Journal: Experimental & Molecular Medicine

Article Title: Extracellular vesicle-mediated delivery of circp53 suppresses the progression of multiple cancers by activating the CypD/TRAP/HSP90 pathway

doi: 10.1038/s12276-025-01506-0

Figure Lengend Snippet: a , GO analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with mitochondrial pathways. b , Go analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with apoptotic process signaling pathways. c , A graphic illustration of circp53–209aa interacting with CypD and releasing CypD from the CypD/TRAP1/HSP90 complex, thereby activating its isomerase activity and inducing mPTP opening. d , A Co-IP assay confirmed the direct interaction between circp53–209aa and CypD in MM cells. e ,Representative confocal images of HA and CypD demonstrate the interaction between circp53–209aa and CypD. f , Statistical analysis of the colocation of circp53-209aa and CypD in H929 and XG1 cells. g , A Co-IP assay showed a weaker interaction between CypD and HSP90 in circp53-OE MM cells compared with Ctrl cells. h , Distribution of calcein in H929 and MM1.S cells. i , Distribution of calcein in XG1 and RPMI 8226 cells. j , IOD quantitative statistics of Calcein in Ctrl and circp53–209aa-OE MM cells. k , ATP levels were significantly decreased in circp53–209aa-OE cells. l , Statistical analysis of ATP levels in different cells ( P < 0.001). m , A model showing the high confidence interval of the CypD protein. n , A model of the circp53–209aa-CypD complex. R175 of circp53–209aa binds to E43, E34 and Q163 of CypD through hydrogen bonds and/or ionic interactions. o , Circp53–209aa-R175 is involved in hydrogen-bond interactions with CypD, and these interactions are disrupted upon mutation of arginine to a histidine, as demonstrated by Co-IP using an HA antibody as bait in circp53-OE cells, followed by WB analysis. p , Circp53–209aa-R175H-OE MM cells were constructed successfully, as shown by WB analysis. q , Circp53–209aa-R175H did not influence the proliferation of MM cells. r , Circp53–209aa interacts with CypD and releases it from the CypD/TRAP1/HSP90 complex, as evidenced by Co-IP. s , Colocalization dynamics of CypD/HSP90 in H929 and MM1.S cells. t , Colocalization dynamics of CypD/HSP90 in XG1 and 8226 cells. u , Statistical analysis of the colocation of CypD and HSP90 in H929 and MM1.S cells. v , Statistical analysis of the colocation of CypD and HSP90 in XG1 and RPMI 8226 cells. w , IOD quantitative statistics of CypD and HSP90 in MM cells. x , Distribution of calcein in circp53-OE and circp53–209aa-R175 MM cells. y , The mPTP was not opened in the circp53–209aa-R175H group by quantitative statistics. The data are presented as mean ± s.d. ( n = 3 cultures for each group, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, no statistical significance).

Article Snippet: The primary antibodies used in this study were diluted at a ratio of 1:1,000, including p53 (10442-1-AP, ProteinTech Group), hemagglutinin (HA) (51064-2-AP, ProteinTech Group), cyclophilin D (CypD) (12716-1-AP, ProteinTech Group), Alix (2171S, Cell Signaling Technology), CD9 (13174S, Cell Signaling Technology), TRAP1 (92345, Cell Signaling Technology), HSP90 (13171-1-AP, ProteinTech Group), BCL-xL (2762s, Cell Signaling Technology), Bax (2774s, Cell Signaling Technology), poly-ADP-ribose polymerase (PARP) (9542S, Cell Signaling Technology), cleaved-Caspase 3 (9661S, Cell Signaling Technology) and β-actin (4970S, Cell Signaling Technology).

Techniques: RNA Sequencing, Protein-Protein interactions, Activity Assay, Co-Immunoprecipitation Assay, Mutagenesis, Construct

Journal: eLife

Article Title: Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation

doi: 10.7554/eLife.75023

Figure Lengend Snippet: MC3T3e1 were stably transfected cells with pCMV6- caPpif vector to express constitutively active CypD K166Q mutant and thus achieve CypD gain-of-function (GOF). Bone marrow stromal cells (BMSCs) from either osteoblast (OB)-specific, inducible 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice or R26 caPpif/+ mice were cultured in osteogenic media for 11–14 days. ( A ) Representative western blot of stably transfected MC3T3e1 cells (EV: pCMV6-empty vector; KQ: pCMV6- caPpif vector; VDAC1: loading control; caCypD: mutant CypD; CypD: endogenous CypD). ( B ) and ( F ) Real-time RT-PCR data of osteogenic markers: CypD GOF MC3T3 cells and OB-specific, inducible 2.3 kb Col1 CreERt2 ;R26 caPpif/+ BMSCs were incapable to complete OB differentiation. ( C ) Diagram showing the caPpif mouse transgene construct. Images show eGFP signal in cell culture confirming recombination induced in vitro. ( D ) Representative western blot of BMSCs from 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice. ( E ) Staining representative image at day 14 for crystal violet, alkaline phosphatase, and alizarin red: CypD GOF (tamoxifen-induced Cre + ) BMSCs showed decreased OB activity and mineralization capacity. ( G ) Representative images from R26 caPpif/+ BMSCs infected with adeno-Cre virus, culture in osteogenic media during 11 days. ( H ) and ( J ) Osteoinduced R26 caPpif/+ BMSCs infected with adeno-Cre collected at day 0 or day 11 showed decreased TMRE ΔF signal for infected (Cre + ) cells compared to non-infected (Cre - ) cells. ( I ) and ( K ) Osteoinduced R26 caPpif/+ BMSCs infected with adeno-eGFP collected at day 0 or day 11 showed no difference in TMRE ΔF signal between infected (Cre + ) and non-infected (Cre - ) cells. Plot shows the actual data points (biological replicates), calculated means, and p value vs. D0 ( B, F ) or Cre - ( H–K ) controls determined by an unpaired t-test. Figure 6—source data 1. Original western blot image of stably transfected MC3T3e1 cells probed for cyclophilin D (CypD). Figure 6—source data 2. Labeled western blot image of stably transfected MC3T3e1 cells probed for cyclophilin D (CypD) (EV: pCMV6-empty vector; KQ: pCMV6- caPpif vector; caCypD: mutant CypD; CypD: endogenous CypD). Figure 6—source data 3. Original western blot image of stably transfected MC3T3e1 cells probed for VDAC1. Figure 6—source data 4. Labeled western blot image of stably transfected MC3T3e1 cells probed for VDAC1 (EV: pCMV6-empty vector; KQ: pCMV6- caPpif vector; VDAC1: loading control). Figure 6—source data 5. Bone marrow stromal cells (BMSCs) from either osteoblast (OB)-specific, inducible 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice or R26 caPpif/+ mice were cultured in osteogenic media for 11–14 days. ( A ) Original western blot image of BMSCs from 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice. Figure 6—source data 6. Bone marrow stromal cells (BMSCs) from either osteoblast (OB)-specific, inducible 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice or R26 caPpif/+ mice were cultured in osteogenic media for 11–14 days. ( A ) Labeled western blot image of BMSCs from 2.3 kb Col1 CreERt2 ;R26 caPpif/+ mice (VDAC1: loading control; caCypD: mutant CypD; CypD: endogenous CypD).

Article Snippet: Transfected construct ( Mus musculus ) , pCMV6- caPpif , Origene , MR202183 , Plasmid construct to transfect and express caCypD.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Cell Culture, Western Blot, Quantitative RT-PCR, Construct, In Vitro, Staining, Activity Assay, Infection, Labeling

Journal: eLife

Article Title: Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation

doi: 10.7554/eLife.75023

Figure Lengend Snippet: ( A ) Experimental design: CypD deletion was induced in 2-month-old OB-specific inducible (2.3 kb Col1 CreERt2 ;Ppif f/f ) CypD loss-of-function (LOF) mice. Virus intra-bone marrow injection performed in the right tibia at 22 months. ( B ) Diagram showing the AAV2-Cre-DIO- caPpif- eGFP construct. The gene of interest is inserted in the vector in antisense orientation and is flanked by double floxed sites. In cells expressing Cre recombinase, the gene of interest and eGFP reporter are flipped and ‘turned-on’. ( C ) eGFP signal captured by IVIS in vivo confirming the successful viral infection and recombination in the tibia of Cre + mice 2 months after intra-bone marrow injection; CypD re-expression decreased osteocalcin immunofluorescence signal ( D ) but it did not change osteoclast activity measured by tartrate-resistant acid phosphatase (TRAP) staining ( E ) (BM: bone marrow). Bone volumes and biomechanical properties of CypD deletion and CypD re-expressing bones were measured by micro-computed tomography (μCT) and a torsion test, respectively. CypD conditional deletion did not affect cortical thickness ( F ) however, it showed protective effect against trabecular bone volume loss in aging ( G ) and improved torsional rigidity ( H ) (PBS: Ppif +/+ X Ppif -/- ). For analysis of bones with AAV-DIO-delivered CypD re-expression, data was normalized to the contralateral PBS-injected limb to account for differences in bone phenotype between animals. Cre + mice showed decreased bone volumetric parameters and mechanical properties when compared to Cre - mice. ( I ) Cortical thickness (Ct Th). ( J ) Bone over total volume (BV/TV). ( K ) Torsional rigidity. ( L ) Yield torque. ( M ) Maximum torque. Plots show the actual data points from six independent mice per group, calculated means, and p value determined by an unpaired t-test. Paired t-test was used when left and right tibia from the same mouse were compared. Specimens' genotype guide: PBS_ Ppif +/+ : wild type; PBS_ Ppif -/- : CypD conditional KO; AAV-DIO_ Ppif +/+ : wild type caCypD rescue; AAV-DIO_ Ppif -/- : CypD conditional KO caCypD rescue.

Article Snippet: Transfected construct ( Mus musculus ) , pCMV6- caPpif , Origene , MR202183 , Plasmid construct to transfect and express caCypD.

Techniques: Injection, Construct, Plasmid Preparation, Expressing, In Vivo, Infection, Immunofluorescence, Activity Assay, Staining, Micro-CT

Journal: Neural Regeneration Research

Article Title: Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

doi: 10.4103/1673-5374.353495

Figure Lengend Snippet: The experimental groups and sample sizes. (A) The timelines for in vivo and in vitro experiments. (B) The sample size in each group. ATP: Adenosine triphosphate; CypD: cyclophilin D; CsA: cyclosporin A; EM: electron microscope; ICH: intracerebral hemorrhage; IF: immunofluorescence; JC1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; OH: oxyhemoglobin; shRNA: short hairpin RNA; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild type.

Article Snippet: CypD (ppif) short hairpin RNA (shRNA) and scrambled shRNA lentiviral particles (Origene, Rockville, MD, USA, Cat# TL713307 V) were used in PC12 cells following the manufacturer’s instructions.

Techniques: In Vivo, In Vitro, Microscopy, Immunofluorescence, shRNA, Western Blot

Journal: Neural Regeneration Research

Article Title: Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

doi: 10.4103/1673-5374.353495

Figure Lengend Snippet: CypD upregulation in degenerated axons. (A) Representative immunofluorescence micrograph of CypD (magenta) around the hematoma in each group. The expression of CypD was much higher in the ICH group compared with that in the Sham group. (B) Representative immunofluorescence micrograph of CypD (magenta) in normal and degenerated axons (green) around the hematomas in Thy1-YFP mice in each group. The expression of CypD was much higher in the degenerated axons in the ICH group compared with that in the normal axons in the Sham group. Scale bar: 20 μm in A, 5 μm in B. (C) Representative western blot and quantification of CypD in tissues around the hematoma 12 and 24 hours after ICH. VDAC is the internal reference. Data are shown as the mean ± SEM ( n = 5 animals for each group). * P < 0.05, ** P < 0.01, vs . Sham group (one-way analysis of variance followed by Tukey’s post hoc t est). CypD: Cyclophilin D; ICH: intracerebral hemorrhage; VDAC: voltage-dependent anion channel.

Article Snippet: CypD (ppif) short hairpin RNA (shRNA) and scrambled shRNA lentiviral particles (Origene, Rockville, MD, USA, Cat# TL713307 V) were used in PC12 cells following the manufacturer’s instructions.

Techniques: Immunofluorescence, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

doi: 10.4103/1673-5374.353495

Figure Lengend Snippet: CypD deficiency and CsA treatment protect against OH-induced mitochondrial injury in vitro . (A) Immunofluorescence images showing mitochondrial calcein fluorescence (green in PC12 cells loaded with calcein-AM and CoCl 2 in each group). The calcein fluorescence in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups was increased compared with that in the OH + scrambled shRNA group. (B) Representative immunofluorescence images showing JC-1poly (magenta), JC-1mono (green) and Hoechst (blue) staining for mitochondrial membrane potential measurement and nuclei in each group. The JC-1poly/JC-1mono fluorescence ratio in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups were significantly increased compared with that in the OH + scrambled shRNA group. (C) Representative immunofluorescence images showing TMRM (magenta), MitoTracker green (green) and Hoechst (blue) staining for mitochondrial membrane potential measurement, mitochondrial structure, and nuclei in each group. The fluorescent absorbance of TMRM in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups were significantly increased compared with those in the OH + scrambled shRNA group. Scale bar: 10 μm, 6 μm (enlarged part in B), 8 μm (enlarged part in C). (D) Representative western blot and quantification of CypD and VDAC in PC12 cells in the Scrambled shRNA and CypD shRNA groups. (E) Quantification of calcein fluorescence at 30 minutes in each group. (F) Quantification of the fluorescence ratio of JC-1poly/JC-1mono in each group. (G) Quantification of the fluorescence absorbance of TMRM in each group. Data are shown as the mean ± SEM ( n = 6 independent cell cultures). ### P < 0.001, vs . scrambled shRNA group; * P < 0.05, *** P < 0.001, vs . OH + scrambled shRNA group (one-way analysis of variance followed by Tukey’s post hoc test). CsA: Cyclosporin A; CypD: cyclophilin D; JC1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; OH: oxyhemoglobin; TMRM: tetramethylrhodamine methyl ester; VDAC: voltage-dependent anion channel.

Article Snippet: CypD (ppif) short hairpin RNA (shRNA) and scrambled shRNA lentiviral particles (Origene, Rockville, MD, USA, Cat# TL713307 V) were used in PC12 cells following the manufacturer’s instructions.

Techniques: In Vitro, Immunofluorescence, Fluorescence, shRNA, Staining, Western Blot

Journal: Neural Regeneration Research

Article Title: Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

doi: 10.4103/1673-5374.353495

Figure Lengend Snippet: Effects of CypD deficiency and CsA treatment on axonal injury after ICH. (A) Representative immunofluorescence micrographs of normal and degenerated axons in each group. (B) EM images of normal and degenerated axons and normal and swollen mitochondria morphology in each group. The mitochondrial and axonal diameters in the ICH CypD–/– and ICH WT + CsA groups were smaller than those in the ICH WT group. Scale bars: 50 μm in A, 2 μm in B. (C) Number of degenerated axons per mm 2 around the hematoma in each group. The number of degenerated axons around the hematoma was significantly decreased in the ICH CypD–/– and ICH WT + CsA groups compared with that in the ICH WT group. (D) Quantification of the diameter of mitochondria in each group. (E, F) The axonal diameters (E) and mean g-ratio of the WM (F) around the hematoma in each group. (G) Quantification of ATP levels in tissue around the hematoma in each group. For data collection, three sections of each mouse and three visual fields around the hematoma of each section were observed. At least 50 degenerated axons and swollen mitochondria were measured for each mouse. Data are shown as the mean ± SEM ( n = 6 animals for each group). ** P < 0.01, *** P < 0.001, vs . ICH WT group (one-way analysis of variance followed by Tukey’s post hoc test). CsA: Cyclosporin A; CypD: cyclophilin D; EM: electron microscopy; ICH: intracerebral hemorrhage; WT: wild type.

Article Snippet: CypD (ppif) short hairpin RNA (shRNA) and scrambled shRNA lentiviral particles (Origene, Rockville, MD, USA, Cat# TL713307 V) were used in PC12 cells following the manufacturer’s instructions.

Techniques: Immunofluorescence, Electron Microscopy

Journal: Neural Regeneration Research

Article Title: Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

doi: 10.4103/1673-5374.353495

Figure Lengend Snippet: CypD deficiency and CsA treatment protect the CST and alleviate motor dysfunction after ICH. (A) Diagram of retrograde tracking of the CST (left) and GFP-positive corticospinal neurons (right, Bregma: –0.7 mm) in each group. The number of retrograde labeled GFP-positive corticospinal neurons in the ICH CypD–/– and ICH WT + CsA groups was greater than that in the ICH WT group. Scale bar: 100 μm. (B) Number of labeled corticospinal neurons in each group. (C, D) The slip ratio of the contralateral limbs in beam walking (C) and ladder rung walking (D) at 3 days post-ICH in each group. Data are shown as the mean ± SEM ( n = 6–8 animals for each group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . ICH WT group (one-way analysis of variance followed by Tukey’s post hoc test). CsA: Cyclosporin A; CST: corticospinal tract; CypD: cyclophilin D; GFP: green fluorescent protein; ICH: intracerebral hemorrhage; WT: wild type.

Article Snippet: CypD (ppif) short hairpin RNA (shRNA) and scrambled shRNA lentiviral particles (Origene, Rockville, MD, USA, Cat# TL713307 V) were used in PC12 cells following the manufacturer’s instructions.

Techniques: Labeling

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Role of the miR-548au-3p/CA12 Axis in Tracheal Chondrogenesis in Congenital Pulmonary Airway Malformations

doi: 10.1155/2023/6428579

Figure Lengend Snippet: Identification of DEGs involved in the pathogenesis of CPAM. (a) Flowchart of the study design and samples at each stage of analysis. (b) Scatterplot of mRNA expression variation between diseased CPAM and normal tissues. (c) Hierarchical cluster of gene expression profiles from microarray assays. Expression of CA12, LONRF3, MAP2, THBS1, and PPID at mRNA (d) and protein (e) levels in lung tissues from CPAM and adjacent normal tissues. ∗∗∗ p < 0.001, compared with normal.

Article Snippet: Primary antibodies for this study were specific for CA12 (Boster, Cat. No. A04063, 1 : 1000), LONRF3 (GeneTex, Cat. No. GTX112150, 1 : 2000), MAP2 (Boster, Cat. No. A01201, 1 : 2000), THBS1 (Boster, Cat. No. PB0471, 1 : 2000), PPID, E-cadherin (Beyotime, Cat. No. AF6759, 1 : 1000), N-cadherin (Beyotime, Cat. No. AF5237, 1 : 800), aggrecan (Abcam, Cat. No. ab3778, 1 : 1000), Col2A1 (Boster, Cat. No. A00517, 1 : 2000), MMP13 (Proteintech, Cat. No. 18165-1-AP, 1 : 3000), ADAMTS4 (Proteintech, Cat. No. 11865-1-AP, 1 : 600), and GAPDH (Abcam, Cat. No. ab9485, 1 : 2000).

Techniques: Expressing, Gene Expression, Microarray