Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: Overexpression of cZFP609 protects chondrocytes from inflammation and degeneration. (A–E) RT‐qPCR of cZFP609 expression. (A) In the articular cartilage (left) and plasma (right) of WT and sm Sirt1 ‐Tg mice ( n = 6). (B) The plasma level of cZFP609 in OA patients and normal control subjects ( n = 20). (C) In the VSMCs of WT and sm Sirt1 ‐Tg mice. (D) In WT and sm Sirt1 ‐Tg VSMC conditional media (CM). (E) Chondrocytes were incubated with WT and sm Sirt1 ‐Tg VSMCs CM for 24 h. (F–H) Western blot analysis of p16, p21, p53, IL‐1β, and MMP13 in chondrocytes. (F) Incubated with WT and sm Sirt1 ‐Tg VSMCs CM after treatment with TNFα (20 ng/mL). (G) Incubated with CM from the cZFP609 siRNA‐transfected sm Sirt1 ‐Tg VSMCs. (H) Transfected with vector or cZFP609 plasmid DNA for 12 h and then treated with TNFα (20 ng/mL) for 48 h. (I) SA‐β‐gal staining and levels. Scale bar = 200 µm. Data and images represent at least three independent experiments by paired t ‐test and Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Clinical Proteomics, Control, Incubation, Western Blot, Transfection, Plasmid Preparation, Staining

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 contributes to mitochondrial homeostasis in chondrocytes. (A) Analysis of GO terms enriched by differentially expressed genes in the category of biological process (BP), cellular component (CC), and molecular function (MF) in chondrocytes treated with TNFα (20 ng/mL) after being transfected with vector or cZFP609. (B) Analysis of KEGG pathways enriched by differentially expressed genes of transcriptomics analysis in the above conditions. (C) Representative images and relative quantification by image Fiji macro tool for the mitochondrial morphology of chondrocytes treated with the indicated conditions. Scale bar = 20 µm. (D) The mitochondrial membrane potential was detected with JC‐1 and quantified by the Fiji image macro tool. Scale bar = 40 µm. (E) Mitochondrial ROS (MitoSOX Red) were detected by incubation with MitoSOX and quantified by the Fiji image macro tool. Scale bar = 20 µm. (F) ATP levels were quantified in chondrocytes, the ATP content was calculated as nmol/mg of protein, and the data are represented as the rate of control. Data and images represent at least three independent experiments using the paired t ‐test and Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Transfection, Plasmid Preparation, Quantitative Proteomics, Membrane, Incubation, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 interacts with BiP in chondrocytes. (A) Analysis of cZFP609‐Protein Interactions by mass spectrometry after RNA‐Protein Pull‐down Assays. (B) HDOCK SERVER predicted the binding site of cZFP609 and BiP. (C) Schematic showing cZFP609, full‐length and truncated BiP. (D) Fluorescence in situ hybridization (FISH) assay for the localization of cZFP609 in chondrocytes. Scale bars = 10 µm. (E, F) RNA immunoprecipitation (RIP) assay (E) and RNA pull‐down assay (F) for the interactions between cZFP609 and BiP in chondrocytes. (G) FISH assay for the co‐localization of cZFP609 and BiP in chondrocytes, and quantified by the Fiji image macro tool. Scale bar = 10 µm. (H and I) RNA pull‐down assay (H) and RIP assay (I) for the interactions between cZFP609 and BiP in chondrocytes in the indicated conditions. (J) Representative western blots for mapping the domains of BiP binding to cZFP609. HEK293T cells were transduced with cZFP609 and with either full‐length Flag‐BiP or its truncations. (K) RIP assay for chondrocytes transfected with the cZFP609 full‐length and deletion mutants. Data and images represent at least three independent experiments by a paired t ‐test. * p < 0.05, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Mass Spectrometry, Binding Assay, Fluorescence, In Situ Hybridization, RNA Immunoprecipitation, Pull Down Assay, Western Blot, Transduction, Transfection, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 inhibits ER stress via stabilizing oligomeric BiP. (A and B) Western blot analysis of XBP‐1S, IRE1α‐p, and ATF4 proteins in chondrocytes treated with the indicated conditions. (C) Co‐IP assay for the interactions of BiP with IRE1α in chondrocytes. (D) Native PAGE analysis of BiP in chondrocytes. (E) Native PAGE analysis of BiP in chondrocytes after cZFP609 pull‐down assay. Data and images represent at least three independent experiments using Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Clear Native PAGE, Pull Down Assay, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 reduces ERMCs via inhibiting the depolymerization of BiP. (A) Representative images of the ERMCs probed with MitoTracker and ER‐resident protein IP3R in chondrocytes treated with the indicated conditions by confocal microscopy and quantified by the Fiji image macro tool. Scale bar = 20 µm. (B) Representative TEM images of ERMCs in chondrocytes treated with the indicated conditions. Mitochondria and ER masks were manually segmented and distinguished in blue and yellow, respectively. ERMCs (distances between 100 and 30 nm) were quantified by the Fiji image macro tool. Scale bar = 500 nm. (C) Native PAGE analysis of BiP in chondrocytes treated with ATP in different concentrations. (D) Native PAGE analysis of BiP in cZFP609 overexpressed chondrocytes exposed to 150 µM ATP after being treated with TNFα (20 ng/mL). (E) Representative images of ERMCs in cZFP609 overexpressed chondrocytes exposed to 150 µM ATP after being treated with TNFα (20 ng/mL), and quantified by the Fiji image macro tool. Scale bar = 20 µm. (F) Representative co‐localization images and relative quantification by image Fiji macro tool in chondrocytes stained with MitoTracker and BiP. Scale bar = 20 µm. Data and images represent at least three independent experiments by paired t ‐test. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Confocal Microscopy, Clear Native PAGE, Quantitative Proteomics, Staining, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 suppresses ferroptosis of chondrocytes. (A) Representative images of BODIPY (581/591) C11 staining in chondrocytes treated with the indicated conditions. The relative fluorescence intensity of R‐Bodipy/Ox‐Bodipy was quantified using Fiji images. Scale bar = 20 µm. (B) Representative images and relative quantification of FerroOrange staining. Scale bar = 40 µm. (C, D) MDA (C) and GSH (D) levels were detected in chondrocytes. (E) Western blot analysis of TFRC, ACSL4, COX2, FTH, and GPX4 proteins in chondrocytes. (F) Representative images from flow cytometry in chondrocytes. The total percentage of apoptosis (red square) is equal to the percentage of early apoptosis (Q2, Annexin V + 7‐AAD + ) plus the percentage of late apoptosis (Q3, Annexin V + 7‐AAD + ). (G) The cell viability of the chondrocytes was determined by CCK‐8 analysis. (H) Representative images and relative quantification of FerroOrange staining in chondrocytes treated with the indicated conditions. Scale bar = 40 µm. (I) Representative images of BODIPY (581/591) C11 staining and relative quantification. Scale bar = 20 µm. Data and images represent at least three independent experiments by Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Staining, Fluorescence, Quantitative Proteomics, Western Blot, Flow Cytometry, CCK-8 Assay, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: Intra‐articular injection of cZFP609 plasmid alleviates OA progression. (A) Schematic of the experimental timeline receiving sham or DMM surgery and intra‐articular cZFP609 plasmid DNA (pDNA) injection in wild‐type mice ( n = 6). (B) The efficiency of cZFP609 was determined by RT‐qPCR in cartilage tissue at sacrifice. (C–F) Safranin‐O/fast green staining (C) and scoring of OA parameters, including OARSI grade (D), chondrocyte number (E), and cartilage thickness (F). Scale bar = 100 µm ( n = 6). (G, H) PWT (G) and PWL (H) were used to evaluate mechanical pain sensitivity and hyperalgesia. (I, J) Immunohistochemical staining of GPX4 and ACSL4 proteins in the cartilage tissue (I) and quantitative analysis (J). Scale bar = 50 µm. (K, L) Western blot (K) and quantification analysis (L) of the protein levels of GPX4, ACSL4, and COX2 in the cartilage tissue. Data and images represent at least three independent experiments. Statistical analyses, paired t ‐tests, and Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: Chondrocytes were lysed in 500 μL lysis buffer and then incubated with 3 μg of biotinylated DNA oligonucleotide probes targeting cZFP609 at 4°C for 2 h. A total of 50 μL of BeyoMag Streptavidin Magnetic Beads (P2151, Beyotime, China) were added to the supernatant after it was centrifuged for 5 min at 12,000 g and further incubated at 4°C for 4 h. After incubation, the beads were washed three times using washing buffer.

Techniques: Injection, Plasmid Preparation, Quantitative RT-PCR, Staining, Immunohistochemical staining, Western Blot, Control