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: 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

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: HR inhibition impairs the chromosomal association of CIP2A and disrupts chromosome conformation. GV oocytes were treated with ETP for 15 min, washed out and matured in the presence of B02 for up to 15 h. ( A ) Representative images of MII chromosomes stained with CIP2A and ACA antibodies. Scale bar, 10 μm. ( B ) Quantification of overall CIP2A intensity. ( C ) Enlarged views of the boxed regions (a–e) in panel (A). Chromosomes are categorized into three groups: intact, fragmented and aggregated. Centric and acentric fragments are marked with arrows and arrowheads, respectively. ( D , E ) Quantification of CIP2A-positive chromatid and CIP2A intensity on each fragment in ETP-treated groups. ( F ) Quantification of the types of chromosome conformations observed in each treatment group. ( G ) Distribution of centric and acentric fragments in B02-treated oocytes with DNA damage. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Inhibition, Staining

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: CIP2A recruitment onto damaged chromosomes is dependent on the HR pathway. Oocytes at the MI stage were treated with ETP in the presence of either DMSO or B02 for 15 min. After ETP washout, oocytes were allowed to recover for 1 h in the presence (+) or absence (−) of B02. ( A ) Representative images of MI oocytes showing CIP2A chromosomal recruitment. Profiling graphs are shown below to illustrate the pole-to-chromosome distribution of CIP2A. Scale bar, 10 μm. ( B , C ) Quantification of MI spindle length and chromosomal CIP2A intensity. ( D ) Representative images of MI chromosomes stained with p-TOPBP1 and ACA antibodies. Scale bar, 10 μm. ( E ) Quantification of p-TOPBP1 intensity. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001; ns, not significant.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Staining

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: Chromosomal recruitment of CIP2A is independent of NHEJ, MMEJ and RAD52-dependent repair pathways. ( A ) Representative images of MI oocytes treated with ETP for 15 min in the presence of SCR7 (NHEJ inhibitor) or ART558 (MMEJ inhibitor). Oocytes were subjected to immunostaining with CIP2A antibody, showing chromosomal recruitment of CIP2A. Scale bar, 10 μm. ( B , C ) Quantification of MI spindle length and chromosomal CIP2A intensity. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001; ns, not significant. ( D ) Representative images of MI oocytes treated with ETP for 15 min in the presence of D-I03 (RAD52 inhibitor). Oocytes were subjected to immunostaining with CIP2A antibody, showing chromosomal recruitment of CIP2A. Scale bar, 10 μm. ( E ) Quantification of chromosomal CIP2A intensity. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001; ns, not significant.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Immunostaining

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: CIP2A depletion leads to chromosome fragmentation after DSB induction. ( A ) Scheme of Trim-away experiments to acutely deplete CIP2A in oocytes at the MI stage. GV oocytes were microinjected with Trim21-mCherry mRNA and cultured for 8 h to reach the MI stage. After confirming mCherry expression, MI oocytes were injected with either IgG control antibody (CTL) or CIP2A antibody (CIP2A-TA). After 1 h incubation to allow CIP2A depletion, oocytes were treated with ETP for 15 min, washed out and matured to the MII stage. ( B ) Representative images of MII oocytes showing chromosome fragmentations and CIP2A intensity. Scale bar, 10 μm. ( C , D ) Quantification of chromosomal CIP2A intensity and chromosome fragmentation. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001. ( E ) Correlation of CIP2A intensity against chromosome fragmentation after ETP treatment. r = −0.9091, P < 0.05. ( F ) Representative images of MII chromosomes illustrating chromosome fragmentation and aggregation. Centric and acentric fragments are marked with arrows and arrowheads, respectively. Scale bar, 10 μm. ( G ) Quantification of the types of chromosome conformations observed in each treatment group. ( H ) Distribution of centric and acentric fragments in CIP2A-depleted oocytes with DNA damage. Data are presented as mean ± SEM of three independent experiments.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Cell Culture, Expressing, Injection, Control, Incubation

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: BRCA1 depletion impairs chromosomal recruitment of CIP2A induced by DNA damage. ( A ) Oocytes at the MI stage were treated with ETP in the presence of either DMSO or B02 for 15 min. Representative images of MI chromosomes stained with BRCA1 antibody. Scale bar, 10 μm. ( B ) Quantification of BRCA1 intensity. ( C ) Representative images of CIP2A chromosomal recruitment in MI oocytes after BRCA1 Trim-away (BRCA1-TA) and ETP treatment. BRCA1 Trim-away was conducted as described for CIP2A Trim-away. Scale bar, 10 μm. ( D ) Quantification of chromosomal CIP2A intensity. ( E ) Representative images of MI chromosomes stained with BRCA1 antibody after CIP2A Trim-away (CIP2A-TA). Scale bar, 10 μm. ( F ) Quantification of BRCA1 intensity. Data are presented as mean ± SEM of three independent experiments; **** P < 0.0001; ns, not significant.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Staining

Journal: Nucleic Acids Research

Article Title: Novel BRCA1–PLK1–CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis

doi: 10.1093/nar/gkae1207

Figure Lengend Snippet: Impaired chromosomal recruitment of CIP2A induced by HR inhibition is restored by PLK1 inhibition. ( A ) Representative images of p-T210-PLK1 at spindle poles of MI oocytes after BRCA1 Trim-away (BRCA-TA). Scale bar, 10 μm. ( B ) Quantification of p-T210-PLK1 intensity at spindle poles. ( C ) Representative images of oocytes showing CIP2A chromosomal recruitment after treatment with ETP, B02 and BI2536. Scale bar, 10 μm. ( D ) Quantification of chromosomal CIP2A intensity. Data are presented as mean ± SEM of three independent experiments. **** P < 0.0001, *** P < 0.0006.

Article Snippet: Oocytes were then matured in IBMX-free M2 medium for 5 h before an injection of CIP2A (Santa Cruz, sc-80662), BRCA1 (Abcam, ab17680) or IgG (Santa Cruz, sc-2027) antibodies at a final concentration of 100 ng/μl.

Techniques: Inhibition