Journal: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 2 | NSUN4 inhibition affects cell growth, apoptosis, stemness, invasiveness, and migratory ability in vitro. (A–H) Various experiments were conducted using different treatment methods and cell types (A549 and SK-MES-1 NSCLC cells), with three groups: Sh-ctrl, sh-NSUN4#1, or sh-NSUN4#2. (A) Evaluation of NSUN4 protein expression by immunoblotting in cells transfected as indicated. (B) Assessment of the number of formed colonies with cells transfected as indicated. (C) Determination of cell apoptotic ratio with cells transfected as indicated by flow cytometry. (D and E) Examination of cell migratory rate and invasiveness with transfected cells. Scale bars: 100 μm. (F) Measurement of sphere formation with transfected NSCLC cells. Scale bars: 100 μm. (G and H) Expression of SOX2, CD133, and KLF4 proteins using immunoblot analysis in transfected A549 and SK-MES-1 NSCLC cells. n = 3 in (A–G). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Inhibition, In Vitro, Expressing, Western Blot, Transfection, Flow Cytometry

Journal: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 5 | NSUN4 affects NSCLC cell malignant phenotypes through CDC20. (A–H) A549 and SK-MES-1 NSCLC cells were subjected to intro- duction with vector + sh-ctrl, NSUN4 expression construct + sh-ctrl, vector + sh-CDC20, or NSUN4 + sh-CDC20. (A) CDC20 protein expression by immunoblotting in cells transfected as indicated. (B) The number of formed colonies by colony formation assay with cells transfected as indicated. (C) Cell apoptotic ratio by flow cytometry with cells transfected as indicated. (D and E) Cell migratory rate and invasiveness by transwell assay with transfected cells. Scale bars: 100 μm. (F) Measurement of sphere formation with transfected NSCLC cells. (G and H) Expression of SOX2, CD133, and KLF4 proteins by immunoblot analysis in transfected A549 and SK-MES-1 NSCLC cells. n = 3 in (A–G). *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Plasmid Preparation, Expressing, Construct, Western Blot, Transfection, Colony Assay, Flow Cytometry, Transwell Assay

Journal: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 6 | NSUN4 depletion hinders the growth of A549 subcutaneous xenografts. (A–E) A549 subcutaneous xenografts were generated by implanting sh-ctrl or sh-NSUN4#2 lentivirus-infected A549 cells. After 30 days, xenografts were harvested. n = 5 for each group. (A) Growth curves of A549 subcutaneous xenografts (n = 3). (B) Representative pictures of A549 subcutaneous xenografts. (C) Tumor average weight was calculated (n = 3). (D) Expression of NSUN4, CDC20, SOX2, CD133, and KLF4 proteins by immunoblot analysis in A549 subcutaneous xenografts (n = 3). (E) Expression of NSUN4, CDC20, SOX2, CD133, and KLF4 proteins by immunohistochemistry in sections of subcutaneous xenografts. **p < 0.01, ***p < 0.001.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Generated, Infection, Expressing, Western Blot, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Outer radial glia promotes white matter regeneration after neonatal brain injury

doi: 10.1016/j.xcrm.2025.101986

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Sox2 , Cell Signaling , Cat# 2748; RRID: AB_823640.

Techniques: Virus, Plasmid Preparation, Recombinant, RNAscope, Blocking Assay, Staining, Knock-Out, In Situ, Multiplex Assay, Sequencing, Software

Journal: Cell Reports Medicine

Article Title: Outer radial glia promotes white matter regeneration after neonatal brain injury

doi: 10.1016/j.xcrm.2025.101986

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Sox2 , Millipore , Cat# AB5603; RRID: AB_2286686.

Techniques: Virus, Plasmid Preparation, Recombinant, RNAscope, Blocking Assay, Staining, Knock-Out, In Situ, Multiplex Assay, Sequencing, Software

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Scheme of genome-wide CRISPR-Cas9 screen in MB157R cells. ( B ) Volcano plot of genes differentially expressed in MB157R cells from the CRISPR-Cas9 screen, n = 3. ( C ) Representative immunoblotting of N1-ICD and SOX2 derived from MB157 and MB157R cells. ( D ) Cell proliferation assay of MB157R cells transfected with siRNA SOX2 or control, n = 3. ( E ) Representative immunoblotting of N1-ICD, SOX2, and EMT/Stemness markers derived from MB157R cells as indicated or ( F ) iSOX2 or iEV MB157 cells (72 h), or SOX2 washout (WO) after 2 or 5 days. ( G ) Cell proliferation assay of MB157 cells as indicated, at day 6, n = 4. ( H ) GSEA of BC-specific signatures for Notch signaling, EMT, SC, luminal, basal, and mesenchymal from RNAseq analysis of iSOX2 compared to iEV MB157 cells, n = 3. ( I ) Quantification and size of tumorspheres with representative images derived from iSOX2 or iEV MB157 cells, n = 3. Scale = 100 µm. Data from biological replicates are represented as mean ± SEM. MAGeCK test ( B ), two-way ANOVA ( D ), one-way ANOVA ( G ), permutation test ( H ) or Student t test ( I ) were used to determine P values. .

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Genome Wide, CRISPR, Western Blot, Derivative Assay, Proliferation Assay, Transfection, Control

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Robust rank aggregation (RRA) of genes negatively selected in CRISPR-Cas9 screen of MB157R cells treated with GSI (10 µM) for 14 days. ( B ) Representative immunoblotting of N1-ICD, SOX2 and EMT/Stemness markers derived from MB157 or MB157R cells as indicated. ( C ) Cell cycle analysis of MB157R cells 72 h after transfection with siRNA SOX2 or Ctrl, n = 5. ( D ) Number and size of tumorspheres derived from MB157R cells with siRNA SOX2 or Ctrl, n = 3. ( E ) Relative mRNA expression of NOTCH1 and its target genes ( CCND1 , MYC , HES1 ) and EMT/Stemness markers in iSOX2 or iEV control MB157 cells, 72 h after DOX induction, n = 3. ( F ) Cell growth inhibition of HCC1599 treated with GSI (1 µM) for 6 days, n = 4. ( G ) Cell proliferation assay of HCC1599 cells treated with GSI (1 µM) for 6 days, normalized to day 0, n = 3. ( H ) Relative mRNA expression of CCND1 , MYC and HES1 in HCC1599 treated with GSI (1 µM) for 24 h, n = 3. ( I ) Representative immunoblotting of N1-ICD and MYC derived from HCC1599 cells treated with GSI (1 µM) for 24 h, n = 3. ( J ) Relative mRNA expression of NOTCH1 and its target genes ( CCND1 , MYC , HES1 ) and EMT/Stemness markers, n = 3 and ( K ) Representative immunoblotting of N1-ICD, SOX2, N-Cadherin and Claudin-3 derived from iSOX2 or iEV control HCC1599 cells, 72 h after DOX induction, n = 3. ( L ) Cell proliferation assay of iSOX2 or iEV control HCC1599 cells treated with GSI (1 µM) or VHC. Cell growth was assessed 6 days post treatment and normalized to day 0, n = 4. ( M ) Cell proliferation assay of HCC1806 cells treated with GSI (10 µM) for 6 days, normalized to day 0, n = 3. ( N ) Cell proliferation assay of HCC1806 cells 72 h after transfection with siRNA SOX2 or Ctrl, normalized to day 0, n = 3. ( O ) Representative immunoblotting of N1-ICD, SOX2 and SLUG derived from HCC1806 cells 72 h after transfection with siRNA SOX2 or Ctrl, n = 3. ( P ) Number and size of tumorspheres derived from HCC1806 cells with siRNA SOX2 or Ctrl, n = 3. Data from biological replicates are represented as mean ± SEM. Student t test ( C – E , H , J , P ) two-way ANOVA ( G , N ) or one-way ANOVA ( L ) were used to determine P value.

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: CRISPR, Western Blot, Derivative Assay, Cell Cycle Assay, Transfection, Expressing, Control, Inhibition, Proliferation Assay

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Overlap of ChIP peaks for RBPJ and SOX2 in MB157R cells ( n = 3). ( B ) Genome-wide ChIP overlapping peaks for RBPJ-SOX2 compared to RBPJ-H3K27ac. ( C ) ChIP peaks for HA-tagged SOX2 or SOX2 and RBPJ on NOTCH1 and ( D ) HES1 promoter in MB157 and MB157R cells as indicated. The y-axis represents reads per million mapped reads. ( E ) Representative immunoblotting of IP using anti-HA (SOX2) or anti-SOX2 antibodies, and IP of HA (SOX2) or SOX2 using anti-RBPJ antibodies in iSOX2 MB157 and MB157R cells. ( F ) Luciferase reporter assay for RBPJ reporter in MB157 cells as indicated, 24 h after iSOX2 or GSI treatment, n = 3. Data from biological replicates are represented as mean ± SEM. Fisher test ( B ) and one-way ANOVA ( F ) were used to determine P values. .

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Genome Wide, Western Blot, Luciferase, Reporter Assay

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Representative immunoblotting of N1-ICD, SOX2 and EMT/Stemness markers derived from HCC1599, MB157, MB157R and HCC1806 cells. ( B ) Relative mRNA expression of SOX2 and EMT/Stemness markers in iN1-ICD or iEV control MB157R cells, 72 h after DOX induction n = 3–4. ( C ) Cell proliferation assay of iN1-ICD or iEV control MB157R cells. Cell growth was assessed 6 days after DOX induction and normalized to day 0, n = 4. ( D ) Relative mRNA expression of SOX2 in iN1-ICD or iEV control HCC1806 cells, 72 h after DOX induction, n = 3. ( E ) Cell proliferation assay of iN1-ICD or iEV control HCC1806 cells. Cell growth was assessed 6 days after DOX induction and normalized to day 0, n = 3. ( F ) Representative immunoblotting of N1-ICD, SOX2 and EMT/Stemness markers derived from iN1-ICD or iEV control HCC1806 cells, 72 h after DOX induction. ( G ) Number and size of tumorspheres with representative pictures derived from iN1-ICD compared to iEV control HCC1806 cells after 14 days, n = 3. Scale = 100 µm. ( H ) Representative immunoblotting of N1-ICD, SOX2 and EMT/Stemness markers and ( I ) Relative mRNA expression of SOX2 in iN1-ICD or iEV control BT-549 cells, 72 h after DOX induction n = 3. ( J ) Number and size of tumorspheres derived from iN1-ICD compared to iEV control BT-549 cells after 14 days, n = 3. ( K ) Relative mRNA expression of HEY/HES family genes in MB157 and MB157R cells, n = 3. ( L ) Relative mRNA expression of HEY2 , HEYL , HES5 and SOX2 in iN1-ICD or iEV control HCC1806 cells 72 h after transfection with siRNA HEY2, HEYL, HES5 or Ctrl n = 3–4. Data from biological replicates are represented as mean ± SEM. Student t test ( B – E , G , I – L ) or one-way ANOVA ( L ) were used to determine P value.

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Western Blot, Derivative Assay, Expressing, Control, Proliferation Assay, Transfection

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Representative immunoblotting of N1-ICD, SOX2 and EMT/Stemness markers derived from iN1-ICD or iEV MB157R cells (72 h), or N1-ICD washout (WO) after 2 or 5 days. ( B ) GSEA of BC-specific signatures for Notch signaling, EMT, Stem Cell, luminal, basal and mesenchymal from RNAseq analysis of iN1-ICD compared to iEV MB157R cells, n = 3. ( C ) Number and size of tumorspheres with representative pictures derived from iN1-ICD or iEV MB157R cells, n = 4. Scale = 100 µm. ( D ) Relative mRNA expression of NOTCH1 , HEY2 , HEYL , HES5 , and SOX2 in MB157R cells as indicated, n = 4–5. ( E ) Representative immunoblotting of HA (N1-ICD), N1-ICD, and SOX2 derived from MB157R cells as indicated. Data from biological replicates are represented as mean ± SEM. Permutation test ( B ), Student t test ( C ) or one-way ANOVA ( D ) were used to determine P values. .

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Scheme of MIND xenograft model setup in NSG mice, adapted from Sflomos et al (Sflomos et al, ). ( B ) Representative pictures of hematoxylin–eosin coloration for indicated MIND xenograft tumors at endpoint, scale = 100 µm. ( C ) Representative images of co-immunofluorescence N1-ICD–SOX2, scale = 50 µm, and ( D ) Quantification of N1-ICD and/or SOX2-positive tumor cells from co-immunofluorescence N1-ICD–SOX2 for indicated xenograft tumors at endpoint, n = 6. ( E ) Representative images of co-immunofluorescence N1-ICD– SOX2 in TMA of human TNBC samples, scale = 100 µm, with proportion of N1-ICD + or SOX2 + TNBC samples, n = 79 ( F ) OS of TNBC patients from METABRIC dataset with NOTCH High/Low signature and SOX2 High/Low expression: NOTCH High /SOX2 High ( n = 40), NOTCH High /SOX2 Low ( n = 38), NOTCH Low /SOX2 High ( n = 35) and NOTCH Low /SOX2 Low ( n = 32). ( G ) Tumor growth of iSOX2 or iEV MB157 xenografts treated with GSI (8 mg/kg) for 1 week, n = 10–11. ( H ) Quantification of N1-ICD, SOX2 and CD49f positive tumor cells in invasive or ductal areas from HE coloration and IHC staining in MB157 xenografts treated with GSI (8 mg/kg), n = 3. ( I ) Tumor growth of MB157 xenografts treated with GSI (8 mg/kg) for 10 weeks, n = 10–11. Data from biological replicates are represented as mean ± SEM. Log-rank test ( F ), two-way ANOVA ( G ), Cochran–Mantel–Haenszel test ( H , left) or Student t test ( H ) were used to determine P values. .

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Immunofluorescence, Expressing, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: ( A ) Tumor growth of HCC1599, MB157, MB157R and HCC1806 MIND xenografts ( n = 9–11). ( B ) Quantification of tumor cells in invasive or ductal areas from HE coloration in HCC1599, MB157, MB157R and HCC1806 MIND xenografts at endpoint ( ~ 1000 mm 3 ) ( n = 3). ( C ) Lung metastasis number in HCC1599, MB157, MB157R and HCC1806 MIND xenografts at endpoint, n = 9–11. ( D ) Representative pictures of N1-ICD, SOX2, CD49f and SLUG IHC stainings for HCC1599, MB157, MB157R and HCC1806 MIND xenograft tumors at endpoint, scale = 100 µm. ( E ) Representative images of co-immunofluorescence staining of N1-ICD – SOX2 for MB157 and MB157R cell lines in vitro, scale = 50 µm. ( F ) RFS of TNBC patients from METABRIC dataset with NOTCH High/Low signature and SOX2 High/Low expression. TNBC patients are divided in 4 groups: NOTCH High /SOX2 High ( n = 40), NOTCH High /SOX2 Low ( n = 38), NOTCH Low /SOX2 High ( n = 35) and NOTCH Low /SOX2 Low ( n = 32). ( G ) Quantification of tumor cells in invasive or ductal areas from HE coloration in MB157-iSOX2 xenografts, n = 3. ( H ) Lung metastasis number in SOX2-expressing or EV control MB157 MIND xenografts treated with GSI (8 mg/kg, 3×/week) or VHC for 1 week, n = 10–11. ( I ) Representative pictures of HE coloration, N1-ICD, SOX2 and CD49f IHC stainings for iSOX2 or iEV control MB157 MIND xenografts treated with GSI (8 mg/kg, 3x/week) or VHC for 1 week, scale = 100 µm. ( J ) Tumor growth of iSOX2 or iEV control HCC1599 MIND xenografts treated with GSI (8 mg/kg, 3×/week) or VHC for 1 week, n = 9–13. ( K ) Lung metastasis number in iSOX2 or iEV control HCC1599 MIND xenografts treated with GSI (8 mg/kg, 3×/week) or VHC for 1 week, n = 9–13. ( L ) Tumor growth of HCC1599 xenografts treated with GSI (8 mg/kg) or VHC for 8 weeks, n = 9–13. Data from biological replicates are represented as mean ± SEM. Log-rank test ( F ), Cochran–Mantel–Haenszel test ( G ), one-way ANOVA ( H , K ) or two-way ANOVA ( J ), were used to determine P value.

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Immunofluorescence, Staining, In Vitro, Expressing, Control

Journal: EMBO Molecular Medicine

Article Title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer

doi: 10.1038/s44321-024-00161-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit polyclonal anti-SOX2 _ WB: 1/2000 , Merck Millipore , Cat#AB5603; RRID:AB_2286686.

Techniques: Recombinant, Plasmid Preparation, Binding Assay, Polymer, SYBR Green Assay, Transfection, Protease Inhibitor, Reporter Assay, Gel Purification, Purification, Ligation, Software

Journal: BMC Cardiovascular Disorders

Article Title: The role and mechanism of NRG1/ErbB4 in inducing the differentiation of induced pluripotent stem cells into cardiomyocytes

doi: 10.1186/s12872-024-04224-z

Figure Lengend Snippet: Identification of target genotypic cell lines. ( A ) Western blot results of ErbB4 protein expression. ( B ) Western blot results of Sox2 and Nanog protein expression. ( C ) Immunofluorescence staining analysis of OCT4 and SSEA-4

Article Snippet: Following washing, the membrane was blocked with BSA for 1 h and then incubated overnight at 4 °C with the primary antibodies of rabbit polyclonal anti-cardiac troponin (cTnT) antibody (1:500, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-Sox2 antibody (1:500, Proteintech, Rosemont, IL, USA), rabbit anti-Nanog antibody (1:500, Proteinteh), rabbit anti-ErbB4 antibody (1:500, Abcam), and rabbit anti-phosphorylated AKT antibody (1:500, Proteintech).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining