Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin inhibits cell proliferation and survival of blastic plasmacytoid dendritic cell neoplasm (BPDCN) cell lines CAL-1 and GEN2.2 BPDCN cells were treated with increasing concentrations of metformin (0–50 mM) for 24 or 48 h. Cell proliferation (A–C) and cell death (A and D) were analyzed using the IncuCyte S3® live-cell analyzer. (A) CAL-1 cells 24 h after treatment were shown (10 X image magnification) with upper images (phase) representing phase-contrast imaging while lower images (Cytotox Red) show fluorescence imaging after staining with cytotox red dye, which allows to count dead cells (labeled in red). Scale bars, 200 μm. (B and C) Metformin inhibits proliferation of CAL-1 and GEN2.2 cells in a concentration-dependent manner. (D) Metformin increases cell death of CAL-1 cells in a concentration-dependent manner. Data are expressed as the mean ± SEM of 8 independent wells per condition. (E) Viable cells were defined by flow cytometry as Annexin-V – /7-AAD – CAL-1 (black) or GEN2.2 (gray) cell lines after treatment with metformin (0–50 mM) for 24 h. Untreated CAL-1 and GEN2.2 cells were arbitrarily assigned to a 100%. Histograms represent the mean ± SEM of three independent experiments. p value is calculated using one-way ANOVA (more than two groups). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: Imaging, Fluorescence, Staining, Labeling, Concentration Assay, Flow Cytometry

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin induces apoptosis in blastic plasmacytoid dendritic cell neoplasm cell line CAL-1 cells were treated with increasing concentration of metformin (0–50 mM) in the presence of IncuCyte® caspase-3/7 Dye for 48 h and were imaged at 10 X magnification in the IncuCyte S3® live-cell analyzer every 2 h. (A) Representative image of caspase-3/7 green fluorescence emitted by CAL-1 cells 24 h after treatment. Scale bars, 200 μm. (B) Histogram represents the mean ± SEM of caspase-3/7 green fluorescent positive CAL-1 cells per well (8 independent wells per condition) and statistically significant differences are indicated. (C) CAL-1 cells were treated, 6 h with increasing concentration of metformin (0–50 mM), and expression of caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were evaluated by western blot analysis and semi-quantified. One representative experiment of two is shown. The expression of proteins was normalized with actin and non-treated condition. Actin was used as control for protein expression. p value is calculated using one-way ANOVA (more than two groups). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: Concentration Assay, Fluorescence, Expressing, Western Blot, Control

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin activates AMPK signaling pathway in blastic plasmacytoid dendritic cell neoplasm cell line CAL-1 (A) Expression levels of AMPK and phosphorylated AMPK ( p -AMPK) in CAL-1 cell line were analyzed by western blotting 6 h after treatment with increasing concentrations of metformin (0–10 mM). One representative experiment out of three is shown. (B) Expression was semi-quantified in CAL-1 cells and normalized to AMPK and, relative to the untreated condition. Actin was used as a loading control. Data are presented as mean ± SEM of the p -AMPK to AMPK ratio from three independent experiments. (C) CAL-1 cells were pretreated or not with 1 μM of AMPK inhibitor CC for 2 h, and then treated or not with 10 mM metformin for 6 h. (D) Cell viability of CAL-1 cells was assessed 24 h after treatment with 20 mM metformin in the presence or absence of 1 μM of AMPK inhibitor CC. Histogram represents the mean ± SEM of three independent experiments. The vehicle corresponds to DMSO used for CC reconstitution. Met, metformin; CC, compound C; ns, not significant. p value is calculated using one-way ANOVA (more than two groups). ∗ p < 0.05.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin inhibits Akt/mTOR signaling pathway in blastic plasmacytoid dendritic cell neoplasm cell lines and patient-derived xenograft cells Three BPDCN cell models: CAL-1 ( n = 3) and GEN2.2 ( n = 3) cell lines as well as three patient-derived xenograft cells (PDX: P224B-M2 [ n = 1], P178M-M1 [ n = 1] and P193B-M1 [ n = 1]) were treated 6 h with or without 5 mM metformin. (A) and (C) one representative experiment out of 3 showing intracellular expression of p-Akt and p-mTOR evaluated by confocal microscopy in CAL-1 and GEN2.2 cells ( n = 3). Scale bars, 10 μm. (B) and (D) Protein expression levels in the CAL-1 and GEN2.2 cell lines and PDX cells were quantified with Zen Blue software. Histograms represent the mean ± SEM and statistically significant differences are indicated. p value is calculated using the Mann-Whitney U test (two groups). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001. Met: metformin. PDX: patient-derived xenografts.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: Derivative Assay, Expressing, Confocal Microscopy, Software, MANN-WHITNEY

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin inhibits the NF-κB p65, STAT3, and STAT5 signaling pathway in blastic plasmacytoid dendritic cell neoplasm cell lines and patient-derived xenografts The intracellular expression levels of p-NF-κB p65, p-STAT3, and p-STAT5 were assessed using confocal microscopy in CAL-1 and GEN2.2 BPDCN cell lines and three different PDX cells incubated with or without 5 mM of metformin for 24 h. (A) A representative experiment out of three showing nucleus expression of p-NF-κB p65 in CAL-1 and GEN2.2 cells. CAL-1 cells were stimulated with TLR7 (using R848, 1 μg/mL) for 6 h to increase NF-κB activation. Scale bars, 10 μm. (B) Histograms represent p-NF-kB p65 expression in CAL-1 and GEN2.2 cell lines as well as in three different PDX cells (P224B-M2 [ n = 1], P178M-M1 [ n = 1] and P193B-M1 [ n = 1]), represented as mean ± SEM. GEN2.2 and PDX cells were incubated with or without 5 mM metformin for 6 h without TLR7 stimulation to assess the effect on basal p-NF-κB expression. (C) and (E) CAL-1 cells were treated or without 5 mM metformin for 24 h followed by stimulation with 10 ng/mL IL-3 for 30 min. A representative experiment out of three showing intracellular expression of p-STAT5 (C) and p-STAT3 (E) in CAL-1 cells. (D) Cumulative data from three independent experiments quantifying p-STAT5 fluorescent intensity in CAL-1 cells and data from one experiment quantifying p-STAT5 fluorescent intensity in one PDX cell (P193B-M1) are shown as mean ± SEM. (F) p-STAT3 expression levels in CAL-1 cells ( n = 3) and one PDX cell (P193B-M1, n = 1) were quantified, and cumulative data from three independent experiments in CAL-1 and from one experiment in PDX cells (P193B-M1) are represented as mean ± SEM. PDX cells were incubated with or without 5 mM metformin for 6 h in the absence of IL-3 stimulation. p value is calculated using the Mann-Whitney U test (two groups). ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Met: metformin. PDX: patient derived xenografts.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: Derivative Assay, Expressing, Confocal Microscopy, Incubation, Activation Assay, MANN-WHITNEY

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: Metformin inhibits mitochondrial respiration in BPDCN cells CAL-1 cells were treated with metformin (0–5 mM) 24 h then the Cell Mito stress test kit was used to measure different parameters of mitochondrial respiration ( n = 12 wells/group). (A) and (B) oxygen consumption rate (OCR) profile, (C) basal respiration, (D) ATP mitochondrial production, (E) and (F) the extracellular acidification rate (ECAR) and (G) spare respiration capacity (which consists in the difference between max respiration and basal respiration, see panel (A) were measured using the Seahorse XF96 Analyzer. B, C, and D correspond to t = 0 in A, and F corresponds to t = 0 in E, i . e ., 24 h after metformin treatment. The sequential addition of oligomycin, FCCP, and a mix of rotenone/antimycin A, was performed at the indicated time points, as described in the Material and Methods section. (A) and (E) represent the whole kinetics of a representative experiment for OCR and ECAR, respectively. Histograms represent the mean ± SEM for 12 wells per condition. p value is calculated using one-way ANOVA (more than two groups). ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques:

Journal: iScience

Article Title: Metformin dual-targets metabolism and survival pathways in BPDCN

doi: 10.1016/j.isci.2026.116323

Figure Lengend Snippet: In vivo metformin tends to limit tumoral progression by BPDCN, to extend mouse survival as well as to limit spleen infiltration by BPDCN and to inhibit NF-κB p65, and STAT5 signaling pathways (A) A diagram describing the in vivo study, NSG mice were irradiated (2.5 Gy) and 18 h later, inoculated intravenously with 0.5 × 10 6 Luciferase-expressing CAL-1 cells (LUC + CAL-1). Treatment was started on day 3 (d3) after the LUC + CAL-1 cell inoculation with metformin (100 mg/kg/day intraperitoneally) given for 5 days/week for 4 weeks ( n = 5 mice). Mice injected with phosphate-buffered saline (PBS) over the 4 weeks were used as the control ( n = 5 mice). (B) The potential toxic side effects of metformin in NSG mice were assessed by monitoring changes in body weight and mouse fur texture every two days. The mice weight and mouse fur texture graphs receiving the indicted treatment are represented. The viability of mouse cells (spleen and lungs, white bars) and CAL-1 line (yellow bars) was assessed with fixable viability Dye eFluor solution using flow cytometry. CAL-1 cells were identified using human CD123 expression, while mouse cells were negative. Of note, CAL-1 cells do not infiltrate mouse lungs. (C) In vivo kinetics of tumor cell growth following the LUC + CAL-1 cells, treated with metformin (Red) or with PBS (Black). Histogram represents the mean ± SEM for (D) luminescence of tumor-bearing mice from day 3 to day 21. (E) Kaplan Meier survival curves of mice receiving the indicted treatment. Overall survival of BPDN-inoculated mice treated with metformin (red) or with PBS (black) is shown. At the end of the in vivo experiments, spleens were collected from the two surviving control mice and the four surviving treated mice and were assessed for LUC + CAL-1 cell quantification and phosphorylated NF-κB p65, and STAT5 expression. Cells were stained with human CD123, as well as intracellular p-NF-κB, and p-STAT5 prior to analysis by flow cytometry. The percentage of (F) CD123, (G) p-STAT5, and (H) p-NF-κB positive cells isolated from spleens of metformin-treated mice and PBS-treated mice, was quantified. Histograms represent the mean ± SEM for one experiment. ns: no significant difference.

Article Snippet: Metformin hydrochloride for in vivo assay (Medchem express (MCE), Stockholm Sweden).

Techniques: In Vivo, Protein-Protein interactions, Irradiation, Luciferase, Expressing, Injection, Saline, Control, Flow Cytometry, Staining, Isolation

Journal: Journal of Orthopaedic Translation

Article Title: Semaglutide alleviates osteoarthritis independent of weight loss via GLP-1R–mediated activation of autophagy through AKT/mTOR inhibition

doi: 10.1016/j.jot.2026.101166

Figure Lengend Snippet: Establishment of zebrafish cartilage injury and mouse OA models and therapeutic evaluation of antidiabetic drugs, including semaglutide. A. The diagrams show the Tg(col2a1a:dendra2-NTR) transgenic line and its labeling of mandibular cartilages, including Meckel's cartilage (M), palatoquadrate cartilage (Pq), and ceratohyal cartilage (Ch). B. The flowchart illustrates the process of cartilage injury and drug treatment in zebrafish: at 3.5 days post-fertilization (dpf), zebrafish larvae were exposed to 12 mM metronidazole (MTZ) for 20 h, followed by drug intervention, with phenotypic analysis performed 3 days later. C. Representative gross morphology of zebrafish in the MTZ/NTR-induced cartilage injury repair model. D. Confocal microscopy analysis showing the repair effects of different antidiabetic drugs on zebrafish cartilage injury (n = 10). The antidiabetic agents included metformin (Met), empagliflozin (Emp), rosiglitazone (Ros), gliclazide (Gli), and semaglutide (Se). Working concentrations were selected based on published doses and further refined in pilot assays to identify the highest effective concentration that did not cause overt toxicity or teratogenic effects. E. Experimental design of Se administration in wild-type (WT) mice with DMM-induced osteoarthritis (OA). F. Body weight changes in WT mice during treatment with low- or high-dose Se following destabilization of the medial meniscus (DMM) surgery (n = 6). G. Mechanical allodynia in each group of WT mice was assessed using the von Frey test during Se treatment after DMM surgery (n = 6). H. Effects of Se on pain-related behavior in OA mice, along with representative gait footprints from each group. I. Quantitative gait analysis based on H-track parameters, expressed as the ratio of right hindlimb to left hindlimb (RH/LH) (n = 6). J. Representative micro-computed tomography (micro-CT) images of knee joints from each group, including sagittal, coronal, and transverse reconstructions, illustrate overall subchondral bone alterations following Se treatment. K. Quantitative analysis of subchondral bone structural parameters derived from micro-CT images in (J), including subchondral bone plate (SBP) thickness, bone volume fraction (BV/TV), and trabecular thickness (Tb.Th) (n = 6). Scale bar represents 100 μm. Data are presented as the means ± SD. ns. Non-significant. ∗P value < 0.05. ∗∗P value < 0.01. ∗∗∗P value < 0.001.

Article Snippet: All antidiabetic agents, including metformin (Met) (5 μM, HY-B0627), empagliflozin (Emp) (1 μM, HY-15409), rosiglitazone (Ros) (1 μM, HY-17386), gliclazide (Gli) (5 μM, HY-B0753), and semaglutide (Se) (0.5 μM, HY-114118), were purchased from MedChemExpress (MCE, USA).

Techniques: Transgenic Assay, Labeling, Confocal Microscopy, Concentration Assay, Micro-CT, Derivative Assay