Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: Primers used to reverse transcriptional quantitative PCR

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques:

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: The enrichment of CSCs from NSCLCs and detection of the expressing levels of PRRX1A/B and EMT hallmarkers in CSCs and NSCLC clinical samples. (A) CSCs from A549 and SPC-A1 cells were enriched. Then, the CD133+ proportion was analyzed by performing flow cytometry. (B) Serial replating assay was performed to detect self-renewal capacity. (C) RT-qPCR was performed to detect the expression patterns of stemness factors, PRRX1A/B and EMT hallmarkers; *, P<0.05, vs. HEB group. (D) RT-qPCR was performed to detect the expression patterns of PRRX1A/B and EMT hallmarkers between CSCs and their parental cells. *, P<0.05, vs. A549 cells; #, P<0.05, vs. SPC-A1 cells. (E) Semi-quantitative Western blot was performed to detect the protein levels of PRRX1A/B and EMT hallmarkers between CSCs and their parental cells; *, P<0.05, vs. A549 cells; #, P<0.05, vs. SPC-A1 cells. (F) Differential expression of PRRX1 between tumor and adjacent normal tissues in NSCLC. (G) PRRX1 protein expression in normal lung tissue and NSCLC specimens. Images were obtained from the Human Protein Atlas online database. (H) Kaplan-Meier curves stratified by PRRX1 mRNA expression. Data was available on GEPIA web server. CSCs, cancer stem-like cells; NSCLC, non-small cell lung cancer; PRRX1, paired-related homeobox 1; EMT, epithelial-mesenchymal transition.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, Western Blot, Quantitative Proteomics

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: The effect of PRRX1A on proliferation, invasive capacity, and stemness in A549- and SPC-A1-CSCs. After overexpression of Flag-PRRX1A, Flag-PRRX1B (A), and knockdown of PRRX1A and PRRX1 (B) in A549- or SPC-A1-CSCs, the efficacy was detected by performing semi-quantitative Western blot. (C) After PI staining, flow cytometric analyses was performed to detect cell cycle phases. *, P<0.05, vs. vector group. (D) Cell viability was measured after CCK-8 staining. *, P<0.05, vs. vector group. Sphere formation of A549-CSCs (E) and SPC-A1 CSCs (F) after overexpression or knockdown of PRRX1A/B was measured. *, P<0.05, vs. pENTR/U6 vector group; #, P<0.05, vs. pENTR/U6 group. (G) Transwell assay was performed to detect the effect of PRRX1A/B on invasive capacity. *, P<0.05, vs. vector group; #, P<0.05, vs. pENTR/U6 group. PRRX1, paired-related homeobox 1; CSCs, cancer stem-like cells.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Knockdown, Western Blot, Staining, Plasmid Preparation, CCK-8 Assay, Transwell Assay

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: The expression patterns of PRRX1A, PRRX1B, and TGF-β in lung cancer tissues. (A) RT-qPCR was performed to assess the expression patterns of stemness factors, PRRX1A, PRRX1B, and EMT hallmarkers. *, P<0.05, vs. adjacent tissues. (B) Expression levels of PRRX1A and TGF-β in lung cancer tissue samples were positively correlated. The correlation between PRRX1 and TGF-β1 (C), E-cadherin (D), N-cadherin (E), and Vimentin (F) were analyzed by using the GEPIA server. PRRX1, paired-related homeobox 1.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: PRRX1A regulates EMT processes via TGF-β/TGF-βR signaling pathway. (A) After overexpression of PRRX1A or PRRX1B, the expression levels of TGF-β1, β2, and β3 were determined by performing RT-qPCR analysis. *, P<0.05, vs. vector group. RT-qPCR assay (B) and semi-quantitative Western blot (C) were performed to detect the mRNA levels of E-cadherin, N-cadherin, and Vimentin after blocking TGF-β/TGF-βR signaling pathway. *, P<0.05, vs. vector group. #, P<0.05, vs. PRRX1A group. (D) Transwell assay was performed to detect the invasive capacity. *, P<0.05, vs. vector group; #, P<0.05, vs. vector + TGF-β1 group; &, P<0.05, vs. PRRX1A group. PRRX1, paired-related homeobox 1; EMT, epithelial-mesenchymal transition.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Blocking Assay, Transwell Assay

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: PRRX1A potentially binds to SOX2 and regulates stemness factors. (A) RT-qPCR was performed to detect the different expression patterns between CSCs and parental cells. *, P<0.05, vs. A549 group; #, P<0.05, vs. SPC-A1 group. (B) After overexpression or knockdown of PRRX1A/B, the stemness factors were quantitatively analyzed. *, P<0.05, vs. pENTR/U6 vector group. (C) Co-immunoprecipitation was performed to detect the binding of Flag-PRRX1A or Flag-PRRX1B to SOX2. d Detection of SOX2 protein level after overexpression of PRRX1A/B. PRRX1, paired-related homeobox 1; CSCs, cancer stem-like cells.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Plasmid Preparation, Immunoprecipitation, Binding Assay

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: PRRX1A partially regulates sphere formation depending on the interaction with SOX2. (A) Sphere formation was performed. (B) The cell-cycle proportion was measured by performing PI staining followed by flow cytometry. *, P<0.05, vs. pENTR/U6 vector group. (C) Tumor formation assay using lentiviral stably transfected CSCs injected into the cutaneous nerve of the back in nude mice; 3 weeks after the injection, tumors were dissected, photographed, and weighed. *, P<0.05; **, P<0.01, vs. pENTR/U6 vector group. PRRX1, paired-related homeobox 1; CSCs, cancer stem-like cells.

Article Snippet: The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Staining, Flow Cytometry, Plasmid Preparation, Tube Formation Assay, Stable Transfection, Transfection, Injection

Journal: Science Advances

Article Title: Artificial cells drive neural differentiation

doi: 10.1126/sciadv.abb4920

Figure Lengend Snippet: The synthesis of PFO monomers (light orange) is controlled by a genetic AND-gate that requires both LuxR and 3OC6 HSL for gene expression. Monomers of PFO assemble into pores in the presence of cholesterol-containing membranes, thereby releasing BDNF. Homodimers of mature BDNF (green) act on the cognate receptor TrkB, activating signaling pathways leading to neural stem cell differentiation and maturation. The figure was not drawn to scale.

Article Snippet: Human TrkB complementary DNA (cDNA) was synthesized by GenScript in two parts, joined in-house by Gibson Assembly, and cloned into a pLenti vector with standard molecular cloning techniques.

Techniques: Gene Expression, Protein-Protein interactions, Cell Differentiation