Journal: Nature Communications

Article Title: CRL4 DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication

doi: 10.1038/s41467-025-64258-5

Figure Lengend Snippet: a , d QIBC plots of MCM4-Halo cells treated with siRNAs as indicated. Lines denote medians; n = 4973 (minimum cells) ( a ) or 5003 (minimum cells) ( d ) per condition. b , e Quantification of QIBC plots; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 4 (2 biological replicates, each with 2 technical replicates) for both ( b , e ). c , f Representative images; the pseudo-color gradient indicates the mean fluorescent intensity (MFI). Scale bar, 20 µm. g Schematic representation of the tandem affinity purification strategy. h , i CRL4 DCAF12 -associated proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Non-related ubiquitin ligases CRL4 DCAF4 in ( h ) or SCF FBXL6 in ( i ) were used as a control. Proteins that were significantly enriched with CRL4 DCAF12 compared to controls are in the upper right quadrants ( n = 2 biological replicates). j Affinity purification (AP) of a panel of CRLs. HEK293T cells were transfected with an empty vector (EV) or StrepII-FLAG-tagged CRL constructs for 48 h and treated with MLN4924 for 6 h before harvesting. Whole-cell lysates were subjected to AP with Strep-TactinXT resin. M denotes a molecular marker. k Affinity purification of N-terminal StrepII-FLAG-tagged DCAF12 (SF-DCAF12) and immunopurification of HA-tagged MCMBP, MOV10, and C-terminal degron-lacking mutants. HEK293T cells were co-transfected with SF-DCAF12 and either an empty vector or specified gene constructs. MLN4924 was added for the final 6 h before harvest. Whole-cell lysates were subjected to affinity purification (AP) with Strep-TactinXT resin or anti-HA immunoprecipitation (IP). The left panel shows 1% input samples, the upper right panel shows Strep-Tactin AP, and the lower right panel shows anti-HA IP. j , k are representatives of three independent replicates with similar results. For ( h , i ), statistical analysis was performed in Perseus (version 1.6.15.0) using a two-sample Student’s t test (two-sided; S ₀ = 0.1) with permutation-based false discovery rate (FDR) correction (FDR = 0.05). P values were calculated by ordinary one-way ANOVA with Dunnett’s test ( b , e ); n.s. (not significant) indicates p > 0.1. (A.U. Arbitrary Units). Source data are provided as a file.

Article Snippet: Primary antibodies used for western blotting were as follows: α-tubulin (mouse, Proteintech, 66031-1-Ig, 1:1,000), β-actin (mouse, Santa Cruz, sc-69879, 1:1,000), CCNA (home-made serum), DDB1 (rabbit, Zymed, 34-2300, 1:1,000), GART (mouse, Santa Cruz, sc-166379, 1:1,000), γH2AX (rabbit, Proteintech, 83307-2-RR, 1:1,000; mouse, Milipore, 05-636, 1:1,000), HA (rabbit, Cell Signaling, 3724, 1:1,000), MCMBP (rabbit, Proteintech, 19573-1-AP, 1:1,000; rabbit, Atlas Antibodies, HPA038481, 1:1,000), MCM2 (rabbit, Proteintech, 10513-1-AP, 1:1,000), MCM2 (rabbit, ABClonal, A1056, 1:1,000), MCM3 (mouse, Santa Cruz, sc-390480, 1:1,000), MCM3 (rabbit, ABClonal, A1060, 1:1,000), MCM4 (rabbit, Proteintech, 13043-1-AP, 1:1,000), MCM5 (rabbit, Proteintech, 11703-1-AP, 1:1,000), MCM6 (rabbit, Proteintech, 13347-2-AP, 1:1,000), MCM7 (mouse, Santa Cruz, sc-9966, rabbit, Proteintech, 11225-1-AP, 1:1,000), p53 (mouse, Santa Cruz, sc-126, 1:1,000), PARP1 (mouse, Proteintech, 66520-1-Ig, 1:1,000), PCNA (mouse, Santa Cruz, sc-56, 1:1,000), pH3 (Ser10) (rabbit, Cell Signaling, 53348, 1:1,000), RPS3A (rabbit, ABClonal, A5885, 1:1,000), RPS6 (mouse, Santa Cruz, sc-74459, 1:1,000), SKP1 (rabbit, Cell Signaling, 12248, 1:1,000).

Techniques: Affinity Purification, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Control, Transfection, Plasmid Preparation, Construct, Marker, Immu-Puri, Immunoprecipitation

Journal: Nature Communications

Article Title: CRL4 DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication

doi: 10.1038/s41467-025-64258-5

Figure Lengend Snippet: a A schematic describing the experimental approach for understanding the role of CUL4-DCAF12 in MCM2-7 assembly (see text for details). b Halo-immunoprecipitation (Halo-IP) of whole cell lysates. Cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. c Quantification of Halo-IP in ( b ); IP products in control cells have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d Halo-IP of nuclear fraction. Cells were treated with siDCAF12 as indicated. IPs in ( b , d ) were done in low salt condition (150 mM NaCl). e Quantification of Halo-IP in ( d ); IP products in control cells have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). f Sum fluorescence intensity (SFI) of PLA foci for the MCMBP-MCM2 antibody pair in U2OS cells treated with control or siRNA against DCAF12 . Lines denote medians; n = 3454 (minimum cells per condition). g Representative images from ( f ). This experiment was independently repeated two times with similar results. Scale bar, 10 µm. h SFI of PLA foci for the CDT1-MCM2 antibody pair in G1/S phase of PLA-positive U2OS cells treated with control or siRNA against DCAF12. Lines denote medians; n = 746 cells per condition. i Representative images from ( h ). This experiment was independently repeated two times with similar results. Scale bar, 10 µm. j Heatmap, derived from mass spectrometry analysis of HEK293T cells co-transfected with StrepII-FLAG-tagged DCAF12, shows log2 intensities of indicated proteins. Cells were treated with either DMSO (CTRL) or 10 µM MG132 for 6 h prior to protein purification. k Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. l , m QIBC plots of the cytoplasm of HCT116 cells stained for StrepII-FLAG-tagged DCAF12 (SF-DCAF12) ( l ) or MCMBP ( m ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n = 1051 (minimum cells) ( l ) or 1105 (minimum cells) ( m ) per condition. P values in ( f , h ) were calculated by two-tailed unpaired t -test. Source data are provided as a file.

Article Snippet: Primary antibodies used for western blotting were as follows: α-tubulin (mouse, Proteintech, 66031-1-Ig, 1:1,000), β-actin (mouse, Santa Cruz, sc-69879, 1:1,000), CCNA (home-made serum), DDB1 (rabbit, Zymed, 34-2300, 1:1,000), GART (mouse, Santa Cruz, sc-166379, 1:1,000), γH2AX (rabbit, Proteintech, 83307-2-RR, 1:1,000; mouse, Milipore, 05-636, 1:1,000), HA (rabbit, Cell Signaling, 3724, 1:1,000), MCMBP (rabbit, Proteintech, 19573-1-AP, 1:1,000; rabbit, Atlas Antibodies, HPA038481, 1:1,000), MCM2 (rabbit, Proteintech, 10513-1-AP, 1:1,000), MCM2 (rabbit, ABClonal, A1056, 1:1,000), MCM3 (mouse, Santa Cruz, sc-390480, 1:1,000), MCM3 (rabbit, ABClonal, A1060, 1:1,000), MCM4 (rabbit, Proteintech, 13043-1-AP, 1:1,000), MCM5 (rabbit, Proteintech, 11703-1-AP, 1:1,000), MCM6 (rabbit, Proteintech, 13347-2-AP, 1:1,000), MCM7 (mouse, Santa Cruz, sc-9966, rabbit, Proteintech, 11225-1-AP, 1:1,000), p53 (mouse, Santa Cruz, sc-126, 1:1,000), PARP1 (mouse, Proteintech, 66520-1-Ig, 1:1,000), PCNA (mouse, Santa Cruz, sc-56, 1:1,000), pH3 (Ser10) (rabbit, Cell Signaling, 53348, 1:1,000), RPS3A (rabbit, ABClonal, A5885, 1:1,000), RPS6 (mouse, Santa Cruz, sc-74459, 1:1,000), SKP1 (rabbit, Cell Signaling, 12248, 1:1,000).

Techniques: Immunoprecipitation, Control, Fluorescence, Derivative Assay, Mass Spectrometry, Transfection, Protein Purification, Staining, Construct, Two Tailed Test

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: Skeletal muscle-specific ablation of LONP1 causes bone loss and mechanical impairments. (A to D) Scheme (A) for proteomic analysis using muscle samples from HU and control mice. Data were obtained from published dataset PXD041190. (B) Heatmap showing differentially regulated proteins in HU muscles ( n = 5). (C) GO analysis from up-regulated proteins in HU muscles ( n = 5). (D) Bar graphs for comparison of normalized protein expression of LONP1 in muscle tissues from HU muscles and controls ( n = 5). (E to N) WT and LONP1 mKO male mice were harvested at 8 weeks old. (E and F) μCT pictures (E) and quantification (F) of trabecular bone parameters in the distal femur metaphysis ( n = 6). (G) Bending test analysis of elastic modulus, maximum force, compressive ultimate stress, and post-yield displacement of femurs ( n = 6). (H and I) μCT pictures (H) and quantification (I) of trabecular bone parameters in the fifth LVs ( n = 6). (J) Pictures of calcein double labeling in femur sections (scale bar, 50 μm) and quantification of MAR ( n = 5). (K) Images of ALP staining in femur sections (scale bar, 50 μm) and quantification of ALP + cell surface per bone surface (ALP + .s/BS) in femurs ( n = 5). (L) TRAP staining pictures in femur sections (scale bar, 50 μm) and quantification of TRAP + cell surface per bone surface (TRAP + .s/BS) in femurs ( n = 5). (M and N) Serum osteocalcin levels (M) and CTX-1 levels (N) ( n = 8). Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by an unpaired 2-tailed Student’s t test.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Control, Muscles, Comparison, Expressing, Labeling, Staining

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: The sudden loss of LONP1 in mature muscles results in bone loss. (A) Diagrammatic drawing demonstrating the generation of tamoxifen-inducible muscle-specific deletion of Lonp1 (LONP1 HSA-MCM ). (B to I) LONP1 f/f and LONP1 HSA-MCM male mice were fed CD and injected by tamoxifen at 6 weeks old and harvested at 15 weeks old. (B) Representative quantification of LONP1 protein expression in skeletal muscles, eWAT, liver, and heart from LONP1 f/f and LONP1 HSA-MCM male mice. Quantification of the LONP1/β-actin ratio was determined by ImageJ ( n = 3). (C and D) μCT pictures (C) and quantification (D) of trabecular bone parameters in the distal femur metaphysis ( n = 5). (E) Bending test analysis of elastic modulus, maximum force, compressive ultimate stress, and post-yield displacement of femurs from LONP1 f/f and LONP1 HSA-MCM mice ( n = 5). (F and H) Pictures of ALP staining and TRAP staining in femur sections (scale bar, 50 μm). (G and I) Serum osteocalcin levels (G) and CTX-1 levels (I) of LONP1 f/f and LONP1 HSA-MCM mice ( n = 8). Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by an unpaired 2-tailed Student’s t test. Ud, undetectable.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Muscles, Injection, Expressing, Staining

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: Mitochondrial proteostasis stress elicits mitochondrial UPR and myokine expression in skeletal muscle. (A) Bubble diagram showing fold changes for dataset (GSE192991) generated from the GC of MCK-ΔOTC mice compared to littermate control (NTG) mice versus fold changes for dataset (GSE166071) generated from the GC of LONP1-mKO mice compared to WT mice. Both up-regulated genes in the 2 groups are represented by pink dots. (B) Venn diagram exhibiting an overlap between secreted factors and both significantly up-regulated genes in LONP1-mKO and MCK-ΔOTC mice. (C) Heatmap analysis of muscle-UPR mt myokine genes up-regulated in LONP1-mKO or MCK-ΔOTC mice compared to littermate controls ( n = 2). (D) Gene expression of muscle-UPR mt myokines in GC muscles in WT and LONP1 mKO mice ( n = 5). (E) Gene expression of muscle-UPR mt myokines in GC muscles in NTG and MCK-ΔOTC mice ( n = 5). (F and G) ALP staining images (F) and quantification (G) of osteoblasts administered with the serum of WT and LONP1 mKO mice ( n = 4). (H and I) TRAP staining images (H) and quantification (I) of osteoclasts administered with the serum of WT and LONP1 mKO mice (scale bar, 100 μm; n = 3). Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by an unpaired 2-tailed Student’s t test.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Expressing, Generated, Control, Gene Expression, Muscles, Staining

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: Skeletal muscle mitochondrial proteostasis stress-induced bone loss is independent of ATF4. (A) Heatmap analysis of muscle-UPR mt myokines and Lonp1 or Atf4 genes in indicated mice (GSE192990; n = 2). (B to J) WT, LONP1 mKO, and LONP1/ATF4 DmKO male mice were harvested at 8 weeks old. (B and C) μCT pictures (B) and quantification (C) of trabecular bone parameters in the distal femur metaphysis ( n = 5 to 6). (D) Bending test analysis of elastic modulus, maximum force, compressive ultimate stress, and post-yield displacement of femurs ( n = 5). (E and F) μCT pictures (E) and quantification (F) of trabecular bone parameters in the fifth LVs ( n = 5 to 6). (G) Pictures of ALP staining in femur sections (scale bar, 50 μm) and quantification of ALP + .s/BS ( n = 5). (H) Pictures of TRAP staining in femur sections (scale bar, 50 μm) and quantification of TRAP + .s/BS ( n = 5). (I and J) Serum osteocalcin levels (I) and CTX-1 levels (J) ( n = 8). Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by one-way ANOVA.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Staining

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: ATF4-independent regulation of FGF21 is linked to bone metabolism in mice and humans. (A) Relative mRNA expression of Fgf21 in WT, LONP1 mKO, and LONP1/ATF4 DmKO mice ( n = 5). (B) Serum FGF21 levels detected by ELISA kit (Proteintech, KE10042) in NTG and MCK-ΔOTC mice ( n = 8). (C) Serum FGF21 levels detected by ELISA kit (Proteintech, KE10042) in WT, LONP1 mKO, and LONP1/ATF4 DmKO mice ( n = 6). (D and E) ALP staining pictures (D) and quantification (E) of osteoblasts administered with the serum of WT and LONP1 mKO mice or combined with an FGF21-blocking antibody [anti-insulin-like growth factor 2 (IGF2), 1 μg/ml; n = 4]. (F and G) TRAP staining images (F) and quantification (G) of osteoclasts administered with the serum of WT and LONP1 mKO mice or combined with an FGF21-blocking antibody (anti-IGF2, 1 μg/ml; scale bar, 100 μm; n = 3). Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by one-way ANOVA.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

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

Journal: Research

Article Title: Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss

doi: 10.34133/research.0465

Figure Lengend Snippet: Serum FGF21 is linked to bone health in humans. (A) Serum FGF21 levels detected by ELISA kit (Mlbio, ml058174) in normal, osteopenia, and osteoporosis persons ( n = 25 to 30). (B to F) Correlations between serum FGF21 levels and T score (B), Z score (C), total BMD (D), lumbar average BMD (E), or femoral neck BMD (F) ( n = 84). (G) Model for the LONP1-dependent mitochondrial proteostasis in facilitating muscle–bone crosstalk. Data are shown as the mean ± SEM. * P < 0.05 versus corresponding controls. P values were determined by Kruskal–Wallis test with Dunn’s corrections test (A). Spearman’s correlation (B to F) analysis was used to determine the correlation.

Article Snippet: Serum osteocalcin (Novus,NBP2-68151), CTX-1 (Cloud-Clone, CEA665Mu), and FGF21 (Proteintech, KE10042) in male LONP1 mKO, LONP1 HSA-MCM , MCK-ΔOTC, and LONP1/ATF4 DmKO mice and serum FGF21 (Mlbio, ml058174) in humans were conducted using enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Enzyme-linked Immunosorbent Assay