Journal: Nature Aging

Article Title: Serum amyloid P secreted by bone marrow adipocytes drives skeletal amyloidosis

doi: 10.1038/s43587-025-00924-z

Figure Lengend Snippet: a – c , Bone tissue sections from 9-month-old AD and WT mice were stained for SA-βGal and with Oil Red O. Representative images are shown in a ; quantification of Oil Red O⁺ BMAds per mm² ( b ) and percent SA-βGal⁺ BMAds ( c ). Scale bar, 50 μm; n = 6. d – g , Bone tissue sections from AD and WT mice were subjected to RNAscope using probes for Cdkn2a (p16) and Cdkn1a (p21). Representative images are shown in d , f , with quantification of p16⁺ ( e ) and p21⁺ ( g ) signal intensity. Scale bar, 50 μm; n = 6. h , RT–qPCR analysis of bone marrow cells from AD and WT mice showing relative mRNA expression levels of p16Ink4a , p19Arf and p21Cip ( CDKN1A ). i – k , Bone tissue sections from AD and WT mice were analyzed by RNAscope using probes for Cdkn2a (p19) and H2ax . Representative images are shown in i , with quantification of p19⁺ ( j ) and H2AX⁺ ( k ) mean signal intensity. Scale bar, 50 μm; n = 3. l – n , Bone tissue sections from 24-month-old (old) and 4-month-old (young) mice were subjected to RNAscope. Representative images are shown in l ; quantification of p19⁺ ( m ) and H2AX⁺ ( n ) signal intensity. Scale bar, 50 μm; n = 3. o – r , Double-immunofluorescence staining of tibial sections from AD and WT mice was performed using antibodies specific to perilipin and p19 ARF or γH2AX. Representative images of perilipin⁺p19⁺ cells ( o ) and perilipin⁺γH2AX⁺ cells ( q ), with quantification shown in p , r , respectively. s , t , Similar staining of bone sections from old and young mice using perilipin- and γH2AX-specific antibodies; representative images and quantification shown in s , t . Scale bar, 50 μm; n = 6. Data are shown as mean ± s.e.m. P < 0.05, P < 0.01, P < 0.001, P < 0.0001; statistical analysis via unpaired two-tailed Student’s t -test (two groups) or two-way ANOVA (four groups). Exact P values are available in the .

Article Snippet: Bone sections were blocked in PBS with 3% BSA for 1 h and then stained overnight (>8 h) with individual primary antibodies specific to p19 ARF (Novus, NB200-111, 1:100), γH2AX (Cell Signaling, 20E3, 1:200), perilipin (Cell Signaling, 9349, 1:200) and perilipin (Sigma, P1873, 1:500) and antibodies recognizing human APP (6E10, Novus, NBP2-62566, 1:200) and human Aβ (clone H31L21, Thermo Fisher Scientific 700254, 1:100).

Techniques: Staining, RNAscope, Quantitative RT-PCR, Expressing, Double Immunofluorescence Staining, Two Tailed Test

Journal: Nature Aging

Article Title: Serum amyloid P secreted by bone marrow adipocytes drives skeletal amyloidosis

doi: 10.1038/s43587-025-00924-z

Figure Lengend Snippet: a , Schematic showing four CEBPα binding sites (F1–F4) in the p19 ARF -encoding gene. ChIP–qPCR assays using CEBPα or control immunoglobulin G (IgG) antibodies were performed on BMAds from AD and WT mice ( b , c ) and from 24-month-old (old) and 4-month-old (young) mice ( d , e ). RNA Pol II was used as a positive control. Representative agarose gels are shown in b , d ; qPCR fold enrichment at binding sites is shown in c , e . f , Schematic showing four predicted CEBPα-binding sites in the p16 INK4a -encoding gene. ORF, open reading frame; TSS, transcription start site. g , ChIP–qPCR assays using anti-CEBPα or control IgG antibodies were performed on BMAds from AD and WT mice. Two independent experiments yielded consistent results; one is shown. Data are presented as mean ± s.e.m. P < 0.05, P < 0.01, P < 0.001, P < 0.0001 (unpaired two-tailed Student’s t -test or two-way ANOVA). Exact P values are available in the .

Article Snippet: Bone sections were blocked in PBS with 3% BSA for 1 h and then stained overnight (>8 h) with individual primary antibodies specific to p19 ARF (Novus, NB200-111, 1:100), γH2AX (Cell Signaling, 20E3, 1:200), perilipin (Cell Signaling, 9349, 1:200) and perilipin (Sigma, P1873, 1:500) and antibodies recognizing human APP (6E10, Novus, NBP2-62566, 1:200) and human Aβ (clone H31L21, Thermo Fisher Scientific 700254, 1:100).

Techniques: Binding Assay, ChIP-qPCR, Control, Positive Control, Two Tailed Test

Journal: Cancers

Article Title: Truncated DAPK Variants Restore Tumor Suppressor Activity and Synergize with Standard Therapies in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers17121910

Figure Lengend Snippet: Mammalian vector-based expression of full-length DAPK1 in HGSOC cells induces apoptosis. ( A ) A mammalian vector was used to express full-length DAPK1 in different ovarian cancer cell lines and primary HGSOC cells. Using Flag, PARP, p53, p21, and β-Actin antibodies, cell lysates were submitted to WB. ( B ) A Caspase-Glo 3/7 assay was used to measure the Caspase-3/7 activity of cell lines and primary cells transfected with recombinant DAPK1-expressing vectors, as depicted in ( A ). p < 0.01 for ** and p < 0.001 for ***. Student’s two-tailed, unpaired t -test. ( C ) Increasing doses of recombinant DAPK1-expressing vectors were used to transfect ovarian cancer cell lines. Cell lysates were submitted to WB using Flag, PARP, Caspase 3, PLK1, p53, p21, p14ARF, pMLC-2, and β-Actin antibodies. ( D ) A Caspase-Glo 3/7 assay was used to measure the dose-dependent Caspase-3/7 activity of cells depicted in ( C ). p < 0.01 for ** and p < 0.001 for ***. Student’s two-tailed, unpaired t -test. (The uncropped blots are shown in ).

Article Snippet: Antibodies were used at the following concentrations: DAPK1 (ab109382) (1:1000; Abcam, Cambridge, United Kingdom), mouse monoclonal PLK1 (F-8:sc-17783) (1:1000; Santa Cruz, Biotechnology Heidelberg, Germany), p53 (sc-126) (1:1000; Santa Cruz), MDM2 (sc-965) (1:1000; Santa Cruz), β-Actin (AC-15:A1978) (1:200.000; Sigma-Aldrich, Taufkirchen, Germany), Flag (M2:A8592) (1:1000; Sigma-Aldrich), p16 (#18769) (1:1000; Cell Signaling, Danvers, MA, USA), Noxa (#14766) (1:1000; Cell Signaling), p14ARF (#74560) (1:1000; Cell Signaling), Caspase-2 (#2224) (1:1000; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741) (1:1000; Cell Signaling), p53-pS20 (#9287) (1:1000; Cell Signaling, Danvers, MA, USA), p21 (#2947) (1:1000; Cell Signaling), PARP (#9542) (1:1000; Cell Signaling, Danvers, MA, USA), Puma (#98672) (1:1000; Cell Signaling, Danvers, MA, USA), Bax (AF820) (1:1000; R&D systems, MN, USA), and HRP-conjugated secondary antibodies (1:5000; GE Healthcare and Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Plasmid Preparation, Expressing, Caspase-Glo Assay, Activity Assay, Transfection, Recombinant, Two Tailed Test

Journal: Cancers

Article Title: Truncated DAPK Variants Restore Tumor Suppressor Activity and Synergize with Standard Therapies in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers17121910

Figure Lengend Snippet: Impact of reactivation of DAPK1 functions in HGSOC cell lines by ΔDAPK1-IVT mRNA or by vector-based expression of full-length DAPK1 on p53 signaling and apoptosis. ( A ) Using Flag, p21, p53, p53pS20, p16, Noxa, Puma, PLK1, p14ARF, and β-Actin antibodies, cell lysates were subjected to WB. ( B ) An immunoprecipitation with p53-specific antibodies based on cell lysates shown in ( A ) was submitted to WB using Flag, MDM2, cleaved MDM2, Caspase-2, p53pS20, and p53 antibodies. ( C ) Interphase localization of ∆DAPK1: OVCAR-8 cells expressing either vector-based ∆DAPK1 or transfected with ∆DAPK1-IVT mRNA were fixed and processed for immunofluorescence using Flag antibodies, with DNA stained using DAPI. Both vector-expressed ∆DAPK1 and mRNA-derived ∆DAPK1 exhibited similar cytoplasmic localization (scale bar = 10 µm). ( D ) Mitotic localization of ∆DAPK1: OVCAR-8 cells expressing ∆DAPK1-IVT mRNA were synchronized into mitosis using nocodazole. After washing off nocodazole, cells were released for 2 h, then fixed and processed for immunofluorescence using Flag and α-tubulin antibodies. The truncated ∆DAPK1 form was observed in the cytoplasm, associating with the mitotic spindle during early mitosis (scale bar = 10 µm). (The uncropped blots are shown in ).

Article Snippet: Antibodies were used at the following concentrations: DAPK1 (ab109382) (1:1000; Abcam, Cambridge, United Kingdom), mouse monoclonal PLK1 (F-8:sc-17783) (1:1000; Santa Cruz, Biotechnology Heidelberg, Germany), p53 (sc-126) (1:1000; Santa Cruz), MDM2 (sc-965) (1:1000; Santa Cruz), β-Actin (AC-15:A1978) (1:200.000; Sigma-Aldrich, Taufkirchen, Germany), Flag (M2:A8592) (1:1000; Sigma-Aldrich), p16 (#18769) (1:1000; Cell Signaling, Danvers, MA, USA), Noxa (#14766) (1:1000; Cell Signaling), p14ARF (#74560) (1:1000; Cell Signaling), Caspase-2 (#2224) (1:1000; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741) (1:1000; Cell Signaling), p53-pS20 (#9287) (1:1000; Cell Signaling, Danvers, MA, USA), p21 (#2947) (1:1000; Cell Signaling), PARP (#9542) (1:1000; Cell Signaling, Danvers, MA, USA), Puma (#98672) (1:1000; Cell Signaling, Danvers, MA, USA), Bax (AF820) (1:1000; R&D systems, MN, USA), and HRP-conjugated secondary antibodies (1:5000; GE Healthcare and Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, Transfection, Immunofluorescence, Staining, Derivative Assay

Journal: Cancers

Article Title: Truncated DAPK Variants Restore Tumor Suppressor Activity and Synergize with Standard Therapies in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers17121910

Figure Lengend Snippet: Effects of DAPK1 reactivation on the survival of primary HGSOC cells. ( A ) Patient-derived organoids were transfected with 2 μg of ΔDAPK1-IVT mRNA. On day 2, following transfection with 2 μg of ΔDAPK1-IVT mRNA, HGSOC organoids are depicted in representative photos ( left panels ), and a live/dead assay was performed. Flag and β-actin antibodies were used to perform WB on the lysates of HGSOC organoids mock or transfected with ΔDAPK1-IVT mRNA ( lower left panel ). The Caspase-Glo 3/7 assay was used to analyze mock- or ΔDAPK1-IVT mRNA-transfected HGSOC organoids ( right panel ; n = 3). * p < 0.05; unpaired, two-tailed Student’s t -test. ImageJ 1.0 was used to calculate the size of the organoids ( middle panel ; n = 20). ** unpaired, two-tailed Student’s t -test; p < 0.01. ( B ) Targeted reactivation of DAPK1 in ovarian cancer cells and resulting changes in cellular signaling. Ovarian cancer lacking DAPK1 exhibits a reduced expression of p14ARF, enabling MDM2 to stably bind to p53, resulting in its ubiquitination and degradation. This degradation impairs the transcription of p53-dependent apoptotic effectors and cell cycle regulators, promoting cancer cell proliferation and survival. In contrast, the reactivation of DAPK1 using an IVT-mRNA triggers multiple pathways that stabilize and activate p53, promoting cell cycle arrest and triggering cell death through the p53-dependent transcription of apoptosis and cell cycle effectors. Furthermore, active DAPK1 promotes Caspase-2 activation, which targets MDM2 for cleavage, thus preventing p53 degradation. Additionally, DAPK1 phosphorylates p53 at S20, further enhancing its activity. Reactivated DAPK1 also indirectly increases p14ARF expression, stabilizing p53 and facilitating the transcriptional activation of apoptosis and cell cycle regulatory genes. Finally, DAPK1 promotes the conversion of LC3A/B-I to LC3A/B-II, and both processes are crucial in inducing autophagy. Hence, the vector-based and IVT-mRNA-dependent reactivation of DAPK1 demonstrates a dual role in promoting p53 stability and autophagy, emphasizing its therapeutic potential in targeting ovarian cancer.

Article Snippet: Antibodies were used at the following concentrations: DAPK1 (ab109382) (1:1000; Abcam, Cambridge, United Kingdom), mouse monoclonal PLK1 (F-8:sc-17783) (1:1000; Santa Cruz, Biotechnology Heidelberg, Germany), p53 (sc-126) (1:1000; Santa Cruz), MDM2 (sc-965) (1:1000; Santa Cruz), β-Actin (AC-15:A1978) (1:200.000; Sigma-Aldrich, Taufkirchen, Germany), Flag (M2:A8592) (1:1000; Sigma-Aldrich), p16 (#18769) (1:1000; Cell Signaling, Danvers, MA, USA), Noxa (#14766) (1:1000; Cell Signaling), p14ARF (#74560) (1:1000; Cell Signaling), Caspase-2 (#2224) (1:1000; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741) (1:1000; Cell Signaling), p53-pS20 (#9287) (1:1000; Cell Signaling, Danvers, MA, USA), p21 (#2947) (1:1000; Cell Signaling), PARP (#9542) (1:1000; Cell Signaling, Danvers, MA, USA), Puma (#98672) (1:1000; Cell Signaling, Danvers, MA, USA), Bax (AF820) (1:1000; R&D systems, MN, USA), and HRP-conjugated secondary antibodies (1:5000; GE Healthcare and Jackson Laboratory, Bar Harbor, ME, USA).

Techniques: Derivative Assay, Transfection, Live Dead Assay, Caspase-Glo Assay, Two Tailed Test, Expressing, Stable Transfection, Ubiquitin Proteomics, Activation Assay, Activity Assay, Plasmid Preparation