Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: CITED2 is the target gene of SEs. (A, B) The hockey map lists target genes that are regulated by enhancers under normoxia and hypoxia. The entire curve consists of many points, each of which represents a gene. The right side of the image represents genes regulated by SEs. (C) The images represent the level of H3K27ac modification upstream of the CITED2 promoter under normoxia and hypoxia. (D) The three gene fragments and promoter fragments were loaded into plasmids for dual luciferase experiments. In the experimental group with three gene segments, the luciferase activity was higher, and the fluorescence intensity was stronger. (E) The ChIP‐PCR experiment was carried out according to the primers of the promoter constructed by the CITED2 gene and these three gene sequences. The results showed that the region upstream of the CITED2 gene promoter, including the promoter, had significant H3K27ac modification. The fifth primer represented the desert region of the gene. (F) RT‐qPCR results of CITED2 mRNA level after treatment of JQ‐1 and i‐BET. Nor: normal, Hyp: hypoxia, SE: super‐enhancer. (Bar = mean ± S.E.M, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Modification, Luciferase, Activity Assay, Fluorescence, Construct, Quantitative RT-PCR

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: Downregulation of CITED2 in the PH model. (A) The results of the in situ hybridisation experiments showed that the mRNA content of CITED2 was depressed in the pulmonary small vessels of model mice exposed to hypoxia for 12 and 21 days. (B, C) The protein expression of CITED2 decreased in mouse pulmonary arteries on days 12 and 21 of hypoxia. (D, E) The protein and mRNA levels of CITED2 in mPASMCs gradually decreased with increasing hypoxia duration. (F) The result of the FISH demonstrated that CITED2 was depressed in mPASMCs. (Bar = mean ± S.E.M, * p < 0.05; ** p < 0.01).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: In Situ, Hybridization, Expressing

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: CITED2 can affect the proliferation of idiopathic PAH patient cells. (A) The overexpression efficiency of the CITED2 was detected in iPAH patient cells. (B) Expression changes of CITED2 in iPAH patient cells. (C) The CCK8 experiment showed that overexpression of CITED2 could affect the growth and viability of cells. (D, E) After overexpression of CITED2, the levels of cell cycle‐related proteins decreased in patients. (F) Overexpression of CITED2 in hypoxia‐treated hPASMCs reduced Ki67 levels in the cells. (G) Flow cytometry results showed that overexpression of CITED2 reduced the proportion of cells in the G2/M phase and S phase and affected cell cycle progression. (H) After overexpression of CITED2, the expression of PCNA in patient cells decreased. (I, J) Overexpression of CITED2 in hypoxia‐treated hPASMCs also affected the expression of cell cycle‐related proteins. (Bar = mean ± S.E.M, * p < 0.05; ** p < 0.01).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Over Expression, Expressing, Flow Cytometry

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: Overexpression of CITED2 at the animal level is able to alleviate PH. (A) The results of gene enrichment analysis in normoxia and hypoxia showed that CITED2 was downregulated in hypoxia, showing a negative correlation. (B–D) Expression of CITED2 mRNA and protein in the pulmonary arteries of model mice. (E) Schematic diagram of lentivirus model mouse construction. (F) The decreased cardiopulmonary function of model mice was effectively reversed by overexpression of CITED2. (G) Right ventricular systolic blood pressure and right ventricular specific gravity were improved in the CITED2 overexpression group. (H, I) H&E and Masson staining of tissue sections of mouse pulmonary arterioles. (K) The expression of PCNA was downregulated in the pulmonary arteries of mice in the lentivirus‐treated group. (J) Tissue immunofluorescence experiments showed that the expression of Ki67 in small pulmonary vessels decreased significantly after CITED2 overexpression. A: artery. (L, M) The expression of cycle‐related proteins in the pulmonary arteries of mice in the lentivirus‐treated group was downregulated. LV: lentivirus. (Bar = mean ± S.E.M, ** p < 0.01; *** p < 0.001).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Over Expression, Expressing, Staining, Immunofluorescence

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: Overexpression of CITED2 can affect the proliferation of smooth muscle cells and hinder cell cycle progression. (A) Western blot results of efficiency detection after overexpression of CITED2. (B) The results of the CCK8 experiment indicated that overexpression of CITED2 could affect the growth and viability of mPASMCs. (C) Overexpression of CITED2 affected the expression of PCNA in cells. (D) After overexpression of CITED2, the proportion of cells in the G2/M and S phase was reduced, which affected cell division. (E, F) The results of the EdU cell fluorescence assay and Ki67 cell fluorescence assay indicated that overexpression of CITED2 could reduce cell proliferation. Red and green represent remarkably proliferating cells. (G, H) Overexpression of CITED2 at the cellular level can affect the expression levels of multiple proteins in the cell cycle. (Bar = mean ± S.E.M, ** p < 0.01; *** p < 0.001).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Over Expression, Western Blot, Expressing, Fluorescence

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FOXJ3 affects the expression of CITED2 at the transcriptional level and then regulates cell proliferation. (A–C) Western blot results of efficiency detection after overexpression and interference with FOXJ3 and CITED2. (D) Motifs of the transcription factor FOXJ3 predicted from gene motifs by segmenting the CITED2 SEs. (E–G) The mRNA and protein levels of CITED2 were affected by FOXJ3. (H, J) The growth viability of cells decreased after overexpression of FOXJ3 but recovered after interference with CITED2 on this basis. Red in EdU experiments represents cells undergoing proliferation. (I, M) Cell proliferation experiments showed that overexpression of FOXJ3 at the cellular level could affect cell proliferation, and this degree of cell proliferation was restored after interference with CITED2. (K, L) Overexpression of FOXJ3 in mPASMCs decreased the expression of cell cycle‐related proteins and kinase proteins, which was recovered after interference with CITED2. (Bar = mean ± S.E.M, ** p < 0.01; *** p < 0.001).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Expressing, Western Blot, Over Expression

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FOXJ3 cooperates with SEs to regulate the transcription of CITED2. (A) Schematic representation of overexpression plasmid construction and transfection. (B–D) In the dual‐luciferase experiment, plasmids containing three SEs motifs were co‐transfected with FOXJ3 plasmids into mPASMCs. When both were present, the luciferase activity was the greatest. (Bar = mean ± S.E.M, * p < 0.05).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: SEs indirectly affect cell proliferation during hypoxia. (A) Schematic diagram of pCMV‐CITED2 and pCMV‐SEs overexpression plasmids. (B, C) The results of EdU and Ki67 immunofluorescence experiments showed that cell proliferation was accelerated under hypoxia and that SEs could indirectly affect cell proliferation by regulating the level of CITED2. Red represents proliferating cells, and green represents Ki67 expression in cells. (D) Western blot results of efficiency detection after overexpression of pCMV‐SEs. (E) Expression of the proliferation indicator PCNA was suppressed in the presence of SEs. (F) When SEs are present, the cell cycle is affected, and cyclin protein expression decreases. (Bar = mean ± S.E.M, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Over Expression, Immunofluorescence, Expressing, Western Blot

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: Differential effects of CITED2 on PASMC proliferation under normoxia and hypoxia. Normally, the H3K27ac modification of the SEs and promoter regions of CITED2 is significant. Downstream proteins influenced by CITED2, such as cyclin proteins, CDKs and PCNA, decreased. SMC proliferation is also diminished. However, the phenomenon is reversed under hypoxia.

Article Snippet: The membranes were blocked with 10% skim milk, the primary antibody used include CITED2 (1:500, BOSTER, BM4825); FOXJ3 (1:500, Affinity, AF0605;1:500, Novus, NBP1‐71861); CyA (1:1000, BOSTER, BM1582); CyB (1:1000, BOSTER, BM4667); CyD (1:1000, BOSTER, BM0771); CDK1 (1:1000, BOSTER, PB0561); CDK2 (1:1000, BOSTER, PB0562); CDK4 (1:1000, BOSTER, PB0563); and PCNA (1:1000, BOSTER, BM0104), overnight at 4°C.

Techniques: Modification

Journal: bioRxiv

Article Title: p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

doi: 10.1101/2024.03.29.587346

Figure Lengend Snippet: p300/CBP degradation compared to bromodomain inhibition leads to stronger suppression of oncogenic gene programs. a. Volcano plot of gene expression in VCaP cells treated for 24 hours with either 100 nM CBPD-409 or 1 µM GNE-049. A set of genes, including CCND1 , CITED2 , and NKX3-1 , are exclusively repressed by CBPD-409. The data represent the log2 fold change (FC) of CBPD-409 relative to GNE-049 plotted against the –log10 p-value for each gene. Based on n=2 independent experiments. Statistical tests were two-tailed t-tests with the assumption of equal variances. b. Uniquely down-regulated genes in CBPD-409 relative to GNE-409 treated VCaP cells analyzed for overlap with MSigDB hallmark gene sets. The top five hallmark gene sets (ranked by p-value) are highlighted in red. c. Immunoblot analysis of indicated proteins in VCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049 for indicated times. d. Immunoblot analysis of labeled proteins and histone marks in VCaP cells pre-treated with different concentrations of thalidomide (Thali) for 1 hour, then treated with CBPD-409 at indicated concentrations for 4 hours. e. Quantitative-PCR (qPCR) of NKX3-1 , CCND1 , CITED2 , and MYC expression in VCaP cells pre-treated with 100 µM thalidomide for 1 hour, then treated with CBPD-409 or GNE-409 for 4 hours. f. Immunoblot analysis of indicated proteins and histone marks in VCaP cells treated with CBPD-409 or CBPD-409-Me (inactive analogue) for indicated times. g. Dose-response curves and IC50 of prostate cancer cells treated with CBPD-409 and GNE-049. Data are presented as mean +/− SD (n = 6). h. Rank-order plot of IC50 values for CBPD-409 across 131 human normal and cancer cell lines following 5 days of treatment. Models of AR-positive prostate cancer, AR-negative prostate cancer, non-neoplastic prostatic cells, multiple myeloma, and neuroblastoma are highlighted in specific colors. The originating tissue lineages are indicated below. PNET, primitive neuroectodermal tumor; EMRS, embryonal rhabdomyosarcoma.

Article Snippet: For immunoblotting, the following antibodies were used: p300 (Invitrogen: MA1-16608); CBP (Invitrogen: PA5-27369); AR (Abcam: ab133273); H2BK5ac (Cell Signaling Technology: 12799S); H2BK20ac (Cell Signaling Technology: 34156S); H2BK12ac (Abcam: ab40883); H2BK16ac (Abcam: ab177427); H3K27ac (Cell Signaling Technology: 8173S); H3K18ac (Active Motif: 39755); H3K4me1 (Abcam: ab8895); H3K4me3 (Active Motif: 39060); H3K27me3(Millipore: 07-449); H2B (Active Motif: 39210); H3 (Cell Signaling Technology: 3638S); H2BK120ac (Active Motif: 39119); Myc (Cell Signaling Technology: 9402S); KLK3/PSA (Dako: A0562); CITED2 (Abcam: ab108345); NKX3-1 (Cell Signaling Technology:83700S); CCND1 (Abcam: ab16663); FOXA1 (Thermo Fisher Scientific: PA5-27157); Vinculin (Cell Signaling Technology: 18799S); GAPDH (Santa Cruz Biotechnology: sc-47724); BRD2 (Bethyl Laboratories: A700-008); BRD3 (Bethyl Laboratories: A302-368A); BRD4 (Bethyl Laboratories: A700-004CF); GSPT1 (Proteintech: 28130-1-ap); Aiolos (Cell Signaling Technology: 15103S); Ikaros (Cell Signaling Technology: 14859S).

Techniques: Inhibition, Expressing, Two Tailed Test, Western Blot, Labeling, Real-time Polymerase Chain Reaction

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) expression was down-regulated in hypoxia-induced pulmonary hypertension (HPH) rat models

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Expressing

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) expression was down-regulated in pulmonary artery smooth muscle cells (PASMCs) in hypoxia

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Expressing

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Effects of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) overexpression on pulmonary artery smooth muscle cells (PASMCs)

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Over Expression

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Effects of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) knockdown on pulmonary artery smooth muscle cells (PASMCs)

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Knockdown

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) reversed Transforming growth factor-beta1 (TGF-β1)-induced proliferation and migration of pulmonary artery smooth muscle cells (PASMCs)

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Migration

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway

doi: 10.22038/IJBMS.2023.74455.16178

Figure Lengend Snippet: Peroxisome proliferatoractivated receptor gamma (PPARγ) cooperates with Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) to inhibit the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs)

Article Snippet: A protein-free quick blocking solution (Boster, AR0041) was used to block the membrane at room temperature for 1 hr, and then primary antibodies against Cited2 (1:500, Affinity), anti-PPARγ (1:2000, Proteintech), and anti-β-actin (1:3000, Affinity) were used to incubate the membranes overnight at 4 ° C. On the next day, HRP-labeled secondary antibody (1:5000, Affinity) was used to incubate the membranes at room temperature for 1 hr, and the chemiluminescence method was used for color development.

Techniques: Migration

Journal: Frontiers in cell and developmental biology

Article Title: CircSNHG5 Sponges Mir-495-3p and Modulates CITED2 to Protect Cartilage Endplate From Degradation.

doi: 10.3389/fcell.2021.668715

Figure Lengend Snippet: FIGURE 5 | MiR-495-3p targets the CITED2 and CITED2 promotes cell proliferation inhibit ECM degeneration. (A) Volcano plot of the significantly upregulated and downregulated mRNAs. (B) Venn diagram demonstrating the intersection of downregulated mRNAs and predicted target mRNAs. (C,D) The expression levels of CITED2 in CEP tissues were measured in 21 patients and 21 controls by qRT-PCR and Western blot (***P < 0.001). (E) Luciferase reporter analysis of either wild-type or mutant CITED2 3′-UTR activity. miR-495-3p was co-transfected with the wild-type or mutant vector. The presented values are the mean ± SEM of three different preparations (***P < 0.001). (F) 3′-UTR region of CITED2 mRNA was found to harbor a binding site for miR-495-3p. (G,H) The expression of CITED2, MMP13, Collagen II, and Aggrecan expression levels was assessed by qRT-PCR and Western blotting (*P < 0.05, **P < 0.01).

Article Snippet: The antibodies used in this study were collagen type II (1:3,000; Abcam, United Kingdom), aggrecan (1:1,000; Abcam, United Kingdom), MMP-13 (1:3,000; Abcam, United Kingdom), CITED2 (1:500; Santa Cruz Biotechnology), GAPDH (1: 5,000; Abcam, United Kingdom), and goat anti-rabbit secondary antibody (1:5,000; Abcam, United Kingdom).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Binding Assay

Journal: Frontiers in cell and developmental biology

Article Title: CircSNHG5 Sponges Mir-495-3p and Modulates CITED2 to Protect Cartilage Endplate From Degradation.

doi: 10.3389/fcell.2021.668715

Figure Lengend Snippet: FIGURE 6 | Overexpression of COL5A1 rescues the functional depletion caused by silencing circSNHG5. (A) The effect of CITED2 overexpression on miR-495-3p knockdown-mediated cell proliferation was measured by CCK-8 assay (*P < 0.05, **P < 0.01). (B,C) CITED2, MMP13, Collagen II, and Aggrecan expression levels were analyzed by qRT-PCR and Western blotting. The presented values are the means ± SD of three different preparations (***P < 0.001). (D) The effect of CITED2 overexpression on circSNHG5 knockdown-mediated cell proliferation was measured by CCK-8 assay (*P < 0.05, **P < 0.01). (E,F) CITED2, MMP13, Collagen II, and Aggrecan expression levels were analyzed by qRT-PCR and Western blotting. The presented values are the means ± SD of three different preparations (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: The antibodies used in this study were collagen type II (1:3,000; Abcam, United Kingdom), aggrecan (1:1,000; Abcam, United Kingdom), MMP-13 (1:3,000; Abcam, United Kingdom), CITED2 (1:500; Santa Cruz Biotechnology), GAPDH (1: 5,000; Abcam, United Kingdom), and goat anti-rabbit secondary antibody (1:5,000; Abcam, United Kingdom).

Techniques: Over Expression, Functional Assay, Knockdown, CCK-8 Assay, Expressing, Quantitative RT-PCR, Western Blot