Journal: bioRxiv

Article Title: SMC1A facilitates gastric cancer cells proliferation, migration and invasion via promoting SNAI2 activated EMT

doi: 10.1101/2021.09.23.461612

Figure Lengend Snippet: (A) The expression of SMC1A was analyzed in cancer tissues from TCGA data. (B) RT-qPCR analysis for SMC1A mRNA in 20 samples of GC tissues and the corresponding adjacent tissues. (C) The overall survival of GC patients was evaluated using Kaplan-Meier Plotter. The expression of SMC1A was examined in human gastric cancer cell lines and the human gastric epithelial cell line GES-1using RT-qPCR method (D) and western blot method (E). **P<0.01, **P<0.001, ***P<0.001.

Article Snippet: The SMC1A cDNA ORF clone (HG18194-UT) and SNAI2 cDNA ORF clone (HG11196-M) were purchased from SinoBiological(Beijing, China), and subcloned into a pCDNA3.1 vector.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: SMC1A facilitates gastric cancer cells proliferation, migration and invasion via promoting SNAI2 activated EMT

doi: 10.1101/2021.09.23.461612

Figure Lengend Snippet: (A) The expression of SMC1A was examined in SMC1A silenced and overexpressed cells by western blot method. CCK-8 assay (B) and Colony formation assay (C) were used to determine the effect of SMC1A knockdown and overexpression on cell proliferation. Matrigel invasion assay (D) and Wound healing assay (F) analyzed the effect of SMC1A knockdown and overexpression on cell invasion and migration respectively. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The SMC1A cDNA ORF clone (HG18194-UT) and SNAI2 cDNA ORF clone (HG11196-M) were purchased from SinoBiological(Beijing, China), and subcloned into a pCDNA3.1 vector.

Techniques: Expressing, Western Blot, CCK-8 Assay, Colony Assay, Over Expression, Invasion Assay, Wound Healing Assay, Migration

Journal: bioRxiv

Article Title: SMC1A facilitates gastric cancer cells proliferation, migration and invasion via promoting SNAI2 activated EMT

doi: 10.1101/2021.09.23.461612

Figure Lengend Snippet: ( A) SNAI2 level was examined in SMC1A silenced and overexpressed cells using western blot method. (B) Proteins level of EMT markers E-cadherin, N-cadherin and Vimentin were detected after SMC1A depletion and overexpression. Immunofluorescence assay detected the expression of E-cadherin (C) and Vimentin (D) in SMC1A silenced and overexpressed cells. (E) The expression of SNAI2 was examined in SNAI2 overexpressed cells by western blot method. (F) Proteins level of EMT markers E-cadherin, N-cadherin and Vimentin were detected in response to the treatment of SMC1A siRNA and SMC1A siRNA+SNAI2.

Article Snippet: The SMC1A cDNA ORF clone (HG18194-UT) and SNAI2 cDNA ORF clone (HG11196-M) were purchased from SinoBiological(Beijing, China), and subcloned into a pCDNA3.1 vector.

Techniques: Western Blot, Over Expression, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: SMC1A facilitates gastric cancer cells proliferation, migration and invasion via promoting SNAI2 activated EMT

doi: 10.1101/2021.09.23.461612

Figure Lengend Snippet: CCK-8 assay (A) and Colony formation assay (B) were performed to analysis cell proliferation in response to the treatment of SMC1A siRNA and SMC1A siRNA+SNAI2. Matrigel invasion assay (C) and Wound healing assay (D) used to investigate cell invasion and migration in response to the treatment of SMC1A siRNA and SMC1A siRNA+SNAI2. *P<0.05.

Article Snippet: The SMC1A cDNA ORF clone (HG18194-UT) and SNAI2 cDNA ORF clone (HG11196-M) were purchased from SinoBiological(Beijing, China), and subcloned into a pCDNA3.1 vector.

Techniques: CCK-8 Assay, Colony Assay, Invasion Assay, Wound Healing Assay, Migration

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 1 Comprensive analysis of SMC1A-cohesin gene at different steps during CRC development. OncoScan was used to obtain both CNVs and LOH in mucosa (n = 16), early adenoma (n = 16) and carcinoma (n = 16) samples. a CNVs profile in mucosa. b CNVs profile in adenomas. c CNVs profile in carcinoma showing the gain of whole chromosomes 7, 13, and X and the loss of chromosome 18. d Mutational screening in colorectal early adenomas and carcinoma allowed us to identify twenty-five SMC1A mutations (16 mutations in carcinomas and 9 in adenomas, see Table 2). Example of representative SMC1A sequencing is reported showing the nucleotide change c.G1966A (leading to p.A656T amino acid change) identified in patient 6. e Diagram of the SMC1A protein with mutations identified in carcinomas (above) and adenomas (below). The protein length is not in scale. f Percentage of subjects (n = 66) analyzed by SMC1A immunohistochemistry and showing strong, moderate and weak intensity. g Examples of representative immunohistochemistry results are shown. It is evident that the expression of SMC1A protein increased during cancer development

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Sequencing, Immunohistochemistry, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 2 Effects of SMC1A mutation and overexpression in vivo. a HCT116 cells, a colorectal cancer cell line with stable karyotype, were stably transfected with both SMC1A wild-type gene and SMC1A c.A2027G mutation. Western blot shows a more marked expression in HCT116 SMC1A wild-type (2) and HCT116 SMC1A c.A2027G (3) when compared with HCT116 cells (1). An antibody against Tubulin was used as loading control. b The effects of HCT116, HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G cells were analyzed in vivo using an immunocompromised mouse model. Time-dependent analysis shows that HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G require 11 and 13 days to form tumors. In contrast, the development of tumors peaked after 18 days with HCT116 cells. c Representative images of tumors formed in the mice with HCT116 cells. d Tumors induced by HCT116 SMC1A wild-type. e Tumors formed in the mice in which HCT116 SMC1A c.A2027G cells were implanted. f Change in tumor weight. g Difference in tumor volume after subcutaneous cell inoculation. h Example of representative histopathological examination performed with hematoxylin and eosin staining. Enlargement 500x. *p < 0.05

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Over Expression, In Vivo, Stable Transfection, Transfection, Western Blot, Expressing, Control, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 3 Gene expression profile in induced tumors. a RNA-seq data shows that HCT116 SMC1A wild-type induced tumors display 401 up- and 343 down-regulated genes, whereas HCT116 SMC1A c.A2027G-induced tumors show 486 up- and 256 down-regulated genes. b Venn diagram showing that HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G share sixty-eight dysregulated genes. c Heatmap of sixty-eight dysregulated genes showing that the transcriptional effects were very small, with fold changes ranging from + 0.8 to −0.8

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RetSat stabilizes mitotic chromosome segregation in pluripotent stem cells

doi: 10.1007/s00018-024-05413-x

Figure Lengend Snippet: RetSat interacts with chromosome condensation components Nudcd2. a . Illustration of immunoprecipitation combined with LC-MS/MS in mitotic mESCs using anti-RetSat antibody. Isotypical IgG and RetSat KO mitotic cells were set up as negative controls. b . Heatmap of potential RetSat interactome based on LC-MS/MS identification results. c . Co-immunoprecipitation (co-IP) in lysis from mitotic RetSat WT mESCs. Isotypic IgG and RetSat KO mitotic cells were set up as negative controls. d . Immunoblotting detection of Smc1a, Smc3 and Nudcd2 in whole-cell lysis from RetSat WT or KO mESCs, respectively. b actin was used as a loading control. e . Immunoblotting detection of Smc1a, Smc3 and Nudcd2 in chromosomal lysis from RetSat WT or KO mESCs, respectively. H2A was used as a loading control

Article Snippet: The following antibodies were obtained from the indicated suppliers: rabbit anti-RetSat polyclonal antibody (Invitrogen, Cat. No. PA5-65443, 1:1000 for immunoblotting), mouse anti-RetSat monoclonal antibody (made in our laboratory, we have isolated and purified a high concentration of single epitope specific RetSat monoclonal antibody at a concentration of ∼ 0.8 mg/ml after fusion culture and screening of hybridoma cells from mice immunized with RetSat antigen, 1:400 for immunoblotting, 1:20 for immunofluorescence and 3 μg for immunoprecipitation), rabbit anti-Aurora B polyclonal antibody (Abcam, Cat. No. ab2254, 1:500 for immunofluorescence), rabbit anti-Oct4 monoclonal antibody (Abcam, Cat. No. ab181557, 1:1000 for immunoblotting), rabbit anti-Sox2 monoclonal antibody (Abcam, Cat. No. ab92494, 1:1000 for immunoblotting), rabbit anti-Nudcd2 polyclonal antibody (Proteintech, Cat. No. 21205-1-AP, 1:1000 for immunoblotting), mouse anti-Smc1a monoclonal antibody (Proteintech, Cat. No. 66987-1-Ig, 1:1000 for immunoblotting), rabbit anti-Smc3 polyclonal antibody (Proteintech, Cat. No. 14185-1-AP, 1:500 for immunoblotting), mouse anti-Gapdh monoclonal antibody (Beyotime, Cat. No. AF0006, 1:1000 for immunoblotting), mouse anti-β-Actin monoclonal antibody (Sigma-Aldrich, Cat. No. A1978, 1:2000 for immunoblotting), rabbit anti-Histone H2A polyclonal antibody (Beyotime, Cat. No. AH419, 1:1000 for immunoblotting), mouse IgG antibody (Beyotime, Cat. No. A7028, 1 μg for immunoprecipitation).

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Lysis, Western Blot, Control

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Twenty-one mutations in SMC1A identified among 29 unrelated CdLS probands. (A) Schematic illustration of SMC1A gene structure and localization of mutations in CdLS probands. Mutations are uniformly distributed along SMC1A coding regions. Labeled blocks represent exons. Mutations of probands are described in both nucleotide and amino acid changes. Probands from our group are marked with “CDL” or “UC”, probands reported previously by Deardroff et al. are underlined with previous labels in the brackets, probands reported by Musio et al. and Borck et al. are marked as “Musio” and “Borck” in Italic, respectively, affected males are labeled with “♂”. (B) SMC1A mutations are matched to the corresponding protein domains. Most of the mutations are located outside the conserved protein domains and no mutation is within functional motifs. The hinge domain is mutation free. The arrows point to the 7 probands with expression analyzed in this report. Amino terminal P-loop NTPase domain: aa 4-148; SMC proteins Flexible Hinge Domain: aa 515-629; carboxy terminal P-loop NTPase domain: aa 1,117-1,220. Motifs with the amino ternimal P-loop NTPas domain are: walker/P-loop (aa 32-39), ATP binding sites (aa 35-36 and aa 38-40), Q-loop/lid (aa 134-137). Motifs within the carboxy terminal P-loop NTPase domain are: ABC transporter signature motif (aa 1,128-1,137), Walker B (aa 1,152-1,157), ATP binding site (aa 1,156-1,157), D-loop (aa 1,160-1,163) and H-loop/switch region (aa 1,185-1,191) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). GenBank NM_006306.2 and NP_006297.2 were used as SMC1A sequence references. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Labeling, Mutagenesis, Functional Assay, Expressing, Binding Assay, Sequencing

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Twenty-one in-frame mutations identified in SMC1A gene among 29 unrelated CdLS probands.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Mutagenesis

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Transcriptional analysis of CdLS probands with SMC1A mutations. (A) RT-PCR shows that both male and female probands express SMC1A mRNA. P: proband; Ma: mother; Fa: father. 107P is a female proband and 017P is a male proband. (B) Quantitative RT-PCR reveals females expressed twice as much SMC1A mRNA as males, and there is no difference between controls and probands. One male proband (017P) and 6 female probands (060P, 074P, 107P, 144P, 174P and 203P) were tested. (C) Quantitative RT-PCR analyzing the 23 samples by their genders further confirms that SMC1A escapes X-inactivation. (D) Quantitative RT-PCR analysis on 5 different families demonstrated remarkable variations between related and un-related individuals. Families 017, 144, 174, 107 and 074 were analyzed. Primer pairs amplifying the 5′ end were used for this analysis. Reverse transcribed cDNAs were tested in triplicates at each dilution for 3 serial dilutions.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Wild type allele is transcribed at a slightly higher level than the mutant allele of SMC1A as demonstrated by allele specific quantitative RT-PCR in proband 074P. Wild type SMC1A allele has a small but significant elevated expression level (p=0.01) as compared to the mutant allele in 074P.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Western blot analysis of SMC1A protein in CdLS probands with SMC1A mutations. RAD21 protein was also tested as an internal protein control. All examined individual expressed comparable amounts of either SMC1A or RAD21. No difference at the protein level is observed between probands (017P, 174P, 60P and 203P) and healthy controls (017Fa, 017Ma, 174Fa and 174Ma). The male proband 017P does not show less SMC1A or RAD21 proteins than others. Similarly, it is hard to identify the dosage alterations between males (017P, 017Fa, 174Fa) and females (017Ma, 174P, 174Ma, 60P and 203P). SMC1A and RAD21 are represented by a ∼160KD and a ∼130 KD protein band, respectively. ACTB is the loading control and appears as a protein band around 40 KD. P: proband; Ma: mother; Fa: father.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Western Blot

Journal:

Article Title: SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome

doi: 10.1002/humu.21095

Figure Lengend Snippet: Classifications of 3 groups of samples based on a 23 gene expression profile. Unsupervised clustering of 21 healthy controls (green dots), 20 severe CdLS probands with NIPBL mutations (red dots) and 7 CdLS probands with SMC1A mutations (blue dots) by Principle Component Analysis (PCA). The separation between the control and severe proband groups indicates that they have different gene expression patterns. Individuals with SMC1A mutations have expression patterns intermediate to these two distinct groups.

Article Snippet: Primer Design SMC1A cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_006306.2","term_id":"30581134","term_text":"NM_006306.2"}} NM_006306.2 ) was used as reference sequence.

Techniques: Expressing