Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; primary, pooled donors; ATCC) were purchased through an authorized distributor in China and originally sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Migration, Activity Assay, Software

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Proposed model of HSPB1-mediated redox regulation of TGF-β1 maturation during post-MI fibrosis. During myocardial fibrosis following myocardial infarction, the expression of HSPB1 is markedly upregulated in the peri-infarct region. Upon activation, HSPB1 exposes its reactive cysteine residue (Cys137), which may interact with critical cysteine sites within pre-pro-TGF-β1, thereby influencing its redox-dependent folding and disulfide bond formation. This interaction potentially interferes with the maturation and secretion of active TGF-β1 into the extracellular space. Reduced secretion of mature TGF-β1 limits Smad2/3 phosphorylation and endothelial-to-mesenchymal transition, ultimately alleviating myocardial fibrosis. The red dashed box highlights the hypothesized redox regulatory interaction between HSPB1 and pre-pro-TGF-β1, which requires further biochemical validation.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; primary, pooled donors; ATCC) were purchased through an authorized distributor in China and originally sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Activation Assay, Residue, Phospho-proteomics, Biomarker Discovery

Journal: Frontiers in Oncology

Article Title: Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

doi: 10.3389/fonc.2021.711207

Figure Lengend Snippet: IGF2BP3 knockdown hampered hypoxia-induced cell migration and angiogenesis in SC. (A) MKN-45 and HGC-27 cells were cultured in normoxic or hypoxic conditions for 24 h Next, the IGF2BP3 protein level was measured by western blot assay. (B) MKN-45 and HGC-27 cells were transfected with si-NC, si-IGF2BP3#1, si-IGF2BP3#2, or si-IGF2BP3#3. Next, the IGF2BP3 mRNA level was measured by RT-qPCR assay at 48 h after transfection. (C–F) MKN-45 and HGC-27 cells were transfected with si-NC or si-IGF2BP3#1 for 48 h and then maintained in hypoxic conditions for another 24 h Cells in the normoxia group were maintained in normoxic conditions for 72 h Cells in the hypoxia group were cultured in normoxia for 48 h and then exposed to hypoxia for an additional 24 h (C, D) Cell migratory potential was assessed by Transwell migration (C) and wound healing (D) assays. (E) VEGF level in cell culture supernatants was detected using a commercial kit. (F) The conditioned medium of MKN-45 and HGC-27 cells were collected after normoxia/hypoxia treatment or/and transfection. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1), followed by the measurement of tube formation ability at 12 h after incubation. * indicate that the difference is significant at 0.05 level. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001 compared with the normoxia group.

Article Snippet: MKN-45 cells and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Knockdown, Migration, Cell Culture, Western Blot, Transfection, Quantitative RT-PCR, Incubation

Journal: Frontiers in Oncology

Article Title: Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

doi: 10.3389/fonc.2021.711207

Figure Lengend Snippet: IGF2BP3 exerted its functions by up-regulating HIF1A. (A) MKN-45 and HGC-27 cells were transfected with pcDNA3.1 or pcDNA-HIF1A. Next, HIF1A mRNA level was measured by RT-qPCR assay at 48 h upon transfection. (B–E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the examination of cell migratory potential (B, C) and VEGF secretion level (D) . (E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the collection of conditioned medium. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1) and tube formation potential was examined at 12 h after incubation. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001.

Article Snippet: MKN-45 cells and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Quantitative RT-PCR, Cell Culture, Incubation

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effects of α 1 -AR stimulation on the expression of mitochondrial energetic molecules, oxidative phosphorylation, and biological functions in skeletal and cardiac muscle cells and liver cells. (a, b) The expression of p-AMPK and PPAR δ in C2C12, HL1, and HepG2 cells was stimulated with 1–30 μ M midodrine for the indicated times. (c) Cytosolic calcium mobilization after midodrine treatment in C2C12 and HL1 cells. Each cell type was pretreated with the calcium reactive dye Fluo-3 AM for 45 min and then stimulated with 30 μ M midodrine for the indicated times. Green fluorescence emitted by Fluo-3 AM was detected using confocal microscopy. (d) The phosphorylation of AMPK α at Thr172 and expression of PPAR δ in C2C12 and HL1 cells after pretreatment with the calcium/calmodulin-dependent protein kinase kinase antagonist STO-609 for 25 min and treatment with midodrine. (e) Fluorescence after using the CytoPainter mitochondrial staining kit in midodrine-treated and control C2C12 cells. Original magnification was 200x. (f) The measured activity of succinate dehydrogenase (SDH) in C2C12 cells. (G) Oxygen consumption rate (OCR) in C2C12 cells treated with midodrine (30 μ M), as measured by a Seahorse XFp analyzer. (h) ATP content in C2C12 cells treated with midodrine (30 μ M) cultured with low-glucose (5.56 mM) medium. (i) Glucose transporter (GLUT) 4 protein expression in C2C12 cells treated with high glucose (HG) and midodrine (HG+Mido), HG and insulin (HG+Insulin), and the control treatment (Ctrl). (j) The uptake of 2-deoxyglucose in C2C12 skeletal muscle cells treated with midodrine. (k) OCR (measured by the Seahorse XFp analyzer) in H9C2 cells treated with midodrine (30 μ M) and cultured with low-glucose (5.56 mM) medium. (l) ATP content in H9C2 cells treated with midodrine (30 μ M). Data are expressed as the mean ± standard deviation of triplicate experiments. AMPK: adenosine monophosphate-activated protein kinase; p-AMPK: phosphorylated AMPK; PPAR δ : peroxisome proliferator-activated receptor delta; PGC-1 α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mGLUT4: GLUT4 expression of the cell membrane; tGLUT4: total cellular expression of GLUT4; Ctrl: an untreated control group; Mido: midodrine-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Phospho-proteomics, Fluorescence, Confocal Microscopy, Staining, Control, Activity Assay, Cell Culture, Standard Deviation, Membrane

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effect of midodrine on the endothelial expression of p-AMPK and p-eNOS in HUVECs; OCR analyses in H9C2 cells; intracellular fat and the expression of PPAR δ , p-AMPK, and PGC-1 α in differentiated 3T3-L1 cells; and the effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. (a) The expression of phosphorylated AMPK (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS) proteins in human umbilical vein endothelial cells (HUVECs) treated with cholesterol and palmitate, and the effects from the addition of GSK0660, a PPAR δ antagonist. Ctrl: the control group; CP: the cholesterol- and palmitate-treated group; CPM: the cholesterol-, palmitate-, and midodrine-treated group. (b) The maximal oxygen consumption rate (OCR) analysis as estimated using a Seahorse XFp analyzer and ATP content measured by ELISA in H9C2 cells. (c) The effect of compound C (1 μ M) on p-AMPK expression and PPAR δ expression. (d) The effect of midodrine on intracellular lipid deposits (Oil Red O staining result) and the protein levels of PPAR δ , AMPK, and PGC-1 α in differentiated 3T3-L1 cells treated with midodrine and GSK0660. (e) The effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. Ctrl: untreated control group; Mido: midodrine-treated group; Mido+GSK0660: midodrine- and GSK0660-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining