Journal: Biomedicines

Article Title: Synergistic Anticancer Activity of HSP70 Inhibitor and Doxorubicin in Gain-of-Function Mutated p53 Breast Cancer Cells

doi: 10.3390/biomedicines13051034

Figure Lengend Snippet: Comparison of the survival rate differences in the breast cancer cell line MDA-MB-231 after co-treatment with various misfolding p53 aggregation inhibitors and doxorubicin. ( A ) Cells were treated with the chemotherapy drug doxorubicin (DOX, 1 μM) and the following concentrations of each misfolding p53 aggregation inhibitor: HSP90 inhibitor (IPI-504, 5 μM), HSP70 inhibitor (VER-155008, 10 μM), LOX inhibitor (BAPN, 100 μM), and NAMPT inhibitor (FK886, 10 μM). After 48 h of drug treatment, cell survival rates were assessed using CCK8 reagent. DMSO only was calculated as 100% to normalize other treatment conditions. Results are presented as mean ± SD, n = 3 (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., p > 0.05). ( B ) Calculation of the drug interaction coefficient (CDI) between doxorubicin and HSP90 inhibitor (IPI-504) in MDA-MB-231 cells. According to the cell survival rate calculations from the CCK8 assay, the CDI value is 1.17, indicating that doxorubicin and IPI-504 have no synergistic cytotoxic effect on MDA-MB-231 cells. ( C ) Calculation of CDI for doxorubicin and HSP70 inhibitor (VER-155008) in MDA-MB-231 cells. Based on the cell survival rate calculations from the CCK8 assay, the CDI value is 0.61, indicating that doxorubicin and VER-155008 exhibit a significant synergistic cytotoxic effect on MDA-MB-231 cells. ( D ) Calculation of CDI between doxorubicin and the LOX inhibitor BAPN in MDA-MB-231 cells. According to the CCK8 assay cell survival rate calculations, the CDI value is 1.14, indicating that doxorubicin and BAPN had no drug synergistic cytotoxic effect on MDA-MB-231 cells. ( E ) Calculation of CDI between doxorubicin and FK886 in MDA-MB-231 cells. Based on the cell survival rate calculations from the CCK8 assay, the CDI value is 0.94, indicating that doxorubicin and FK886 had a synergistic cytotoxic effect on MDA-MB-231 cells.

Article Snippet: Cells were treated with DMSO alone, 1 μM Doxorubicin (Sigma-Aldrich, St. Louis, MO, USA), 5 μM IPI-504 (HSP90 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA), 10 μM VER-155008 (HSP70 inhibitor, MedChemExpress), 100 μM BAPN (LOX inhibitor, MedChemExpress), 5 μM FK886 (NAMPT inhibitor, MedChemExpress), or 20 μM z-VAD-FMK (pan-caspase inhibitor, MedChemExpress).

Techniques: Comparison, CCK-8 Assay

Journal: Bone Research

Article Title: The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair

doi: 10.1038/s41413-024-00320-0

Figure Lengend Snippet: The HIF-1α/PLOD2 pathway regulates cell motility and osteogenic differentiation of HO progenitor cells ex vivo. a The pyridinoline cross-link content was determined by HPLC of cell-matrix harvested 4-weeks treatment of cells with ascorbate-2-phosphate in the presence of PLOD2i. Pyridinoline cross-linking was significantly decreased in the cell/matrix of MPCs treated with PLOD2i for 4 weeks. The concentration of hydroxylysine pyridinoline (HP) cross-linking residues is expressed as moles/mole of collagen. * P < 0.05. Mann-Whitney unpaired t test, two-tailed ( n = 3). b Collagen deposited by cultured MPCs was immunostained with a collagen α1[I] C-telopeptide antibody. The collagen matrix in MPCs treated with PLOD2i appears disorganized, while vehicle (DMSO) control appears to have aligned fibrillar collagen strands ( n = 2, representative panels shown). The graph shows the quantification of anisotropy. Error bars represent mean ± SD. **** P < 0.000 1. Mann-Whitney unpaired t -test, two-tailed ( n = 20). c Representative images of alkaline phosphatase (ALP) staining. MPCs were subjected to osteogenic differentiation for 7 days in the presence of PLOD2i or vehicle (DMSO). The graph quantifies ALP-staining intensity by measuring colorimetric absorbance at 570 nm wavelength. The results are expressed as the mean ± SD. **** P < 0.000 1. Mann-Whitney unpaired t -test, two-tailed ( n = 4). d Real-time qPCR analysis of Alpl expression. MPCs isolated from a wild-type BT mouse were subjected to osteogenic differentiation for 7 days in the presence of a PLOD2 inhibitor or vehicle (DMSO). Error bars represent mean ± SD. ** P < 0.01, versus vehicle control. Mann-Whitney unpaired t -test, two-tailed ( n = 3). e Representative images of in vitro mineralization of MPCs. MPCs were subjected to osteogenic differentiation in the presence of PLOD2i or vehicle (DMSO). After 6 weeks in culture, mineralization was visualized by Alizarin Red S (ARS) staining. ARS stain was quantified by measuring colorimetric absorbance at 407 nm wavelength. The results are expressed as the mean ± SD. **** P < 0.000 1. Mann-Whitney unpaired t -test, two-tailed ( n = 8). f Real-time qPCR analysis of Hk2 expression. MPCs isolated from a wild-type BT mouse were cultured in normoxic or hypoxic conditions in the presence of a PLOD2 inhibitor. Error bars represent mean ± SD. *** P < 0.001, versus vehicle control. Mann-Whitney unpaired t -test, two-tailed ( n = 3). g Representative 3-D reconstruction μCT images of injured limbs from BAPN-treated mice and vehicle (5% sucrose)-treated mice 9 weeks post-BT. Ectopic bone formation was indicated by pseudo-coloring (orange) the bone. h Quantification of total, bone-associated, and distal HO. Error bars represent mean ± SD. ** P < 0.01; *** P < 0.001. Mann-Whitney unpaired t -test, two-tailed ( n = 10/group)

Article Snippet: For the BAPN study, drinking water for mice was prepared by adding BAPN (3-Aminopropionitrile, stabilized, AC351750250, Thermo Scientific Chemicals) to sterilized water containing 5% sucrose (S0389, Sigma).

Techniques: Ex Vivo, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Cell Culture, Control, Staining, Expressing, Isolation, In Vitro

Journal: bioRxiv

Article Title: The epithelial adherens junction component PLEKHA7 regulates ECM remodeling and cell behavior through miRNA-mediated regulation of MMP1 and LOX

doi: 10.1101/2024.05.28.596237

Figure Lengend Snippet: (A) Measurements of LOX and (B) MMP1 enzymatic activities in Caco2-WT and PLEKHA7-KO cells, and after treatment of PLEKHA7-KO cells with LOX (BAPN; 150 µM final) and MMP (Doxycycline Hyclate; 0.2 µg/mL final) inhibitors, respectively. (C) Immunostaining of collagen I and fibronectin of HFF-1-derived decellularized ECM and upon plating it with Caco2-WT, PLEKHA7-KO cells, and PLEKHA7-KO cells treated with the LOX inhibitor BAPN, as above. (D) Quantification of images from C, using the TWOMBLI macro on Fiji , from n=6 fields representative of two biological replicates; *p<0.05, one-way ANOVA. (E) Total collagen assay for the insoluble crosslinked-collagen fraction of Caco2-WT, PLEKHA7-KO cells, and PLEKHA7-KO cells treated with the LOX inhibitor BAPN, as above, from n=2 biological replicates; **p<0.01, one-way ANOVA.

Article Snippet: Cells were grown to 80% confluency in 100 mm cell culture dishes and treated with either Lysyl Oxidase (LOX) inhibitor BAPN (3-Aminopropionitrile fumarate salt; Millipore Sigma -A3134-5G, at 150 μM final concentration, or the MMP inhibitor Doxycycline Hyclate CAS 24390-14-5 (Santa Cruz-sc-204734B) at 0.2 μg/mL final concentration, for 24 hours before collecting protein or performing assays.

Techniques: Immunostaining, Derivative Assay, Collagen Assay

Journal: bioRxiv

Article Title: The epithelial adherens junction component PLEKHA7 regulates ECM remodeling and cell behavior through miRNA-mediated regulation of MMP1 and LOX

doi: 10.1101/2024.05.28.596237

Figure Lengend Snippet: (A) Caco2 with non-target shRNA (shNT) and with shRNA mediated PLEKHA7 knockdown (shPLEKHA7), were plated on Matrigel - coated coverslips using Ibidi 2-well cell inserts till full confluency. Once confluent, inserts were removed and cell growth into the “wound” area (indicated within yellow lines) was imaged every 10 hours and (B) quantified (* p=0.04, from n=2 biological replicates, Student’s t- test). (C, D) Caco2-WT, PLEKHA7-KO, and PLEKHA7-KO cells treated with the MMP inhibitor (Doxycycline hyclate: 0.2 µg/mL) or the LOX inhibitor (BAPN: 150 µm), were plated on CIM transwell plates and cell invasion was measured in real time using the xCELLigence real time cell analysis system. All wound closure and invasion experiments were stopped at <32 hours to avoid effects of proliferation. *p<0.05, from n=3 biological replicates, one-way ANOVA with Bonferroni correction.

Article Snippet: Cells were grown to 80% confluency in 100 mm cell culture dishes and treated with either Lysyl Oxidase (LOX) inhibitor BAPN (3-Aminopropionitrile fumarate salt; Millipore Sigma -A3134-5G, at 150 μM final concentration, or the MMP inhibitor Doxycycline Hyclate CAS 24390-14-5 (Santa Cruz-sc-204734B) at 0.2 μg/mL final concentration, for 24 hours before collecting protein or performing assays.

Techniques: shRNA, Knockdown, Cell Analysis

Journal: eBioMedicine

Article Title: Patient-derived microphysiological model identifies the therapeutic potential of metformin for thoracic aortic aneurysm

doi: 10.1016/j.ebiom.2022.104080

Figure Lengend Snippet: In vivo validation of metformin's effect on TAA development. (a) Young wild type C57BL/6J mice were treated with saline only, BAPN + saline and BAPN + metformin for 4 weeks. Hemothorax, indicative of aortic dissection or rupture, presented in more than half of the mice treated with BAPN + saline and only in 1 mouse treated with BAPN + metformin. (b) Survival analysis of the 3 groups (N=29; log-rank test, P=0.108). (c) Aortic dilation is evident across all sections in BAPN-treated mice, and metformin significantly delayed dilation of the aortic arch (n=5–12 including 5–12 biological replicates; ANOVA). (d) Hematoxylin and eosin (H&E), Verhoeff's Van Gieson, and Alcain blue staining of the mouse aortic tissues showed that metformin reduced the excessive inflammatory cell invasion and elastin breakage (n=5–9 including 4 biological replicates; ANOVA). Scale bar: 100 μm. (e) Metformin partially recovered the NAD + pool that was depleted by BAPN treatment (n=6 including 3 biological replicates; ANOVA). (f) qRT-PCR analysis suggested elevated expressions of Acta2, Eln and multiple mitochondrial genes after metformin treatment (n=3 including 3 biological replicates; ANOVA). (g) Flowchart illustrating the screening of patients. (h, i) Comparisons of the raw and body surface area-indexed ascending aortic diameters showed that the patients who received metformin for glucose control had smaller aortic sizes than the diabetic patients without metformin and the non-diabetic patients (N=274). (j, k) Univariate and multivariate linear regression models demonstrated that the use of metformin was an independent factor associated with lower raw and indexed ascending aortic diameters (marked green). Age, male sex, hypertension, and aortic regurgitation were also significant risk factors for ascending aortic diameter. Values are presented in means ± standard deviations for all panels. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After 4 weeks of treatment, TAA was identified in 10 (83.3%) mice treated with BAPN + saline, and in 7 (58.3%) mice treated with BAPN + metformin (chi-square test, P=0.371).

Techniques: In Vivo, Biomarker Discovery, Saline, Dissection, Staining, Quantitative RT-PCR, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: RBPJ is upregulated in aortic macrophages in TAD. ( A ) Re-analysis of scRNA-seq data, showing the expression of Notch pathway-related genes in myeloid cells in aortic tissues and corresponding PBMCs from human TAD patients (CRA003013). ( B ) Representative immunofluorescence images stained with CD68 (red) and RBPJ (green) in aortic sections of patients with TAD and control individuals. The images represent 3 control and 4 TAD samples with similar results. ( C ) The expression of Notch pathway-related genes in aortic macrophages from the mouse TAD model as determined by scRNA-seq (CRA003013). ( D ) Immunofluorescence staining for macrophages (F4/80, red) and RBPJ (green) in aortic sections from mice treated with BAPN, with H 2 O-treated mice as a control. The number of F4/80 + RBPJ + macrophages (MACs) was quantified and compared ( n = 6). ( E ) Mice were treated with 0.25% BAPN or H 2 O. Aortic sections were stained by immunofluorescence for macrophages (F4/80, red) and NICD (cleaved Notch1) (green). The number of F4/80 + macrophages with or without NICD signal was quantified and compared ( n = 6). Student’s t -test was performed for statistical analysis. Data are expressed as mean ± SEM, *** P < 0.001

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: Myeloid-specific Rbpj knockout attenuates TAD progression. ( A , B ) Rbpj cKO and control mice were treated with BAPN for 28 days and the survival of mice was evaluated (A). The aortic size was monitored by ultrasonic imaging (B), and the maximum diameter of the thoracic aorta was quantitatively compared ( n = 7). ( C ) Aortic samples were collected and photographed. Arrows indicate the TAD pathologic sites. ( D , E ) H&E staining (D) and EVG staining (E) of aortic samples from the control and Rbpj cKO mice treated with BAPN. In ( E ), the integrity of elastic fiber was evaluated with the elastin score and quantitatively compared ( n = 7). ( F ) Immunofluorescence of aortic samples from the control and Rbpj cKO mice treated with BAPN. Samples were stained for α-SMA and F4/80, and thick and thin arrows indicate vSMCs and macrophages, respectively. The number of vSMCs was quantified ( n = 7). Student’s t -test was performed for statistical analysis in (B, E, F), and Kaplan-Meier method was used to estimate survival functions and log-rank test (Mantel-Cox) was used in (A). Data are expressed as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Knock-Out, Control, Imaging, Staining, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: Myeloid-specific Rbpj ablation reduces M1-like while increases M2-like macrophages in the aorta of TAD mice. ( A-C ) Rbpj cKO and control mice were treated with BAPN for 28 days. Aorta samples were collected and subjected to immunofluorescence staining for NOS2 and F4/80 ( A ), CD206 and F4/80 ( B ), and MMP2 and F4/80 ( C ). The M1-like (F4/80 + NOS2 + ) ( A ), M2-like (F4/80 + CD206 + ) ( B ), and MMP2 + ( C ) macrophages were quantitatively compared ( n = 7). ( D ) Aortic macrophages from Rbpj cKO and control mice fed with BAPN were analyzed by flow cytometry. The M1-like (CD86 hi CD206 low ) and M2-like (CD86 low CD206 hi ) macrophages (CD11b + F4/80 + ) were quantitatively compared ( n = 7). Student’s t -test was performed for statistical analysis. Data are expressed as mean ± SEM, ** P < 0.01, *** P < 0.001

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Control, Immunofluorescence, Staining, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: RBPJ deficiency attenuates M1-like macrophage polarization and glycolysis. ( A-C ) BMDMs were cultured statically or subjected to cyclic stretch with 10% strain at 1 Hz for 24 h, and analyzed by RNA-seq. Differentially expressed genes were analyzed by a heatmap showing inflammation-related genes ( A ), KEGG ( B ) and GO ( C ) enrichment, and top 20 enrichments are shown. ( D ) GSEA with metabolism-related gene sets including the HIF1α pathway, glycolysis, and TCA cycle. ( E , F ) BMDMs were prepared from the Rbpj cKO and control mice, and subjected to cyclic stretch for 24 h. The mRNA level of glycolysis-related molecules was determined by qRT-PCR ( n = 4) ( E ). The protein level of GLUT1, HIF1α and PKM2 was determined by immunoblotting ( n = 4) ( F ). ( G ) Mice were fed with water or BAPN for 28 days. Aortic sections were stained with immunofluorescence for F4/80 and HIF1α. The number of F4/80 + HIF1α + macrophages was quantified and compared ( n = 6). ( H ) Rbpj cKO and control mice were treated with BAPN for 28 days. Aortic sections were stained with immunofluorescence for F4/80 and HIF1α. The number of F4/80 + HIF1α + macrophages was quantified and compared ( n = 7). Student’s t -test was performed for statistical analysis. Data are expressed as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Cell Culture, RNA Sequencing, Control, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: RBPJ deficiency down-regulates PDK1, which can be induced by mechanical stress. ( A , B ) BMDMs were prepared from the Rbpj cKO and control mice, and subjected to stretching force for 24 h. The mRNA level of Pdp1 , Pdp2 , Pdk1 , Pdk2 , Pdk3 , and Pdk4 was determined by qRT-PCR ( n = 5) ( A ). The protein level of PDP1, PDK1 and PDK3 was determined by immunoblotting ( n = 6) ( B ). ( C ) Mice were treated with water or BAPN. Aortic sections were stained with immunofluorescence for F4/80 and PDK1. The number of F4/80 + PDK1 + macrophages was quantified and compared ( n = 6). ( D ) Rbpj cKO and control mice were treated with BAPN for 28 days. Aortic sections were stained with immunofluorescence for F4/80 and PDK1. The number of F4/80 + PDK1 + macrophages was quantified and compared ( n = 7). Student’s t-test was performed for statistical analysis. Data are expressed as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Control, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The transcription factor RBPJ is required for inflammatory macrophage activation in thoracic aortic dissection by mediating mechanotransduction-induced glycolysis

doi: 10.1007/s00018-025-05908-1

Figure Lengend Snippet: DCA alleviates TAD progression. ( A ) Mice were given 0.25% BAPN for 4 weeks, and treated with DCA (170 mg/kg/day) or saline. The diameter of aortic arch was monitored weekly using ultrasound imaging ( n = 12). ( B ) Mouse survival was recorded and analyzed ( n = 12). ( C ) Aortic samples were collected and photographed. Arrowheads indicate the TAD pathologic sites. ( D ) H&E staining of aortic samples. ( E ) EVG staining of aortic samples. The integrity of elastic fiber was evaluated with the elastin score and quantitatively compared ( n = 12). ( F ) Immunofluorescence of aortic samples. Samples were stained with F4/80 and NOS2, and arrows indicate NOS2 + macrophages, which were quantitatively compared. ( G ) Aortic samples were collected as indicated, and analyzed by flow cytometry for CD86 + macrophages. The mean fluorescence intensity (MFI) was compared between the two groups ( n = 3). ( H ) A schematic illustration of RBPJ-mediated metabolic remodeling in regulating aortic macrophages during TAD progression. Student’s t -test was performed for statistical analysis in ( A , E , F , G ), and Kaplan-Meier method was used to estimate survival functions and log-rank test (Mantel-Cox) was used in ( B ). Data are expressed as mean ± SEM, * P < 0.05 ** P < 0.01 and *** P < 0.001

Article Snippet: To induce TAD, three-weeks-old male mice were given water containing 0.25% BAPN (Tokyo Chemical Industry Co., LTD, Tokyo, Japan) for 4 weeks, with mice drinking water as a control [ ].

Techniques: Saline, Imaging, Staining, Immunofluorescence, Flow Cytometry, Fluorescence