Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: RT‐qPCR primers.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Sequencing

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: High expression of NOLC1 in LUAD. (A) TCGA analysis of NOLC1 expression in LUAD tissues. (B) Kaplan–Meier curve analysis of survival in LUAD patients with different NOLC1 expression. (C) RT‐qPCR detection of NOLC1 expression in LUAD cells. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Expressing, Quantitative RT-PCR

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: NOLC1 promotes stemness of LUAD cells. (A) Correlation analysis of NOLC1 with mRNAsi. (B) RT‐qPCR detection of NOLC1 expression. (C) Colony formation assay. (D) Cell invasion assay. (E) Sphere formation assay. (F) Western blot detection of CD133 and OCT4 expression. (G) Determining the IC 50 value of cells to cisplatin using CCK‐8 assay. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Quantitative RT-PCR, Expressing, Colony Assay, Invasion Assay, Tube Formation Assay, Western Blot, CCK-8 Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: NOLC1 promotes stemness of LUAD cells through NOTCH pathway. (A) GSEA. (B) Western blot detection of Hes1, NOTCH1, and Gli1 expression. (C) Colony formation assay. (D) Cell invasion assay. (E) Sphere formation assay. (F) Western blot detection of D133 and OCT4 expression. (G) Determining the IC 50 value of cells to cisplatin using CCK‐8 assay. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Western Blot, Expressing, Colony Assay, Invasion Assay, Tube Formation Assay, CCK-8 Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: FOXA1 activates NOLC1 transcription. (A) Correlation analysis of FOXA1 and NOLC1. (B) Prediction of binding sites between FOXA1 and NOLC1. (C) RT‐qPCR detection of FOXA1 expression in LUAD. (D) ChIP assay. (E) Dual‐luciferase reporter assay. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: FOXA1 transcriptionally activates NOLC1 to promote stemness of LUAD cells. (A) RT‐qPCR detection of FOXA1 expression. (B) Colony formation assay. (C) Cell invasion assay. (D) Sphere formation assay. (E) Western blot detection of CD133 and OCT4 expression. (F) Determining the IC 50 value of cells to cisplatin using CCK‐8 assay. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: Quantitative RT-PCR, Expressing, Colony Assay, Invasion Assay, Tube Formation Assay, Western Blot, CCK-8 Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: FOXA1 activates NOLC1 transcription through NOTCH pathway to promote cell stemness in lung adenocarcinoma

doi: 10.1002/kjm2.12930

Figure Lengend Snippet: NOLC1 promotes tumor growth and enhances stemness of LUAD in vivo. (A) Growth curve and representative images of LUAD tumors ( n = 3). (B) Measurement of LUAD tumor weights. (C) Western blot detection of NOLC1, CD133, OCT4, Hes1, NOTCH1, and Gli1 expression. * indicates p < 0.05.

Article Snippet: RiboBio (China) created and produced the small interfering RNAs (si‐NOLC, si‐FOXA1) and short hairpin RNA (sh‐FOXA1) that target NOLC1 and FOXA1, the NOLC1 overexpression plasmid (oe‐NOLC1), and the matching controls.

Techniques: In Vivo, Western Blot, Expressing

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 2. The SUMOylation inhibitor TAK-981 decreases SUMOylated coilin and increases CB number in HeLa cells. (A) HeLa cells were transfected either with or without His–SUMO-1 for 48 h. Cells were treated with either DMSO or TAK-981 (0.1 µM) for 24 h post-transfection, as indicated. Protein was subject to Ni-NTA pulldown. Pulldown was visualized on a western blot probed with anti-coilin and anti-SUMO-1 antibodies. SUMOylated coilin is denoted by a bracket. Blots shown are representative of three experiments. (B) Immunofluorescence of HeLa cells treated with either DMSO or TAK-981 (0.1 µM) for 24 h. Coilin is shown in red and Nopp140 is shown in green. DAPI (blue) is used to visualize the nucleus. Scale bars: 20 μm. (C) SuperPlot showing number of CBs in HeLa cells treated with either DMSO or TAK-981. A total of 322 cells were counted from four biological replicates for both DMSO and TAK-981 treatments. Horizontal line, mean; error bars, s.d.; larger points show the mean for each biological repeat and points represent individual data points. ****P<0.0001 (two-tailed unpaired Student’s t-test).

Article Snippet: Nopp140 was detected with 1:50 anti-NOCL1 (Nopp140) polyclonal antibody (11815- 1-AP; Proteintech).

Techniques: Transfection, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 5. Coilin SUMO mutants have altered interaction with Nopp140. (A) HeLa cells were transfected with either GFP-C2 empty vector, WT coilin–GFP (CG WT), coilin–GFP mutant I (CG I) or coilin–GFP mutant L (CG L) for 24 h, as indicated. Lysate was subject to immunoprecipitation (IP) with anti-GFP antibody. IP and input (20 μl of total lysate was run as input) samples were run on SDS-PAGE followed by western transfer and probing for Nopp140 (upper panel) or GFP (lower panel). (B) Quantification of A and repeat experiments, showing Nopp140 signal normalized to recovered coilin–GFP mutant I or coilin– GFP mutant L, with the mutant I ratio set to 1. Bars show the mean, error bars represent s.d. and points represent individual data points. *P<0.05 (two-tailed unpaired Student’s t-test). (C) Schematic of coilin, showing the NTD, C-terminal domain (CTD) and intrinsically disordered region (IDR), with lysine residues that were mutated to arginine residues denoted as red pins and amino acid residue numbers marked. Self-interaction region, Nopp140 interaction region, nuclear localization signals (NLS) and a nucleolar localization signal (NoLS) are shown, as are SIMs.

Article Snippet: Nopp140 was detected with 1:50 anti-NOCL1 (Nopp140) polyclonal antibody (11815- 1-AP; Proteintech).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, SDS Page, Western Blot, Two Tailed Test, Residue

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 6. Coilin SUMO mutants display dysregulated CB formation in HFF cells. (A) SuperPlot of number of CBs in HFF cells when transfected with different mutants for 24 h. Three biological repeats were scored. n=125 cells scored for WT coilin–GFP (CG), n=115 cells scored for coilin–GFP K496R (K496R), n=105 cells scored for coilin–GFP mutant K (Mut K), n=106 cells scored for coilin–GFP mutant I (Mut I) and n=117 cells scored for coilin–GFP mutant L (Mut L). ****P<0.0001; **P<0.01; ns, not significant (two-tailed unpaired Student’s t-test). (B) SuperPlot of CB size (pixels) in HFF cells when transfected with different mutants for 24 h. CB size was measured from three biological repeats. n=405 CBs measured for WT coilin–GFP, n=369 CBs measured for coilin–GFP K496R, n=379 CBs measured for coilin–GFP mutant K, n=419 CBs measured for coilin–GFP mutant I and n=533 CBs measured for coilin–GFP mut L. *P<0.05, **P<0.01, ****P<0.0001 (two-tailed unpaired Student’s t-test). In A and B, horizontal lines mark the mean, error bars represent s.d., larger points show the mean for each biological repeat and points represent individual data points. (C) Representative immunofluorescence images of HFF cells transfected with WT coilin–GFP (CG) or coilin–GFP mutant L (Mut L) for 24 h and stained for SMN. GFP signal is shown in green. SMN staining is in red. DAPI (blue) is used to visualize the nucleus. Arrowheads indicate some of the CBs. Scale bars: 20 μm. (D) Proposed schematic of the involvement of coilin and Nopp140 SUMOylation in CB formation. SA, self-association domain.

Article Snippet: Nopp140 was detected with 1:50 anti-NOCL1 (Nopp140) polyclonal antibody (11815- 1-AP; Proteintech).

Techniques: Transfection, Mutagenesis, Two Tailed Test, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 2. The SUMOylation inhibitor TAK-981 decreases SUMOylated coilin and increases CB number in HeLa cells. (A) HeLa cells were transfected either with or without His–SUMO-1 for 48 h. Cells were treated with either DMSO or TAK-981 (0.1 µM) for 24 h post-transfection, as indicated. Protein was subject to Ni-NTA pulldown. Pulldown was visualized on a western blot probed with anti-coilin and anti-SUMO-1 antibodies. SUMOylated coilin is denoted by a bracket. Blots shown are representative of three experiments. (B) Immunofluorescence of HeLa cells treated with either DMSO or TAK-981 (0.1 µM) for 24 h. Coilin is shown in red and Nopp140 is shown in green. DAPI (blue) is used to visualize the nucleus. Scale bars: 20 μm. (C) SuperPlot showing number of CBs in HeLa cells treated with either DMSO or TAK-981. A total of 322 cells were counted from four biological replicates for both DMSO and TAK-981 treatments. Horizontal line, mean; error bars, s.d.; larger points show the mean for each biological repeat and points represent individual data points. ****P<0.0001 (two-tailed unpaired Student’s t-test).

Article Snippet: The primary antibodies usedwere anti-β-actinmouse monoclonal antibody (1:5000; 8H10D10; Cell Signaling Technology, Danvers, MA, USA); anti-NSMCE2 polyclonal antibody (1:1000; 13627-1- AP; Proteintech, Rosemont, IL, USA), anti-GFP monoclonal antibody (1:500; 11814460001; Roche, Germany), anti-SUMO-1 polyclonal antibody (1:1000; 10329-1-AP; Cell Signaling Technology, Danvers, MA, USA), antiSUMO-2/3 polyclonal antibody (1:1000; 11251-1-AP; Cell Signaling Technology, Danvers, MA, USA), anti-coilin polyclonal antibody (1:1000; sc-32860; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NOCL1 (Nopp140) polyclonal antibody (1:1000; 11815-1-AP; Proteintech) and Jo u rn al o f Ce ll Sc ie n ce anti-myc (1:1000; sc-40; Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 5. Coilin SUMO mutants have altered interaction with Nopp140. (A) HeLa cells were transfected with either GFP-C2 empty vector, WT coilin–GFP (CG WT), coilin–GFP mutant I (CG I) or coilin–GFP mutant L (CG L) for 24 h, as indicated. Lysate was subject to immunoprecipitation (IP) with anti-GFP antibody. IP and input (20 μl of total lysate was run as input) samples were run on SDS-PAGE followed by western transfer and probing for Nopp140 (upper panel) or GFP (lower panel). (B) Quantification of A and repeat experiments, showing Nopp140 signal normalized to recovered coilin–GFP mutant I or coilin– GFP mutant L, with the mutant I ratio set to 1. Bars show the mean, error bars represent s.d. and points represent individual data points. *P<0.05 (two-tailed unpaired Student’s t-test). (C) Schematic of coilin, showing the NTD, C-terminal domain (CTD) and intrinsically disordered region (IDR), with lysine residues that were mutated to arginine residues denoted as red pins and amino acid residue numbers marked. Self-interaction region, Nopp140 interaction region, nuclear localization signals (NLS) and a nucleolar localization signal (NoLS) are shown, as are SIMs.

Article Snippet: The primary antibodies usedwere anti-β-actinmouse monoclonal antibody (1:5000; 8H10D10; Cell Signaling Technology, Danvers, MA, USA); anti-NSMCE2 polyclonal antibody (1:1000; 13627-1- AP; Proteintech, Rosemont, IL, USA), anti-GFP monoclonal antibody (1:500; 11814460001; Roche, Germany), anti-SUMO-1 polyclonal antibody (1:1000; 10329-1-AP; Cell Signaling Technology, Danvers, MA, USA), antiSUMO-2/3 polyclonal antibody (1:1000; 11251-1-AP; Cell Signaling Technology, Danvers, MA, USA), anti-coilin polyclonal antibody (1:1000; sc-32860; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NOCL1 (Nopp140) polyclonal antibody (1:1000; 11815-1-AP; Proteintech) and Jo u rn al o f Ce ll Sc ie n ce anti-myc (1:1000; sc-40; Santa Cruz Biotechnology).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, SDS Page, Western Blot, Two Tailed Test, Residue

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 6. Coilin SUMO mutants display dysregulated CB formation in HFF cells. (A) SuperPlot of number of CBs in HFF cells when transfected with different mutants for 24 h. Three biological repeats were scored. n=125 cells scored for WT coilin–GFP (CG), n=115 cells scored for coilin–GFP K496R (K496R), n=105 cells scored for coilin–GFP mutant K (Mut K), n=106 cells scored for coilin–GFP mutant I (Mut I) and n=117 cells scored for coilin–GFP mutant L (Mut L). ****P<0.0001; **P<0.01; ns, not significant (two-tailed unpaired Student’s t-test). (B) SuperPlot of CB size (pixels) in HFF cells when transfected with different mutants for 24 h. CB size was measured from three biological repeats. n=405 CBs measured for WT coilin–GFP, n=369 CBs measured for coilin–GFP K496R, n=379 CBs measured for coilin–GFP mutant K, n=419 CBs measured for coilin–GFP mutant I and n=533 CBs measured for coilin–GFP mut L. *P<0.05, **P<0.01, ****P<0.0001 (two-tailed unpaired Student’s t-test). In A and B, horizontal lines mark the mean, error bars represent s.d., larger points show the mean for each biological repeat and points represent individual data points. (C) Representative immunofluorescence images of HFF cells transfected with WT coilin–GFP (CG) or coilin–GFP mutant L (Mut L) for 24 h and stained for SMN. GFP signal is shown in green. SMN staining is in red. DAPI (blue) is used to visualize the nucleus. Arrowheads indicate some of the CBs. Scale bars: 20 μm. (D) Proposed schematic of the involvement of coilin and Nopp140 SUMOylation in CB formation. SA, self-association domain.

Article Snippet: The primary antibodies usedwere anti-β-actinmouse monoclonal antibody (1:5000; 8H10D10; Cell Signaling Technology, Danvers, MA, USA); anti-NSMCE2 polyclonal antibody (1:1000; 13627-1- AP; Proteintech, Rosemont, IL, USA), anti-GFP monoclonal antibody (1:500; 11814460001; Roche, Germany), anti-SUMO-1 polyclonal antibody (1:1000; 10329-1-AP; Cell Signaling Technology, Danvers, MA, USA), antiSUMO-2/3 polyclonal antibody (1:1000; 11251-1-AP; Cell Signaling Technology, Danvers, MA, USA), anti-coilin polyclonal antibody (1:1000; sc-32860; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NOCL1 (Nopp140) polyclonal antibody (1:1000; 11815-1-AP; Proteintech) and Jo u rn al o f Ce ll Sc ie n ce anti-myc (1:1000; sc-40; Santa Cruz Biotechnology).

Techniques: Transfection, Mutagenesis, Two Tailed Test, Immunofluorescence, Staining