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: Construction of expression plasmid and transfection 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 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: Construction of expression plasmid and transfection 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: Construction of expression plasmid and transfection 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: Construction of expression plasmid and transfection 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: Construction of expression plasmid and transfection 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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

doi: 10.1073/pnas.1801242115

Figure Lengend Snippet: Single-molecule motility of KIF15 and Eg5 under load. Velocity (A) and randomness (B) are shown as functions of applied load under saturating ATP conditions (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 62–212). Velocity (C) and randomness (D) are shown as functions of the ATP concentration (open circles; mean ± SE) for KIF15 (blue; n = 78–247; F = −2 pN) and Eg5 (red; n = 22–92; F = 0 pN and F = −4 pN). Solid lines in all panels represent global fits to the four-state model of Fig. 3. Eg5 data (but not model fits) are replotted from a study by Valentine et al. (20).

Article Snippet: Separately, a truncated Eg5 gene coding for the first 513 aa of full-length human Eg5 (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"NP_004514","term_id":"13699824"}} NP_004514 ) with a C-terminal 6× His-tag was generated by PCR amplification from an mCherry-Kinesin11-N-18 plasmid (no. 55067; Addgene).

Techniques: Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

doi: 10.1073/pnas.1801242115

Figure Lengend Snippet: Mechanochemical cycles of KIF15 and Eg5. (A) Minimal four-state kinetic representation of the kinesin mechanochemical cycle, based on a study by Milic et al. (50). (B) Transitions in the four-state scheme are assigned to specific biomechanical events by the partial NL-docking model. The first kinetic transition of this scheme, [1] → [2], represents reversible ATP binding to the nucleotide-free (Ø) MT-bound head of a 1-HB kinesin in the ATP-waiting state. This transition, associated with the rates for ATP binding (k1[ATP]) and ATP release (k−1), is followed by the single, load-dependent transition of the kinesin cycle, [2] → [3]. This mechanical transition, modeled by a force-dependent rate, k2(F), corresponds to partial docking of the NL to the MT-bound head. ATP hydrolysis then completes NL docking, which enables the tethered head to bind the MT, releasing ADP in the process, and ultimately advancing the dimer by ∼8 nm. In the minimal model, these events have been combined into a single transition, [3] → [4], associated with the rate constant, k3. This composite transition implies passage through a 1-HB, posthydrolysis state, [3‡], from which either (i) the tethered head binds the MT, [3‡] → [4], thereby allowing the motor to continue stepping along the MT, or (ii) the bound head prematurely hydrolyzes ATP, leading to dissociation from the MT, [3‡] → [4off]. Finally, the trailing head undergoes transition [4] → [1], associated with the rate constant, k4, which entails releasing Pi and detaching from the MT, thereby returning the dimer to the 1-HB ATP-waiting state. The four-state scheme also incorporates a back-stepping transition, kback. In both panels, the nucleotide states of each head (purple/pink) are indicated as the dimer moves along the MT (green). ADP•Pi indicates heads in the posthydrolysis state. (C) Model parameters (rates and distances; mean ± SE) obtained by global fits to velocity and randomness measurements for KIF15 (blue) and Eg5 (red), based on the kinetic scheme in A (also Fig. 2 and SI Appendix, Fig. S2 A and B).

Article Snippet: Separately, a truncated Eg5 gene coding for the first 513 aa of full-length human Eg5 (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"NP_004514","term_id":"13699824"}} NP_004514 ) with a C-terminal 6× His-tag was generated by PCR amplification from an mCherry-Kinesin11-N-18 plasmid (no. 55067; Addgene).

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

doi: 10.1073/pnas.1801242115

Figure Lengend Snippet: Single-molecule KIF15 and Eg5 processivity. (A) KIF15 RL (bars; mean ± SE; n = 47–144) under a +2-pN load in the presence of ATP or ATPγS at the indicated concentrations. No statistically significant change occurred in response to varying the ATP concentration or replacing it by ATPγS. (B) Measurements of RL as a function of applied load at saturating ATP (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 11–277). Linear fits (solid lines) are shown. The Eg5 data (but not the fits) are replotted from a study by Valentine and Block (54) (also SI Appendix, Fig. S2C.). (C) MT dissociation rates as functions of applied load (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 11–277). Dissociation rates as functions of load were modeled (solid lines) by dividing the fit values of the velocity (Fig. 2A) for KIF15 (blue) or Eg5 (red) by the fit values of the RL at the corresponding forces in B.

Article Snippet: Separately, a truncated Eg5 gene coding for the first 513 aa of full-length human Eg5 (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"NP_004514","term_id":"13699824"}} NP_004514 ) with a C-terminal 6× His-tag was generated by PCR amplification from an mCherry-Kinesin11-N-18 plasmid (no. 55067; Addgene).

Techniques: Concentration Assay, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

doi: 10.1073/pnas.1801242115

Figure Lengend Snippet: MT gliding by KIF15 and Eg5 in the presence of inhibitors. (A) Cartoon representation of the gliding assay (not to scale): MTs (green) are transported by kinesin motors (purple) attached via their stalks to a glass coverslip (blue) (Materials and Methods). The chemical structures of KIF15-IN-1 (B), ispinesib (C), and filanesib (D) are shown. (E) MT-gliding velocities (open circles; mean ± SE) generated by KIF15 in the presence of KIF15-IN-1 (blue; n = 14–31) and Eg5 in the presence of ispinesib (red; n = 9–30) or filanesib (dark red; n = 16–27) as functions of inhibitor concentration. Solid lines are exponential fits. (F) Table of IC50 values (mean ± SE) from E. (G) MT-gliding velocities (mean ± SE) in the presence (shaded bars) or absence (open bars) of inhibitors, at the indicated concentrations, by Eg5 (red; n = 12–25), KIF15 (blue; n = 19–23), or both motors (purple; n = 16–35) (also SI Appendix, Fig. S3). (H) MT-gliding velocities (open circles; mean ± SE) generated by mixtures of motors. The KIF15-to-Eg5 motor ratio is expressed as the percentage of KIF15. Velocities were measured in the presence or absence of inhibitors at the concentrations indicated: no inhibitors (gray; n = 15–56), ispinesib (red; n = 12–52), filanesib (dark red; n = 21–37), KIF15-IN-1 (blue; n = 21–32), or KIF15-IN-1 with either ispinesib (light purple; n = 14–20) or filanesib (dark purple; n = 15–19). Sigmoidal (solid lines) and linear fits (dashed lines) are shown. Shaded backgrounds match the corresponding data in G and H, where inhibitor concentrations were chosen to be sufficient to fully inhibit the target motor, based on the data in E.

Article Snippet: Separately, a truncated Eg5 gene coding for the first 513 aa of full-length human Eg5 (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"NP_004514","term_id":"13699824"}} NP_004514 ) with a C-terminal 6× His-tag was generated by PCR amplification from an mCherry-Kinesin11-N-18 plasmid (no. 55067; Addgene).

Techniques: Gliding Assay, Generated, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

doi: 10.1073/pnas.1801242115

Figure Lengend Snippet: Cell growth under combined inhibition of KIF15 and Eg5, quantified by mCherry fluorescence. Synergistic effects of KIF15 and Eg5 inhibitors were investigated with a HeLa cell proliferation assay in the presence of KIF15-IN-1 and either ispinesib (A–E) or filanesib (F–J). (A and F) Number of viable HeLa cells after 72 h of treatment (open circles; mean ± SE; n = 3) with pairwise combinations of KIF15 and Eg5 inhibitors (Materials and Methods), reported as a percentage relative to the control (no inhibitor). (B and G) Cell proliferation data from A and F, presented as a grid displaying the observed inhibition for each pairwise combination of drug doses (note that 0% cell viability corresponds to 100% inhibition). (C and H) Expected level of inhibition for a pair of inhibitors that act independent of one another (Bliss independence; Materials and Methods). (D and I) Grid displaying numerical differences between the observed and expected levels of inhibition (assuming independence) for each pairwise combination of drug doses, where positive values indicate synergy. (E and J) Strongest enhancement in the level of inhibition of viable cell number (bars; mean ± SE; n = 3), beyond that expected for no synergy (dotted horizontal lines), occurred when 20 μM KIF15-IN-1 was paired with 1 nM ispinesib (E; ****P = 3 × 10−6; one-tailed t test) or with 1 nM filanesib (J; ****P = 8 × 10−5; one-tailed t test) (also SI Appendix, Fig. S4).

Article Snippet: Separately, a truncated Eg5 gene coding for the first 513 aa of full-length human Eg5 (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"NP_004514","term_id":"13699824"}} NP_004514 ) with a C-terminal 6× His-tag was generated by PCR amplification from an mCherry-Kinesin11-N-18 plasmid (no. 55067; Addgene).

Techniques: Inhibition, Fluorescence, Proliferation Assay, One-tailed Test