Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

Iwashita, Shintaro; Suzuki, Takehiro; Kiriyama, Yoshimitsu; Dohmae, Naoshi; Ohoka, Yoshiharu; Song, Si‐Young; Nakashima, Kentaro

doi:10.1042/bsr20194012

Cited by 3 publications

(3 citation statements)

References 35 publications

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“…Freshly cultured cells were subjected to protein extraction with RIPA (Beyotime, #P0013B) supplied with a proteinase inhibitor cocktail (Promega, #G6521). Western blot was performed as described previously [ 16 , 17 ]. The following primary antibodies were used: Anti-CRAD antibody (Sigma, #HPA043249) and anti-GAPDH antibody (Santa Cruz Biotechnology, #sc-32233).…”

Section: Methodsmentioning

confidence: 99%

Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4

et al. 2020

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Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related mortality globally. However, the mechanism underlying NSCLC is not fully understood. Here we investigated the role of cancer-related regulator of actin dynamics (CRAD) in NSCLC. We showed that CRAD was up-regulated in human NSCLC tissues and lung cancer cell lines. Lentivirus-mediated knockdown of CRAD repressed the proliferation and colony growth of A549 and H1299 cells. Apoptosis was enhanced by CRAD silencing in both cells, implicating that CRAD might maintain the survival of lung cancer cells. Microarray and bioinformatic assay revealed that CRAD directly or indirectly regulated diverse genes, including those involved in cell cycle and DNA damage repair. qRT-PCR and Western blot results confirmed the dysregulated genes as shown in microarray analysis. Claudin 4 was up-regulated in CRAD silenced A549 cells. The knockdown of Claudin 4 blocked the effects of CRAD on the expression of cell cycle and apoptosis effectors and enhanced the viability of A549 cells with CRAD down-regulation. Taken together, our findings demonstrate that CRAD acts as an oncogene in NSCLC at least partly through repressing Claudin 4.

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Section: Methodsmentioning

confidence: 99%

Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4

et al. 2020

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“…The cfdp1 locus in zebrafish was initially misassigned to the RLTPR (RGD motif, leucine rich repeats, tropomodulin domain and proline-rich containing) gene and the role of BCNT/CFDP1 in the heart has not been investigated as yet. Thus, while there are sparse studies assessing the role of CFDP1, mainly in the chromatin remodeling complex via the maintenance of chromosome organization [23,24], its role in heart development and physiology remains to be addressed and evaluated, especially since GWAS studies have revealed the association of CFDP1 with CAD in humans [25].…”

Section: Introductionmentioning

confidence: 99%

Cfdp1 Is Essential for Cardiac Development and Function

Giardoglou

Deloukas

Dedoussis

et al. 2023

Cells

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Cardiovascular diseases (CVDs) are the prevalent cause of mortality worldwide. A combination of environmental and genetic effectors modulates the risk of developing them. Thus, it is vital to identify candidate genes and elucidate their role in the manifestation of the disease. Large-scale human studies have revealed the implication of Craniofacial Development Protein 1 (CFDP1) in Coronary Artery Disease (CAD). CFDP1 belongs to the evolutionary conserved Bucentaur (BCNT) family, and to date, its function and mechanism of action in Cardiovascular Development are still unclear. We utilized zebrafish to investigate the role of cfdp1 in the developing heart due to the high genomic homology, similarity in heart physiology, and ease of experimental manipulations. We showed that cfdp1 was expressed during development, and we tested two morpholinos and generated a cfdp1 mutant line. The cfdp1−/− embryos developed arrhythmic hearts and exhibited defective cardiac performance, which led to a lethal phenotype. Findings from both knockdown and knockout experiments showed that abrogation of cfdp1 leads to downregulation of Wnt signaling in embryonic hearts during valve development but without affecting Notch activation in this process. The cfdp1 zebrafish mutant line provides a valuable tool for unveiling the novel mechanism of regulating cardiac physiology and function. cfdp1 is essential for cardiac development, a previously unreported phenotype most likely due to early lethality in mice. The detected phenotype of bradycardia and arrhythmias is an observation with potential clinical relevance for humans carrying heterozygous CFDP1 mutations and their risk of developing CAD.

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“…Mouse BCNT gene, cp27 was suggested to mediate early organogenesis and high level of its expression was demonstrated in developing mouse teeth, heart, lung and liver 18–20 . Thus, while there are sparse studies accessing the role of cfdp1 in chromatin remodeling complex via the maintenance of chromosome organization 21,22 , little is known about not only its function and the mechanism it is involved in but also its role in heart physiology and morphogenesis. Recently, it was shown that the zebrafish gazami mutants carry a point mutation in the 3’ end of the gene, resulting in a truncated protein 23 .…”

Section: Introductionmentioning

confidence: 99%

Cfdp1 is Essential for Cardiac Development and Function

Giardoglou

Deloukas

George

et al. 2022

Preprint

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Cardiovascular diseases (CVDs) are the prevalent cause of mortality worldwide and account for the most common noncommunicable disease. CVDs describe a wide spectrum of disorders affecting the proper function, physiology and morphogenesis of the heart and blood vessels. The risk of developing cardiovascular diseases is modulated by a combination of environmental and genetic effectors. Thus, it is highly important to identify candidate genes and elucidate their role in the manifestation of the disease. Large-scale human studies have revealed the implication of Craniofacial Development Protein 1 (CFDP1) in coronary artery disease (CAD). CFDP1 belongs to the evolutionary conserved Bucentaur (BCNT) family and up to date, its function and mechanism of action in Cardiovascular Development is still unclear. In this study, we utilize zebrafish to investigate the role of cfdp1 in the developing heart due to the high genomic homology, similarity in heart physiology and the ease of experimentally manipulation. We showed that cfdp1 is expressed during development and at 120 hours post fertilization its expression is restricted to the region of the heart and the head. We then generated a cfdp1-null zebrafish line using CRISPR-Cas9 system which led to a lethal phenotype since knockout embryos do not reach adulthood. cfdp1-/- embryos develop arrhythmic hearts and defective cardiac performance exhibiting statistically significant differences in heart features including End Diastolic Volume, Cardiac Output, Ejection Fraction and Stroke Volume. Myocardial trabeculation is also impaired in cfdp1-/- embryonic hearts, implying its regulatory role also in this developmental process. Findings from both knockdown and knockout experiments showed that abrogation of cfdp1 leads to downregulation of Wnt signaling in embryonic hearts during valve development but without affecting Notch activation in this process.

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Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

Cited by 3 publications

References 35 publications

Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4

Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4

Cfdp1 Is Essential for Cardiac Development and Function

Cfdp1 is Essential for Cardiac Development and Function

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