino-acid residues used to distinguish Hcp2 and Hcp3 in the cryo-EM map are indicated by arrows. f Close-up of cryo-EM densities identifying key side chains distinguishing Hcp2 (Tyr55, Ile106) and Hcp3 (Ser53, Arg103), thereby confirming the presence of a 4:2 Hcp2-Hcp3 stoichiometry in the in vitro assembled heterohexamers. g Cryo-EM map of the Hcp2 4 -Hcp3 2 heterohexamer (top and side views), with Hcp2 shown in dark sea green and Hcp3 in violet. h Cartoon representation of the Hcp2 4 -Hcp3 2 heterohexamer, shown with C2 symmetry. Individual Hcp subunits and their corresponding chain IDs are labeled. i – k Molecular details of the three distinct subunit interfaces between Hcp2 and Hcp3 (red, blue, yellow boxes in h ), stabilized by hydrophobic and main-chain polar interactions. Interacting residues are shown in stick, and polar interactions are indicated by black dashed lines. For ( a – c ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: In Vivo, Western Blot, In Vitro, SDS Page, Staining, Purification, Sequencing, Cryo-EM Sample Prep, Labeling

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Top (left) and side (right) views of the cryo-EM map of the Bte1-Hcp2 4 -Hcp3 2 complex. Proteins are color-coded: Bte1 (steel blue and cyan), Hcp2 (dark sea green), Hcp3 (violet). b Ribbon model of the Bte1-Hcp2 4 -Hcp3 2 ternary complex. The Hcp ring was modeled using the apo Hcp2 4 -Hcp3 2 structure to resolve low-density regions. Bte1 was built using its resolved N-terminal region and a ModelAngelo-built C-terminal helical domain. The composite Bte1-Hcp2-Hcp3 model was generated for illustrative purposes. c Crystal structure of Bte1 (gold) fitted into the Bte1-Hcp2 4 -Hcp3 2 cryo-EM density (gray). In this conformation, helix α3 sterically clashes with the Hcp2-Hcp3 complex, whereas helices α5 and α6 are positioned at the ring-ring stacking interface of the Hcp tube, thereby potentially interfering with tube assembly. d Structural comparison of crystal structure of Bte1 (gold) versus cryo-EM-resolved Bte1 (steel blue) and ModelAngelo-bulit Bte1 C-terminal helical domain (Cyan) reveals conformational rearrangements in α3, α5, and α6. e Top views 2D class averages of the two stoichiometries of the in vitro purified Bte1-Hcp2-Hcp3 complex obtained from cryoSPARC. Bte1 density and the unique α-helix in Hcp2 are boxed in blue and red, respectively. f The N-terminal region of Bte1 inserts into the central channel of the Hcp2-Hcp3 complex and interacts with a pair of adjacent Hcp2-Hcp3 subunits. Two interaction regions are highlighted by yellow and red boxes, respectively. g Electrostatic surface analysis of the Hcp2-Hcp3 and Bte1 interaction interface. The molecular surfaces are colored by electrostatic potential (red, negative; blue, positive). Key regions of electrostatic complementarity, which are critical for the binding interaction, are highlighted with black dashed boxes. The electrostatic potential scale ranges from − 6 kT/e (red) to + 6 kT/e (blue). h Close-up views of the Bte1-Hcp2 4 -Hcp3 2 interface. Two polar interaction regions are highlighted (yellow and red dashed boxes from f ). Black dashed lines indicate polar contacts. i In vitro pull-down analysis to detect the interactions of Bte1 with Hcp2-Hcp3 complex or its indicated mutants. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Mutation of Hcp2 N30A or Hcp3 R100A within the complex abolished Bte1 binding. j In vitro co-culture assays using wild-type NCTC9343 (WT), Δ hcp2 - hcp3 , or plasmid-based hcp2-hcp3 point mutant complementation strains as killers, with the Bte1-sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was assessed via 10-fold serial dilution and plating on chloramphenicol-supplemented BHI. k Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), Δ hcp2-hcp3 , or plasmid-based hcp2-hcp3 point mutant complementation strains. DnaK serves as a cytoplasmic loading control. l Sequence alignment of Hcp2 and Hcp3 homologs from multiple Bacteroides strains indicates that critical Bte1-interacting residues (Asn30 in Hcp2 and Arg100 in Hcp3) are not conserved. For ( i – k ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: Cryo-EM Sample Prep, Generated, Comparison, In Vitro, Purification, Binding Assay, SDS Page, Staining, Mutagenesis, Co-Culture Assay, Plasmid Preparation, Serial Dilution, Western Blot, Expressing, Control, Sequencing

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Maximum-likelihood phylogenetic analysis of Hcp2-Hcp3 fused proteins from 38 B. fragilis strains, revealing 4 distinct subtypes. Strains encoding the three V1 effectors characterized in this study are highlighted in red. V1/V2-associated effectors are indicated next to each strain. Hcp2 and Hcp3 are tightly linked to V1, but not V2, effectors. Identified effectors are labeled by name; uncharacterized ones are noted as putative. Gradient bars reflect sequence identity to B. fragilis NCTC9343 Hcp2 and Hcp3. Bootstrap values (> 70%) are shown at nodes. b In vitro co-culture assays using NCTC9343 (WT), Δ tssC , Δ hcp2-hcp3 , or indicated plasmid-based complementation strains (Δ hcp23::phcp23 NCTC9343 , Δ hcp23::phcp23 GS086 and Δ hcp23::phcp23 HMW616 ) as killers, with Bte1-sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was quantified by 10-fold dilution plating on chloramphenicol-supplemented BHI. c Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), its isogenic deletion mutants (Δ tssC or Δ hcp2 - hcp3 ) and the indicated plasmid-based complementary strains (Δ hcp23::phcp23 NCTC9343 , Δ hcp23::phcp23 GS086 or Δ hcp23::phcp23 HMW616 ). d In vitro pull-down analysis to detect the interactions between Bte1 or Bfe1 and the Hcp2-Hcp3 complex from either NCTC9343 or GS086. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Each Hcp2-Hcp3 complex selectively bound a particular V1 region effector. e , f In vitro pull-down analysis to detect the interaction of Bte1 ( e ) or Bfe1 ( f ) with different Hcp2-Hcp3 combinations: Hcp2 NCTC9343 -Hcp3 NCTC9343 , Hcp2 GS086 -Hcp3 GS086 , Hcp2 NCTC9343 -Hcp3 GS086 , and Hcp2 GS086 -Hcp3 NCTC9343 . The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Complexes containing Hcp3 NCTC9343 bound Bte1 but not Bfe1. In contrast, those containing Hcp3 GS086 bound Bfe1 but not Bte1. g Surface representation of the Bte1-bound Hcp2-Hcp3 complex colored by sequence conservation. The surface is colored by sequence identity from cyan (0% variable) to pink (100% conserved). The global sequence identity values derived from individual Hcp2 and Hcp3 homologs across different strains are indicated. For ( b – f ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: Labeling, Sequencing, In Vitro, Co-Culture Assay, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, SDS Page, Staining, Derivative Assay