anti-cip2a sc-80660  (Santa Cruz Biotechnology)

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: CIP2A expression in OA cartilage and chondrocytes. A Representative images of HE staining of human knee cartilage, n = 6, scale bar = 100 μm. B Representative images of CIP2A-specific IHC staining of human knee cartilage, n = 6, scale scale bar = 100 μm. C Quantitative statistical plot of the ratio of CIP2A-specific IHC staining positive cells in human knee cartilage, n = 6. D Representative images of HE staining of mouse knee joints, n = 6, scale bar = 100 μm. E Representative images of CIP2A-specific IHC staining of mouse knee joints, n = 6, scale bar = 100 μm. F Quantitative statistical plot of the ratio of CIP2A-specific IHC staining positive cells in mouse knee joints, n = 6. G Quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) statistical plot of CIP2A expression in chondrocytes treated with 5 ng/mL IL-1β for different time points, n = 3. H qRT-PCR statistical plot of CIP2A expression in chondrocytes treated with different concentrations of IL-1β for 24 h, n = 3. I Representative WB results of CIP2A expression in chondrocytes treated with 5 ng/mL IL-1β for different time points, n = 3. J Representative WB results of CIP2A expression in chondrocytes treated with different concentrations of IL-1β for 24 h, n = 3. * P < 0.05, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: Expressing, Staining, Immunohistochemistry, Quantitative RT-PCR

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: Effect of CIP2A on ECM anabolism and catabolism of chondrocytes in vitro. A – F qRT-PCR statistical plots of CIP2A and anabolic–catabolic marker expression in chondrocytes transfected with CIP2A siRNA (si-CIP2A) or scrambled siRNA (si-NC) for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. G Representative WB results of CIP2A and anabolic–catabolic marker expression in chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. H – M qRT-PCR statistical plots of CIP2A and anabolic–catabolic marker expression in chondrocytes transfected with CIP2A overexpression plasmid (p-CIP2A) or control empty plasmid (p-NC) for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. N Representative WB results of CIP2A and anabolic–catabolic marker expression in chondrocytes transfected with p-CIP2A or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: In Vitro, Quantitative RT-PCR, Marker, Expressing, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: Effect of CIP2A on chondrocyte inflammation in vitro and ubiquitination modification of CIP2A. A qRT-PCR statistical plot of inflammation-related factor expression in chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. B qRT-PCR statistical plot of inflammation-related factor expression in chondrocytes transfected with p-CIP2A or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. C Representative WB results of inflammation-related factor expression in chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. D Representative WB results of inflammation-related factor in chondrocytes transfected with p-CIP2A or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. E Representative WB results of CIP2A and NF-κB signaling pathway proteins expression in chondrocytes transfected with si-CIP2A or si-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. F – H Quantitative statistical plots of the ratio of phosphorylated proteins to total proteins of NF-κB signaling pathway in chondrocytes transfected with si-CIP2A or si-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. I Representative WB results of CIP2A and NF-κB signaling pathway proteins expression in chondrocytes transfected with p-CIP2A or p-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. J – L Quantitative statistical plots of the ratio of phosphorylated proteins to total proteins of NF-κB signaling pathway in chondrocytes transfected with p-CIP2A or p-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. M Representative WB results of CIP2A expression in chondrocytes treated with MG132 or DMSO for 2 h followed by 5 ng/mL IL-1β exposure for 6 h, n = 3. N Representative WB results of endogenous CoIP validation of CIP2A and Ub interaction in chondrocytes, n = 3. Chondrocytes were treated with MG132 or DMSO for 2 h followed by 5 ng/mL IL-1β exposure for 6 h. O , P Representative WB results of exogenous CoIP validation of CIP2A and K48/K63-Ub interaction in chondrocytes, n = 3. Chondrocytes were transfected with the FLAG-tagged CIP2A overexpression plasmid and HA-tagged K48/K63-Ub overexpression plasmid for 48 h and then treated with MG132 for 2 h followed by 5 ng/mL IL-1β exposure for 6 h. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: In Vitro, Ubiquitin Proteomics, Modification, Quantitative RT-PCR, Expressing, Transfection, Biomarker Discovery, Over Expression, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: Effect of CIP2A on OA progression in vivo. CIP2A-targeted adenovirus was injected into the knee-joint cavity of the mouse surgical models for 7 consecutive weeks. A Schematic diagram of animal experiment. B Representative images of HE and SO/FG staining of the knee joints at 8 weeks after surgery, during which Ad-shCIP2A or Ad-shControl was injected once a week, n = 6, scale bar = 100 μm. C Representative micro-CT three-dimensional reconstruction images of knee joints at 8 weeks after surgery, n = 6, scale bar = 1 mm. D Knee OARSI score at 8 weeks after surgery, n = 6. E Knee osteophyte score at 8 weeks after surgery, n = 6. F Representative images of IHC staining of CIP2A, ECM anabolic–catabolic markers, and inflammation-related factors of knee joints at 8 weeks after surgery, n = 6, scale bar = 100 μm. G – L Quantitative statistical plots of IHC staining positive cell ratio of CIP2A, ECM anabolic–catabolic markers, and inflammation-related factors in knee joints at 8 weeks after surgery, n = 6. M Representative images of HE and SO/FG staining of the knee joints at 8 weeks after surgery, during which Ad-CIP2A or Ad-Control was injected once a week, n = 6, scale bar = 100 μm. N Representative micro-CT three-dimensional reconstruction images of knee joints at 8 weeks after surgery, n = 6, scale bar = 1 mm. O Knee OARSI score at 8 weeks after surgery, n = 6. P Knee osteophyte score at 8 weeks after surgery, n = 6. Q Representative images of IHC staining of CIP2A, ECM anabolic–catabolic markers, and inflammation-related factors of knee joints at 8 weeks after surgery, n = 6, scale bar = 100 μm. R – W Quantitative statistical plots of IHC staining positive cell ratio of CIP2A, ECM anabolic–catabolic markers, and inflammation-related factors in knee joints at 8 weeks after surgery, n = 6. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: In Vivo, Injection, Staining, Micro-CT, Immunohistochemistry, Control

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: The effect of CIP2A on cartilage is related to its downstream CEMIP. A Heatmap of differential genes identified by RNA-seq of chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. B Volcano plot of differential genes identified by RNA-seq of chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. C Pathway enrichment analysis of RNA-seq of chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h. D NF-κB signaling pathway in GSEA enrichment analysis of RNA-seq of chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h. E Pathway network of RNA-seq of chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h. F Volcano plot of interacting proteins identified by CoIP-MS of chondrocytes transfected with p-CIP2A for 24 h followed by 5 ng/mL IL-1β exposure for 24 h. CIP2A was the target protein. G Network of interaction proteins identified by CoIP-MS of chondrocytes transfected with p-CIP2A for 24 h followed by 5 ng/mL IL-1β exposure for 24 h. CIP2A was the target protein. H Venn diagram depicting overlap of RNA-seq and CoIP-MS results. The Venn diagram was generated using the free online analysis tool OmicShare ( https://www.omicshare.com/tools ). I Representative WB results of endogenous CoIP validation of CIP2A and CEMIP interaction in chondrocytes, n = 3. CIP2A was the target protein of CoIP. J Representative WB results of exogenous CoIP validation of CIP2A and CEMIP interaction in chondrocytes, n = 3. Chondrocytes were transfected with the MYC-tagged CEMIP overexpression plasmid for 24 h and then treated with 5 ng/mL IL-1β for 24 h. CEMIP was the target protein of CoIP. K qRT-PCR statistical plot of CEMIP expression in chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. L qRT-PCR statistical plot of CEMIP expression in chondrocytes transfected with p-CIP2A or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. M qRT-PCR statistical plot of CEMIP expression in chondrocytes treated with TD52 or DMSO for 24 h followed by 5 ng/mLIL-1β exposure for 24 h, n = 3. N Representative WB results of CEMIP expression in chondrocytes transfected with si-CIP2A or si-NC for 24 h followed by 5 ng/mLIL-1β exposure for 24 h, n = 3. O Representative WB results of CEMIP expression in chondrocytes transfected with p-CIP2A or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. P Representative WB results of CEMIP expression in chondrocytes treated with TD52 or DMSO for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: RNA Sequencing, Transfection, Generated, Biomarker Discovery, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: Effect of CEMIP on ECM anabolism–catabolism and inflammation of chondrocytes in vitro. A Representative WB results of CEMIP expression in chondrocytes treated with 5 ng/mL IL-1β for different time points, n = 3. B qRT-PCR statistical plot of CEMIP expression in chondrocytes treated with 5 ng/mL IL-1β for different time points, n = 3. C Representative WB results of CEMIP expression in chondrocytes treated with different concentrations of IL-1β for 24 h, n = 3. D qRT-PCR statistical plot of CEMIP expression in chondrocytes treated with different concentrations of IL-1β for 24 h, n = 3. E – K , V qRT-PCR statistical plots of CEMIP, CIP2A, anabolic–catabolic markers, and inflammation-related factors expression in chondrocytes transfected with CEMIP siRNA (si-CEMIP) or scrambled siRNA (si-NC) for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. L , U Representative WB results of CEMIP, CIP2A, anabolic–catabolic markers, and inflammation-related factors expression in chondrocytes transfected with si-CEMIP or si-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. M – S , X qRT-PCR statistical plots of CEMIP, CIP2A, anabolic–catabolic markers, and inflammation-related factor expression in chondrocytes transfected with CEMIP overexpression plasmid (p-CEMIP) or control empty plasmid (p-NC) for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. T , W Representative WB results of CEMIP, CIP2A, anabolic–catabolic markers, and inflammation-related factors expression in chondrocytes transfected with p-CEMIP or p-NC for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. Y Representative WB results of CEMIP and NF-κB signaling pathway proteins expression in chondrocytes transfected with p-CEMIP or p-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. Z Quantitative statistical plots of the ratio of phosphorylated proteins to total proteins of NF-κB signaling pathway in chondrocytes transfected with p-CEMIP or p-NC for 48 h followed by 5 ng/mL IL-1β exposure, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: CIP2A synergizes with its downstream CEMIP to regulate chondrocytes degeneration and inflammation in vitro. A – H qRT-PCR statistical plots of CIP2A, CEMIP, anabolic–catabolic markers, and inflammation-related factors expression in chondrocytes transfected with si-CIP2A or si-NC with or without p-CEMIP for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. I , J Representative WB results of CIP2A, CEMIP, anabolic–catabolic markers, and inflammation-related factors expression in chondrocytes transfected with si-CIP2A or si-NC with or without p-CEMIP for 24 h followed by 5 ng/mLIL-1β exposure for 24 h, n = 3. K – M Representative immunofluorescence images of anabolic–catabolic markers and inflammation-related factors in chondrocytes transfected with si-CIP2A or si-NC with or without p-CEMIP for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. N – P Semiquantitative statistical plots of mean fluorescence intensity of immunofluorescence detection of anabolic–catabolic markers and inflammation-related factor expression in chondrocytes transfected with si-CIP2A or si-NC with or without p-CEMIP for 24 h followed by 5 ng/mL IL-1β exposure for 24 h, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: CIP2A promotes inflammation and exacerbates osteoarthritis by targeting CEMIP

doi: 10.1186/s11658-025-00748-0

Figure Lengend Snippet: Schematic diagram of the role of CIP2A in OA cartilage. In response to stimuli, CIP2A is ubiquitinated and interacts with CEMIP and PP2A. On one hand, the CIP2A/CEMIP/PP2A axis promotes ECM degradation by mediating the disorder of ECM anabolism and catabolism. On the other hand, it activates NF-κB signaling pathway, upregulates proinflammatory factors, and promotes inflammation. CIP2A leads to cartilage destruction and inflammation, accelerating OA. The schematic diagram created with BioRender.com

Article Snippet: The CIP2A inhibitor TD52 was purchased from MedChemExpress (HY-135699, Shanghai, China).

Techniques:

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: mRNA-seq of rat tissues. To explore the related molecular mechanism of bronchiolitis obliterans, high-throughput analysis was conducted using the lung tissues of rats. (A) DEGs in DA group rats relative to the control are shown using a volcano plot. DEGs were defined as an absolute value of log2FC >1.5 and adjusted-P<0.001. (B) Quantitative PCR was carried out to verify the results of mRNA-seq. Four DEGs were randomly selected for verification. (C) Gene Ontology analysis revealed that apoptosis, inflammation, fibrosis, EMT and epithelium-associated items were enriched by DEGs. (D) Venn diagram shows that 47 DEGs commonly existed in the six datasets. (E) Heatmap of 47 DEGs demonstrated that most genes were functional proteins. (F) CIP2A was located on chromosome 11 and was increased in DA-treated rats. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; FC, fold change; mRNA-seq, mRNA-sequencing; SLC1A6, solute carrier family 1 member 6; ERN2, endoplasmic reticulum to nucleus signaling 2; RNASE2, ribonuclease A family member 2; TPSB2, tryptase β2.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: High Throughput Screening Assay, Control, Real-time Polymerase Chain Reaction, Functional Assay, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A expression is increased in DA group rats. (A) Quantitative PCR and western blotting, and (B) immunohistochemistry (scale bar: 50 µm) were used to detect the increased expression of CIP2A in samples from the DA group, which was initially determined by mRNA-sequencing. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A attenuates the injuries induced by DA in the bronchi and lungs of rats. To evaluate the effect of CIP2A inhibition on bronchiolitis obliterans, Eth, a CIP2A inhibitor, was injected subcutaneously into DA group rats. (A) Animal experimental timeline. Representative (B) H&E and (C) Masson's trichrome-stained lung slices (scale bar: 200 µm). Quantitative analysis of severity is shown. (D) Bronchoalveolar lavage fluid total cell count, and macrophage, neutrophil, lymphocyte and eosinophil counts in the control rats, DA-exposed rats and Eth-treated DA-exposed rats. (E) Western blotting and (F) immunohistochemistry were used to measure the protein expression levels of CIP2A in lung tissues. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; H&E, hematoxylin and eosin.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Inhibition, Injection, Staining, Cell Counting, Control, Western Blot, Immunohistochemistry, Expressing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting cell proliferation regulating inhibitor of protein phosphatase 2A weakens the epithelial-mesenchymal transition and inflammation induced by DA in the bronchi and lungs of rats. (A) E-cadherin and (B) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). (C) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (D) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. n=6. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Immunofluorescence, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A reduces damage caused by DA in human primary bronchial epithelial cells. To explore the cellular mechanisms, an in vitro cellular model of bronchiolitis obliterans was employed. (A) Eth (6 µM) showed the best inhibitory effect on CIP2A expression. (B) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (C) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. (D) E-cadherin and (E) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). n=3. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A blocks the activation of NF-κB signaling in HPBECs. The expression of CIP2A, and phosphorylation of IKBα and expression of p65 were assessed by western blotting. n=3. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; HPBECs, human primary bronchial epithelial cells; IκBα, inhibitor of NF-κB α; NF-κB, nuclear factor-κB; p-, phosphorylated.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A promotes BO by activating the NF-κB pathway. BO is caused by DA and mediated by CIP2A. CIP2A deficiency inhibits fibrosis, inflammation and EMT in BO models through suppressing the NF-κB pathway. Solid and dashed arrows both indicate promotion. The solid arrow reveals the molecular mechanism through which the dashed arrow operates. BO, bronchiolitis obliterans; DA, diacetyl; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; EMT, epithelial-mesenchymal transition; NF-κB, nuclear factor-κB.

Article Snippet: After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at 4°C, the slices were incubated with CIP2A antibody (1:100; cat. no. bs-5948R; BIOSS) overnight at 4°C and with Goat Anti-Rabbit IgG/HRP (1:100; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) for 45 min. Diaminobenzidine was used as a chromogenic substrate and the nuclei were counterstained using hematoxylin at room temperature for 3 min.

Techniques:

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) RPE1 TP53 -/- cells were either left untreated or treated with aphidicolin (APH, 200 nM, 20 h) or ionizing radiation (IR, 0.25 Gy). Cells were stained for DAPI (blue), CIP2A (green) and ψH2AX (red). Scale bar represents 10 µm. (B) Quantification of co-localizing CIP2A and ψH2AX foci per mitotic cell for cells as treated as described in A. Individual values and medians of n>30 cells per condition are shown. (C) RPE1 TP53 -/- cells and CIP2A -/- clones were either left untreated or treated with APH (200 nM, 24 h) or IR (3 Gy) and analyzed 72 h later. Micronuclei per cell were quantified. The bars represent the mean and standard error of the mean (SEM) from three biologically independent experiments with n≥64 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test. (D, E) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h, panel D) or IR (0.25 Gy, panel E). Representative images of three classes of mitotic structures are shown. Left panels show the confocal overview images, and the right three panels show STED images of a single CIP2A-TOPBP1 complex. Cells were stained for DAPI (blue), CIP2A (green) and TOPBP1 (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig 1G, H. (F) Quantification of CIP2A-TOPBP1 structures per cell per mitotic phase for cells treated as described in panel D. Number of cells quantified per mitotic phase from three biologically independent experiments: prophase (n=9), prometaphase (n=9), metaphase (n=19), anaphase (n=18), telophase (n=29). The numbers of CIP2A-TOPBP1 structures per telophase may be an overestimation of the actual number, as only cells with CIP2A-TOPBP1 structures were measured. Bars represent mean and SEM of three experiments. (G) Quantification of CIP2A-TOPBP1 structures per cell per mitotic phase for cells treated with IR as described in panel E. Number of cells quantified per mitotic phase from three biologically independent experiments: prophase (n=11), prometaphase (n=10), metaphase (n=14), anaphase (n=13), telophase (n=19). Bars represent mean and SEM of three biologically independent experiments. (H) Quantification of unstructured, loop-containing and filamentous structures per mitotic phase for indicated treatments, as observed with STED microscopy for cells treated as described in from panels D and E. Percentages compared to the total structures are indicated. Mean, SEM and n, which reflects the total number of observed structures per phase, from three biologically independent experiments are shown per mitotic phase and per treatment. (I) Quantification of the size of unstructured, loop-containing and filamentous structures per mitotic phase for indicated treatments, as observed with STED microscopy for cells treated as described in panels D and E. Structure size was determined by measuring the length of the longest axis through the entire structure as shown in Suppl. Fig. 1J. Individual values, the average of medians per experiment, along with SEM, are plotted per mitotic phase, ‘n’ represents the total number of observed structures per phase from three biologically independent experiments.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Clone Assay, Two Tailed Test, Microscopy

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A, B) Mass spectrometry analysis of mitotic CIP2A interactors in RPE1 TP53 -/- parental versus CIP2A -/- cl#1 cells upon treatment with IR (panel A, 5 Gy) or with APH (panel B, 200 nM, 20 h). Proteins highlighted in red are enriched after IR or APH treatment. (C) Left panel: representative images of RPE1 TP53 -/- cells stained for DAPI (blue), CIP2A (green) and XPF (red) in either untreated conditions, or upon treatment with APH (200 nM, 20 h) or IR (0,25 Gy). Scale bar represents 10 µm. Right panel: quantification of the percentage of CIP2A foci per mitotic cell that co-localize with XPF. Bar represents the median of one experiment with n>35 cells per experimental condition. (D) RPE1 TP53 -/- and CIP2A -/- cl#1 cells were treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (green) and XPF (red). Scale bar represents 10 µm. (E) Representative images of RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells treated with APH (200 nM, 20 h). Cells were stained for DAPI (blue), CIP2A (red) and SLX4 (green). Scale bar represents 10 µm. (F) RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells were left untreated or treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (red) and MUS81 (green) in untreated condition or after treatment with APH (200 nM, 20 h). Scale bar represents 10 µm. (G) RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells were left untreated or treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (red) and ERCC1 (green), in untreated conditions or after treatment with APH (200 nM, 20 h). Scale bar represents 10 µm. (H) Quantification of mitotic XPF foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in D. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on median values per experiment. (I) Quantification of mitotic SLX4 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either untreated or treated as described in panel E. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (J) Quantification of mitotic MUS81 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in panel F. Individual values, medians and interquartile range of three experiments with n≥27 cells per experiment are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (K) Quantification of mitotic ERCC1 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in panel G. Individual values, medians and interquartile range of three experiments with n≥28 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Mass Spectrometry, Staining

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A-C) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (green) and SLX4 (red, panel A), ERCC1 (red, panel B), MUS81 (red, panel C). A representative image of each of the CIP2A structure organizations (unstructured, loop-containing, filamentous) are shown. Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 3A-C. (D-F) Quantification of the percentage of mitotic CIP2A structures that co-localize with SLX4 (panel D), ERCC1 (panel E) or MUS81 (panel F) as observed with STED microscopy for each of the CIP2A structure organizations (unstructured, loop-containing, filamentous) for cells treated as described in panels A-C. Bars represent means and SEM of three experiments with n>75 structures per experiment. (G) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (green) and ψH2AX (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 3D. (H) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h), pulsed with EdU during mitotic entry and stained for DAPI (blue), CIP2A (green) and EdU (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig 3E. (I) Quantification of mitotic CIP2A structures that are positive for ψH2AX as observed by STED microscopy for cells treated as described in panel G. Bars represent means and SEM for three experiments with n>75 structures per experiment. (J) Quantification of mitotic CIP2A structures that are either negative (0 foci) or positive for 1, 2 or more than 2 EdU foci as observed by STED microscopy for cells treated as described in panel I. Pie chart represents the means of three experiments with n>75 structures per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) RPE1 TP53 -/- , CIP2A -/- cl#1 cells and CIP2A-V5 reconstituted CIP2A -/- cl#1 cells were treated with APH (200 nM, 20 h), pulsed with EdU during mitotic entry and stained for DAPI (blue) and EdU (red) Scare bar represents 10 µm. (B) Quantification of EdU foci per mitotic cell in untreated RPE1 TP53 -/- cells, CIP2A -/- cl#1 cells, and CIP2A -/- cl#1 cells reconstituted with full CIP2A WT-V5 or cells treated as described in panel A. Individual values, medians and interquartile range of three experiments with n>28 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (C) Wildtype or CIP2A -/- MDA-MB-231 cells were treated with APH (200 nM, 20 h) and pulsed with EdU during mitotic entry. Representative images of MDA-MB231 cells stained for DAPI, CIP2A, SLX4, EdU are shown. Scale bar represents 10 µm. (D) Quantification of EdU and SLX4 foci per mitotic cell for cells treated as described in panel C. Individual values, medians and interquartile range of three experiments with n≥29 cells per experimental condition are plotted. P-values were calculated using two-tailed unpaired t-test on the medians per experiment. (E) Wildtype or CIP2A -/- HCC38 cells, and doxycycline-treated BT549 cells with doxycycline-inducible scramble (shSCR) or CIP2A (shCIP2A) shRNA were treated with APH (200 nM, 20 h), and pulsed with EdU during mitotic entry. Quantification of EdU foci per mitotic cell in HCC38 and BT549. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental conditions are plotted. P-values are calculated using two-tailed unpaired t-test on the medians per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Two Tailed Test, shRNA

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) CIP2A is predicted to form a stable homodimer, involving a globular domain with a long coiled-coil. Two predicted binding sites of TOPBP1 are indicated, along with an uncertain, and possibly flexible, position of the N-terminal regions onto the coiled-coil shaft. (B) A cartoon and surface representation of the predicted CIP2A:CIP2A dimer structure with two copies of TOPBP1 755-860, and a magnification of predicted TOPBP1:CIP2A binding interfaces. (C) Schematic representation of full length (1-905) CIP2A (WT), the reported dimerization site (L533), the PLK1 phosphorylation site (S904), the globular domain (1-617), the alpha-helical coiled-coil (618-876), and the unstructured C-terminal domain (877-905). Schematic representation of V5-tagged CIP2A variants: CIP2A lacking the unstructured C-terminal domain (‘CIP2A-ΔC’), CIP2A with only globular domain (‘CIP2A-Glob’), CIP2A lacking the alpha-helical coiled-coil (‘CIP2A-ΔCC’) and CIP2A lacking the globular domain (‘CIP2A-ΔGlob’). (D) Quantification of V5 foci and TOPBP1 foci per mitotic cell in RPE1 TP53 -/- CIP2A -/- cl#1 cells reconstituted with indicated CIP2A-V5 cDNAs, treated with APH (200 nM, 20 h). Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (E) Quantification of micronuclei per cell in parental RPE1 TP53 -/- cells, CIP2A -/- cl#1 cells and CIP2A -/- cl#1 cells reconstituted with indicated CIP2A cDNAs, treated with APH (200 nM, 20 h). Individual values, medians and interquartile range of at least three experiments with n≥85 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test. (F) CIP2A -/- cells reconstituted with CIP2A-WT, CIP2A-S904A or CIP2A-ΔC were treated with APH (200 nM, 20 h), and stained for DAPI (blue), V5 (red) and SLX4 (green). Scale bar represents 10 µm. (G) Quantification of SLX4 foci per mitotic cell in CIP2A -/- cl#1 reconstituted with indicated mutants for cells treated as described in panel F. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (H) Quantification of MUS81 foci per mitotic cell in CIP2A -/- cl#1 reconstituted with either CIP2A-WT, CIP2A-S904A or CIP2A-ΔC after treatment with APH (200 nM, 20 h). Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (I) Representative STED microscopy images of DAPI (blue), V5 (green) and TOPBP1 (red) structures in CIP2A -/- cells reconstituted with CIP2A-WT for the three classes of structures (unstructured, loop-containing, and filamentous) after APH treatment (200 nM, 20 h). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 7D (J) Representative STED microscopy images of DAPI (blue), V5 (green) and TOPBP1 (red) structures in CIP2A -/- cells reconstituted with CIP2A-ΔC for the three classes of structures (unstructured, loop-containing, and filamentous) after APH treatment (200 nM, 20 h). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 7E. (K) Quantification of unstructured, loop-containing or loop-like, and filamentous or filament-like structures in CIP2A -/- cells reconstituted with either CIP2A-WT or CIP2A-ΔC. Bars represent the mean and SEM of three experiments with n>15 structures per experimental condition. P-values were calculated using two-tailed unpaired t-test.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Binding Assay, Phospho-proteomics, Comparison, Staining, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) Representative images of RPE1 TP53 -/- PAC -/- cells and RPE1 TP53 -/- PAC -/- BRCA1 -/- cells, either left untreated or treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (red) and SLX4 (green). Scale bar represents 10 µm. (B) Representative images of DLD1 WT cells and DLD1 BRCA2 -/- cells, either left untreated or treated with APH (200 nM, 20 h), and stained for DAPI (blue), CIP2A (red) and SLX4 (green) in. Scale bar represents 10 µm. (C) Quantification of CIP2A and SLX4 foci per mitotic cell for cells treated as described in panel A. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. Quantification of CIP2A foci co-localizing with SLX4 for cells treated as described in panel A. Median values per experiment are plotted. Bars represent the mean and SD of three experiments with n>30 cells per experimental condition. (D) Quantification of CIP2A and SLX4 foci per mitotic cell for cells treated as described in panel B. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. Quantification of the total number of CIP2A foci that co-localize with SLX4 for cells treated as described in panel B. Median values per experiment are plotted. Bars represent the mean and SD of three experiments with n>30 cells per experimental condition. (E) Representative images of clonogenic survival assays of doxycycline-treated parental RPE1 TP53 -/- and CIP2A -/- cl#1 and CIP2A -/- cl#1 cells, reconstituted with CIP2A-WT or CIP2A-ΔC with doxycycline-inducible luciferase (shLUC) or BRCA2 (shBRCA2) shRNA. (F) Quantification of colony survival for cells treated as described in panel E, normalized to doxycycline-treated shLUC-expressing cell lines. Bars represent mean survival and SD of three experiments. P-values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test. (G-H) Representative images of DLD1 BRCA2 -/- cells transfected with indicated siRNAs and stained for DAPI. Scale bar represents 10 µm. Arrows indicate either micronuclei (panel G), or nucleoplasmic bridges (panel H). (I) Quantification of number of micronuclei per cell for DLD1 WT cells or DLD1 BRCA2 -/- cells, transfected with indicated siRNAs. Bars represent the mean with SEM of 6 experiments with at least n>280 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test. (J) Quantification of number of nucleoplasmic bridges per cell for DLD1 WT cells or DLD1 BRCA2 -/- cells transfected with indicated siRNAs. Bars represent the mean with SEM of 6 experiments with at least n>280 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Luciferase, shRNA, Expressing, Comparison, Transfection, Two Tailed Test

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: mRNA-seq of rat tissues. To explore the related molecular mechanism of bronchiolitis obliterans, high-throughput analysis was conducted using the lung tissues of rats. (A) DEGs in DA group rats relative to the control are shown using a volcano plot. DEGs were defined as an absolute value of log2FC >1.5 and adjusted-P<0.001. (B) Quantitative PCR was carried out to verify the results of mRNA-seq. Four DEGs were randomly selected for verification. (C) Gene Ontology analysis revealed that apoptosis, inflammation, fibrosis, EMT and epithelium-associated items were enriched by DEGs. (D) Venn diagram shows that 47 DEGs commonly existed in the six datasets. (E) Heatmap of 47 DEGs demonstrated that most genes were functional proteins. (F) CIP2A was located on chromosome 11 and was increased in DA-treated rats. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; FC, fold change; mRNA-seq, mRNA-sequencing; SLC1A6, solute carrier family 1 member 6; ERN2, endoplasmic reticulum to nucleus signaling 2; RNASE2, ribonuclease A family member 2; TPSB2, tryptase β2.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: High Throughput Screening Assay, Control, Real-time Polymerase Chain Reaction, Functional Assay, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A expression is increased in DA group rats. (A) Quantitative PCR and western blotting, and (B) immunohistochemistry (scale bar: 50 µm) were used to detect the increased expression of CIP2A in samples from the DA group, which was initially determined by mRNA-sequencing. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Sequencing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A attenuates the injuries induced by DA in the bronchi and lungs of rats. To evaluate the effect of CIP2A inhibition on bronchiolitis obliterans, Eth, a CIP2A inhibitor, was injected subcutaneously into DA group rats. (A) Animal experimental timeline. Representative (B) H&E and (C) Masson's trichrome-stained lung slices (scale bar: 200 µm). Quantitative analysis of severity is shown. (D) Bronchoalveolar lavage fluid total cell count, and macrophage, neutrophil, lymphocyte and eosinophil counts in the control rats, DA-exposed rats and Eth-treated DA-exposed rats. (E) Western blotting and (F) immunohistochemistry were used to measure the protein expression levels of CIP2A in lung tissues. n=6. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; H&E, hematoxylin and eosin.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: Inhibition, Injection, Staining, Cell Counting, Control, Western Blot, Immunohistochemistry, Expressing

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting cell proliferation regulating inhibitor of protein phosphatase 2A weakens the epithelial-mesenchymal transition and inflammation induced by DA in the bronchi and lungs of rats. (A) E-cadherin and (B) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). (C) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (D) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. n=6. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: Immunofluorescence, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A reduces damage caused by DA in human primary bronchial epithelial cells. To explore the cellular mechanisms, an in vitro cellular model of bronchiolitis obliterans was employed. (A) Eth (6 µM) showed the best inhibitory effect on CIP2A expression. (B) Western blotting was used to measure the protein expression levels of α-SMA, fibronectin, Snail and iNOS. (C) Enzyme-linked immunosorbent assay kits were used to analyze the levels of IL-1β, IL-6 and TNF-α. (D) E-cadherin and (E) Vimentin levels were detected by immunofluorescence (scale bar: 50 µm). n=3. α-SMA, α-smooth muscle actin; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; Eth, ethoxysanguinarine; IL, interleukin; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A blocks the activation of NF-κB signaling in HPBECs. The expression of CIP2A, and phosphorylation of IKBα and expression of p65 were assessed by western blotting. n=3. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; HPBECs, human primary bronchial epithelial cells; IκBα, inhibitor of NF-κB α; NF-κB, nuclear factor-κB; p-, phosphorylated.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: CIP2A promotes BO by activating the NF-κB pathway. BO is caused by DA and mediated by CIP2A. CIP2A deficiency inhibits fibrosis, inflammation and EMT in BO models through suppressing the NF-κB pathway. Solid and dashed arrows both indicate promotion. The solid arrow reveals the molecular mechanism through which the dashed arrow operates. BO, bronchiolitis obliterans; DA, diacetyl; CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; EMT, epithelial-mesenchymal transition; NF-κB, nuclear factor-κB.

Article Snippet: The following antibodies (Proteintech Group, Inc.) were used in the present study: CIP2A monoclonal antibody (cat. no. 67843-1-Ig; 1:10,000), α-smooth muscle actin (α-SMA) polyclonal antibody (cat. no. 14395-1-AP; 1:1,000), fibronectin polyclonal antibody (cat. no. 15613-1-AP; 1:10,000), Snail polyclonal antibody (cat. no. 13099-1-AP; 1:10,000), inducible NO synthase (iNOS) polyclonal antibody (cat. no. 22226-1-AP; 1:500), NF-κB inhibitor α (IκBα) polyclonal antibody (cat. no. 10268-1-AP; 1:20,000), phosphorylated-IκBα (p-IκBα; Ser32/36) recombinant antibody (cat. no. 82349-1-RR; 1:20,000), p65 polyclonal antibody (cat. no. 10745-1-AP; 1:3,000), HRP-conjugated Affinipure goat anti-rabbit IgG(H+L) (cat. no. SA00001-2; 1:10,000), HRP-conjugated Affinipure rabbit anti-goat IgG(H+L) (cat. no. SA00001-4; 1:10,000), HRP-conjugated Affinipure goat anti-mouse IgG(H+L) (1:10,000), Histone-H3 polyclonal antibody (cat. no. 17168-1-AP; 1:2,000) and β-actin monoclonal antibody (cat. no. 66009-1-Ig; 1:20,000). β-actin was used as an internal control and Histone-H3 was used as a nuclear loading control.

Techniques:

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 5. AMPK activation with sodium salicylate mediates c-Myc ubiquitinylation and nuclear depletion. (A) Exposure of HCT116 colon cancer cells to 1 or 3 mM salicylate for 48 h (lanes 2 and 3, respectively) elicited the loss of c-Myc expression accompanied by CIP2A, but no change in the levels of S62 phosphorylated c-Myc. Cells were also treated with a combination of salicylate (1 or 3 mM) and compound C (dorsomorphin 100 nM) for 8 h (lanes 5 and 6, respectively). (B) Salicylate induced phosphorylation of PP2A Y307. Samples were analyzed with an anti-pY307-PP2Ac antibody, and the blots were reprobed with an anti-PP2Ac antibody. (C) Sodium salicylate induced AMPK-mediated ubiquitinylation of c-Myc. HEK293 cells were transiently transfected with c-Myc-HA (lanes 1 through 4) or a mock construct (HA-vector, lane 5). Cells were then either left untreated (lanes 1 and 5) or treated with compound C (lane 2), sodium salicylate (lane 3), or a combination of both (lane 4), for three hours. C-Myc-HA-tagged protein was then immunopurified and its ubiquitination levels analyzed with a ubiquitin antibody by western blot (upper panel, ubiquitin). Immunoblots of the corresponding cell lysates (ubiquitin antibody) and purified proteins (HA antibody) are shown (lower

Article Snippet: The mouse monoclonal antibody against CIP2A (2G103B5) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Transfection, Construct, Plasmid Preparation, Ubiquitin Proteomics, Western Blot, Purification

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 6. Model of potential mode of action of salicylate on c-Myc. Salicylate decreases the expression of MYC mRNA at the transcriptional level. In addition, salicylate activates AMPK both through directly binding to the ADaM site and changes in the mitochondrial membrane potential, which lead to an increased AMP:ATP ratio. Activated AMPK can directly bind to c-Myc and phosphorylate it at the bHLH-LZ region (S373 and T400), disrupting DNA binding. Additionally, phosphorylation of PP2A at Y307 and downregulation of CIP2A further compound the regulatory loop, decreasing c-Myc levels and affecting the c-Myc transcriptional program. Modulation of miR-34 defines another regulatory loop, further linking AMPK activation and c-Myc expression.

Article Snippet: The mouse monoclonal antibody against CIP2A (2G103B5) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Binding Assay, Membrane, Phospho-proteomics, Activation Assay