Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Aldosterone suppressed mitochondrial DNA and protein in a dose-dependent manner. ( A ) The effects of different dosages of aldosterone on H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (10 −10 , 10 −9 , 10 −8 and 10 −7 M) and vehicle (equal volume of DMSO) for 72 h. The mitochondrial DNA copy number was quantified by qPCR. ( B ) The effects of different durations of aldosterone treatment on H9c2 cells. H9c2 cells were treated with 10 −7 M aldosterone and the mitochondrial copy number was quantified by qPCR at 0, 8, 24, 48 and 72 h. ( C ) The effects of different durations of aldosterone treatment on SOD2. H9c2 cells were treated with 10 −7 M aldosterone, and the expression of SOD2 was determined by ELISA. ( D ) The dose effect of aldosterone on mitochondrial COX IV protein. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV was stained with anti-COX IV antibodies. The fluorescence intensity of COX IV was measured using a fluorescence microscope. ( E ) H9c2 cells were treated with different concentrations of aldosterone (10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV was stained with anti-COX IV antibodies (red), and nuclear DNA was stained with DAPI (blue). The representative images were captured using a fluorescence microscope; magnification ×400. ( F ) The effects of aldosterone on mitochondria (COX IV) and cytosolic (α-Tubulin and GAPDH) protein in H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV, α–Tubulin and GAPDH were determined using Western blot analysis. # p < 0.05 and * p < 0.01, compared between the two groups indicated by the line underneath.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Microscopy, Western Blot

Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Aldosterone-associated reduction in mitochondrial DNA and protein via MR and MAPK/P38 pathway. ( A , B ) Role of MRs and glucocorticoid receptors on mitochondrial DNA and SOD2 protein. H9c2 cells were treated with 10 −7 M eplerenone (an MR antagonist), 10 −7 M mifepristone (a glucocorticoid receptor antagonist) and vehicle (equal volume of DMSO) for 1 h prior to 10 −7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. ( C , D ) Role of signaling mediators on mitochondrial DNA and SOD2. H9c2 cells were treated with 5 µg/mL SB203580 (an MAPK/p38 inhibitor), 50 µg/mL PD98059 (an MEK/ERK inhibitor), 50 µg/mL LY294002 (a PI3K/ AKT inhibitor) or vehicle (equal volume of DMSO) for 1 h prior to 10 −7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. * p < 0.01, compared between the two groups indicated by the line underneath.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Schematic of the signaling of aldosterone-induced cardiac mitochondrial dysfunction in H9c2 cells. Aldosterone-induced cardiac mitochondrial dysfunction through MR/MAPK/p38 and ROS pathways. Mitochondrial DNA, SOD2, COX IV protein and ATP production were suppressed.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: Materials Today Bio

Article Title: Interferon-stimulated gene 15 small interfering RNA-loaded polarized mesoporous silica nanocarriers remodel the immune microenvironment to ameliorate post-myocardial infarction heart failure

doi: 10.1016/j.mtbio.2025.102631

Figure Lengend Snippet: pMSNC@ISG15 siRNA alleviates cardiomyocyte injury. Note: (A) Schematic diagram of the experimental workflow for simulating the immune microenvironment of Post-MI HF via PRCM and BMDM co-culture; (B–C) Western blot analysis of ISG15 protein expression in a CoCl 2 -induced hypoxia model; (D) RT-qPCR analysis of ISG15 mRNA levels in the same model; (E) ELISA quantification of pro-inflammatory cytokines (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10, TGF-β) in co-culture supernatants; (F) Assessment of CK and CK-MB release; (G) Measurement of LDH release; (H) Detection of cTnI levels; (I) CCK-8 assay evaluating cell viability. All cell-based experiments were performed in triplicate. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicate statistical significance between groups.

Article Snippet: Inflammatory cytokines (IL-10, TGF-β, TNF-α, and IL-1β) were quantified using ELISA kits from R&D Systems (catalog numbers R1000, DB100C, RTA00-1, and RLB00-1, respectively).

Techniques: Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, CCK-8 Assay

Journal: Materials Today Bio

Article Title: Interferon-stimulated gene 15 small interfering RNA-loaded polarized mesoporous silica nanocarriers remodel the immune microenvironment to ameliorate post-myocardial infarction heart failure

doi: 10.1016/j.mtbio.2025.102631

Figure Lengend Snippet: Modulation of macrophage phenotype by pMSNC@ISG15 siRNA. Note: (A) Live/Dead fluorescence staining showing the viability of PRCMs and BMDMs treated with pMSNC@ISG15 siRNA at various time points (bar = 25, 50 μm); (B) Experimental workflow illustrating the regulatory effect of ISG15 siRNA-loaded pMSNCs on M2 macrophage activation; (C–D) Flow cytometry analysis of pro-inflammatory (M1) and anti-inflammatory (M2) macrophage subsets; (E–F) IF staining of macrophage markers iNOS and Arg-1 (bar = 12, 25 μm); (G–J) ELISA quantification of anti-inflammatory cytokines (IL-10, TGF-β) and pro-inflammatory cytokines (TNF-α, IL-1β). All cell-based experiments were performed in triplicate. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicates statistical significance between groups.

Article Snippet: Inflammatory cytokines (IL-10, TGF-β, TNF-α, and IL-1β) were quantified using ELISA kits from R&D Systems (catalog numbers R1000, DB100C, RTA00-1, and RLB00-1, respectively).

Techniques: Fluorescence, Staining, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Materials Today Bio

Article Title: Interferon-stimulated gene 15 small interfering RNA-loaded polarized mesoporous silica nanocarriers remodel the immune microenvironment to ameliorate post-myocardial infarction heart failure

doi: 10.1016/j.mtbio.2025.102631

Figure Lengend Snippet: Therapeutic effect of pMSNC@ISG15 siRNA in preventing Post-MI HF. Note: (A) Schematic diagram illustrating the experimental procedure, in which drug-loaded functionalized silica nanoparticles deliver ISG15 siRNA to suppress immune cell activation in the cardiac microenvironment and reverse post-MI HF; (B–D) Echocardiographic evaluation of cardiac function across different treatment groups; (E) ECG analysis of cardiac function in each group; (F) ELISA quantification of anti-inflammatory cytokines (IL-10, TGF-β) and pro-inflammatory cytokines (TNF-α, IL-1β) in treated rats; (G) Measurement of LDH release to assess myocardial injury across groups; (H) Detection of cTnI levels as a marker of myocardial damage; (I) DHE fluorescent probe assay to measure ROS levels in myocardial tissue from each treatment group. Each group included 10 animals. ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicate statistical significance between groups.

Article Snippet: Inflammatory cytokines (IL-10, TGF-β, TNF-α, and IL-1β) were quantified using ELISA kits from R&D Systems (catalog numbers R1000, DB100C, RTA00-1, and RLB00-1, respectively).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Marker

Journal: bioRxiv

Article Title: Targeting the Alk4 pathway protects against age-related bone loss

doi: 10.1101/2025.10.24.684408

Figure Lengend Snippet: Primary bone marrow stromal cells (BMSCs) cultures were treated with sera samples extracted from 3-, 6-, and 12-month-old female C57BL/6J mice. Proteins from BMSCs were extracted to perform western immunoblotting and densitometry analysis ( A ) of pSmad2, Smad2/3, and Actin. ( B ) The same experiment was performed with sera treated with anti-TGFβ antibody (Ab). Circulating levels of Activin A ( C ) and TGFβ1 ( D ) measured by ELISA in sera samples from 3-, 6-, and 12-month-old female C57BL/6J mice. Statistical analyses include 1-way ANOVA (A,C) and 2-way ANOVA (B) with multiple comparisons, p-values shown when < 0.05.

Article Snippet: Serum Activin A and TGFβ1 levels were determined by Quantikine ELISA Kits (R&D Systems, Activin A #DAC00B, TGFβ1 #DB100C).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Targeting the Alk4 pathway protects against age-related bone loss

doi: 10.1101/2025.10.24.684408

Figure Lengend Snippet: Western immunoblots and densitometry analysis ( A ) performed on protein lysates extracted from the marrow compartment of 3-month-old Alk4 fl/fl and Alk4 cKO female mice. Histomorphometric measurements were performed in the proximal tibiae of 3-month-old Alk4 fl/fl and Alk4 cKO female mice to measure osteoblast number ( B ), osteoblast coverage ( C ), erosion surface ( D ), osteoclast coverage ( E ), Osteocyte coverage ( F ), mineral apposition rate ( G ), and bone formation rate ( H ). Abbreviations: Osteoblasts (Ob.), Bone perimeter (B. Pm), Bone surface (BS), osteoclast (Oc.), osteocyte (Ot.), Mineral apposition rate (MAR), Bone formation rate (BFR). Serum levels of CTX ( I ) and P1NP ( J ) measured by ELISA in 3-month-old Alk4 fl/fl and Alk4 cKO female mice. Statistical analyses include unpaired Student t -test, p-values shown when < 0.05.

Article Snippet: Serum Activin A and TGFβ1 levels were determined by Quantikine ELISA Kits (R&D Systems, Activin A #DAC00B, TGFβ1 #DB100C).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Targeting the Alk4 pathway protects against age-related bone loss

doi: 10.1101/2025.10.24.684408

Figure Lengend Snippet: 24-month-old C57BL6J male mice were injected with 5mg/kg body weight of Alk4-Fc or vehicle (PBS). Weekly DXA measurements (as % change from baseline) for total body weight ( A ), fat mass ( B ), lean mass ( C ), and BMD ( D ) over the course of the study. α denotes p-value < 0.05 when running Student t -test on BMD endpoint measurements. Endpoint muscle weights for gastrocnemius (gastroc, E ), quadriceps (quad, F ), and triceps ( G ). Endpoint microCT analyses were performed in distal femurs to measure trabecular bone volume (bone volume /total volume: BV/TV, H ) and trabecular BMD ( I ). Cortical area fraction (cortical area / total area: CA/TA, J ) was measured at the midshaft diaphysis. For vertebral (Vt.) bone, analysis was performed in the L5 vertebral body to measure trabecular bone volume ( K ) and trabecular BMD ( L ). Circulating levels of Activin A ( M ) were measured by ELISA in sera samples at the end of the study. Statistical analyses include unpaired Student t -test, p-values shown when < 0.05.

Article Snippet: Serum Activin A and TGFβ1 levels were determined by Quantikine ELISA Kits (R&D Systems, Activin A #DAC00B, TGFβ1 #DB100C).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Developing single molecule methods for measuring the pathway proteins ERK, AKT, cyclin d and p70s6k in localized colon cancer in relation to mutation status

doi: 10.1101/695809

Figure Lengend Snippet: The average number of enzyme per bead (AEB) against concentration is shown. The calibrators (n=8) were run in duplicate and the mean value for each point of the calibrators is shown.

Article Snippet: The biotinylated detector antibodies and the calibrators were tAKT (DYC1775), pAKT (DYC887B), tERK (DYC1230C), pERK (DYC1018B) (R&D Systems), pp70s6k (DYC896) and cyclin d (ab218793) (abcam).

Techniques: Concentration Assay