fgf2  (R&D Systems)

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Flow chart of the experimental design and verification of FGF2 deletion. a Diagram of experimental design. Bone marrow precursor cells were collected from the tibia and femur of WT and FGF2 KO mice aged 8–12 weeks. They were differentiated into BMDM for 7 days using 100ng/ml M-CSF. Macrophages in C57BL/6 male mice were depleted using clodronate liposomes. BMDM from WT and KO mice were injected via the tail vein into mice to reconstitute macrophages after 2 days. Mice were treated with Sham or CLP, and the samples were analyzed 24 h later. b Serum FGF2 protein levels were assessed by ELISA ( n = 3–5). c FGF2 gene expression levels were measured in the lungs, spleen, and liver using real-time PCR ( n = 3). d Western blot analysis of FGF2 protein levels in lung tissue extractions ( n = 3). e Immunofluorescence staining of FGF2 in BMDM from WT and FGF2 KO mice ( n = 3). f The levels of FGF2 gene expression were quantified in the lung tissue of mice subjected to elimination-reconstruction procedures. Bar is 50 μm. * p < 0.05 vs. WT or vs. Clo + WT

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Liposomes, Injection, Enzyme-linked Immunosorbent Assay, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Effect of FGF2 deficiency on BMDM apoptosis and polarization. a – c FGF2 deletion increased BMDM apoptosis. a Apoptosis in BMDM deprived of FBS for 24 h was assessed by flow cytometry ( n = 4). b - c Percentage of PI + Annexin V + and PI- Annexin V + BMDM after starvation. d - k FGF2 deletion in BMDM promoted M1 polarization. d - g Flow cytometric analysis of macrophage markers in BMDM treated with LPS or IL4, including CD86, iNOS, CD206, and Arg1 ( n = 3). h - k The levels of CD86, iNOS, CD206 and Arg1 in BMDM after treatment with LPS or IL4. N represents no treatment; * p < 0.05, vs. WT; Ψ p < 0.05, vs. N + WT; Ω p < 0.05, vs. N + FGF2 KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Flow Cytometry

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Deficiency of FGF2 in BMDM resulted in the upregulation of M1 markers and proinflammatory cytokine expression and increased nuclear translocation of NF-KB p65. a - j BMDM were treated with LPS or IL4, and real-time PCR was used to determine the expression of M1 and M2 markers and cytokines ( n = 3). k , l P65 nuclear translocation was detected and quantitatively analyzed by immunofluorescence ( n = 3). m The expression of MMP9 in WT and FGF2 KO BMDM treated with LPS or IL4 were determined with real-time PCR. Bar in the first three rows is 50 μm, while bar in the fourth rows is 20 μm, * p < 0.05 vs. WT; Ψ p < 0.05 vs. N + WT; Ω p < 0.05 vs. N + KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Expressing, Translocation Assay, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Transcriptome sequencing was used to analyze BMDM from WT and FGF2 KO mice treated with or without LPS. a Heat map of DEGs in BMDM from WT and FGF2 KO mice stimulated with or without LPS (Average TPM each group, n = 3). b KEGG enrichment analysis identified the top 20 altered signaling pathways in the four groups (WT, FGF2 KO, WT + LPS, and FGF2 KO + LPS). c Construction of a regulatory network to modulate PPI involving FGF2 and LPS using DEGs. d KEGG pathway network based on similarity in gene expression profiles. e Volcano plot showing DEGs of WT + LPS and FGF2 KO + LPS. f Heat map of DEGs in BMDM of WT + LPS and FGF2 KO + LPS. g Top 20 KEGG pathways for DEGs in BMDM from WT + LPS and FGF2 KO + LPS. h KEGG pathway annotation of differentially expressed genes between WT + LPS and FGF2 KO + LPS. i , j Differently regulated pathways in the GSEA. k Inflammation and cytokine gene heat map for WT + LPS and FGF2 KO + LPS. l , m TPM changes in the gene groups compared to WT weights. * p < 0.05 vs. WT; Δ p < 0.05 vs. WT + LPS; # p < 0.05 vs. FGF2 KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Sequencing, Protein-Protein interactions, Gene Expression

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: FGF2 deletion in macrophages aggravates lung injury in CLP mice. a - c Clodronate liposomes were injected intravenously to deplete macrophages, followed by quantification of F4/80 + spleen cells using flow cytometry to measure macrophage clearance. * p < 0.05 vs. control. d - f Levels of the inflammatory cytokines IL1β, TNFα, and IL6 in bronchoalveolar lavage fluid (BALF) from the four groups were quantified via ELISA. The four groups were Clo + WT + Sham, Clo + WT + CLP, Clo + FGF2 KO + Sham, and Clo + FGF2 KO + CLP. g The lung wet-to-dry weight ratios were measured in the four groups. h - i The total cells and total protein in BALF were evaluated in the four groups. j The OD value of Evans blue in the lung tissues from the four groups. k - l HE staining of lung tissue from the four groups and their Smith score. (m-o) Blood gas analysis was performed using abdominal aortic blood from the four groups. Bar is 250 μm. * p < 0.05 vs. WT; Δ p < 0.05 vs. WT + LPS; # p < 0.05 vs. FGF2 KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Liposomes, Injection, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Mice reconstituted with FGF2 KO macrophages and subjected to CLP demonstrate increased M1 polarization in lung tissue. a - f The presence and levels of CD206, CD86, and F4/80 markers on macrophages within lung tissue were identified and quantitatively assessed using immunofluorescence staining. Bar is 20 μm. * p < 0.05, vs. WT; Δ p < 0.05 vs. WT + LPS; # p < 0.05 vs. FGF2 KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Immunofluorescence, Staining

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Mice reconstituted with FGF2 KO macrophages and subjected to CLP exhibit increased proinflammatory gene expression and apoptosis. a - c The expression of the inflammatory factors CXCL1, IL1β, and IL6 in lung tissue was detected by real-time PCR ( n = 3 per group). d TUNEL staining was used to evaluate apoptosis in the lung tissue. e - f The apoptosis-associated genes BCL2 and Bax were identified by western blot analysis. Bar is 20 μm. * p < 0.05, vs. WT; Δ p < 0.05, vs. WT + LPS; # p < 0.05 vs. FGF2 KO

Article Snippet: Briefly, BMDM were stimulated with 10 ng/ml LPS or 10 ng/ml IL4 for 24 h. After fixation with 4% paraformaldehyde (Cat No.BL539A, Labgic, Beijing, China) for 10 min, the cells were blocked with normal goat serum (Cat No. A7007, Beyotime, Jiangsu, China) for 30 min at room temperature, followed by incubation with either FGF2 antibody (1:100, Cat No. MA00121, Boster, Wuhan, China) or P65 antibody (1:500, Cat No.8242, CST, USA) for 1 h at 37 °C.

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of primers for transcript analysis

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques:

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of antibodies for WB, IF, and flow cytometry analysis

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Flow Cytometry, Recombinant

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by late-onset AD. A Scheme illustrating the sample collection of NSCs from two iPSC clones of the same donor. Quantification of B transcript and D protein amounts of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare three healthy iPSCs (WISCi004-B, WAi001-B, and MLUi009-A) versus four AD iPSCs (MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; healthy: n = 9, AD: n = 12). E Representative image of WB membranes. C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to health status ( N = 3 differentiations; healthy: n = 9, AD: n = 12). Results are shown as mean ± SEM (* p = 0.05)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Clone Assay, Multiplex Assay, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Detection of marker proteins for cellular aging in NSCs through immunofluorescence (IF). IF analysis showed cellular localization of APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Protein of interest is shown in green versus DNA staining in blue (scale bar, 100 μm)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Marker, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by APOE genotype. A Transcript and B protein expression analysis of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare two iPSCs carrying APOE3 (WISCi004-B and MLUi009-A) versus five iPSCs carrying APOE4 (WAi001-B, MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to APOE status ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). Results are shown as mean ± SEM (* p = 0.05). D Manipulation of APOE gene expression by APOE siRNAs and APOE3 plasmids in NSCs generated from reference cell line WISCi004-B on 7 d. No morphological changes are shown by phase contrast imaging up to 4 d after transfection. WB analysis showed the strongest APOE protein repression by APOE siRNAs and strongest APOE induction by APOE3 plasmids on 4 d. Representative images of stained WB membranes are shown (scale bar, 100 μm). Transcript analysis of aging markers in NSCs on 4 d after E APOE inhibition and F APOE induction. Results are shown as mean ± SEM (* p = 0.05). For APOE inhibition, MLUi009-A (healthy matched control, APOE3 carrier) was differentiated into NSCs and transfected with siRNAs on 7 d ( N = 3 differentiations; mock: n = 3, siRNA: n = 3). For APOE induction, the MLUi007-H from (AD, APOE4 carrier) was differentiated into NSCs and transfected with APOE3 plasmids at 7 d ( N = 3 differentiations; mock: n = 3, APOE3 plasmids: n = 3)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Expressing, Multiplex Assay, Quantitative RT-PCR, Generated, Imaging, Transfection, Staining, Inhibition

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Significant mRNA expression changes observed in aging markers

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Expressing, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Proposed mechanisms for APOE and its impact on aging markers for regulating NSC plasticity. Observed effects on PTEN, ATG7, FGF2, and SIRT1 may reflect the impact of APOE on RTK signaling including FGF2 as key signaling molecule, SIRT1 as transcriptional regulator, and PTEN and ATG7 as target genes. RTKs: receptor tyrosine kinases; JAKs: Janus kinases; AKT: protein kinases B; gray: Golgi apparatus; green arrows: APOE signaling; violet arrows: aging markers signaling via JAKs, RTKs, and Notch

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques:

Journal: Molecular neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity.

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Fig. 4 Detection of marker proteins for cellular aging in NSCs through immunofluorescence (IF). IF analysis showed cellular localization of APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Protein of interest is shown in green versus DNA staining in blue (scale bar, 100 μm)

Article Snippet: 1 3 Mouse IgG anti-human ACTB WB: 1:40,000 Merck A5441 Mouse IgG anti-human APOE WB: 1:500; IF: 1:500 Bio-Techne NB110-60531 Mouse IgG anti-human ATG7 WB: 1:1000; IF: 1:100 Bio-Techne MAB6608 Mouse IgG anti-human COL1A1 IF: 1:100 Merck C2456 Rabbit IgG anti-human CDH1 IF: 1:100 Abcam Ab40772 Mouse IgG anti-human FGF2 WB: 1:100 Santa Cruz sc-136255 Mouse IgG anti-human FGF2 IF: 1:100 Bio-Techne NBP1-47749 Rabbit IgG anti-human MSI1 IF: 1:100 Merck AB5977 Mouse IgG anti-human NES IF: 1:100 Santa Cruz Sc-23927 Rabbit IgG anti-human NEUROG3 IF: 1:100 Abcam Ab38548 Mouse IgG anti-human OCT4 (POU5F1) IF: 1:100 Santa Cruz Sc-5279 Mouse IgG anti-human PAX6 IF: 1:100 Santa Cruz Sc-53108 Goat IgG anti-human PRRX1 IF: 1:100 Bio-Techne NBP1-06067 Rabbit IgG anti-human p21m (CDKN1A) WB: 1:1000; IF: 1:800 Cell Signaling Technology 2947 Mouse IgG anti-human p16 (CDKN2A) IF: 1:100 Origene TA500036 Mouse IgM anti-human PTEN WB: 1:100; IF: 1:200 Thermo Fisher Scientific MA5-12278 Mouse IgG anti-human SIRT1 WB: 1:1000; IF: 1:1000 Bio-Techne NBP1-51641 Goat IgG anti-human SOX2 IF: 1:100 Santa Cruz Sc17320 Mouse IgG anti-human SOX17 IF: 1:100 Santa Cruz Sc-130295 Mouse IgM anti-human SSEA1 IF: 1:100 Santa Cruz Sc-21702 Mouse IgG anti-human STAT3 WB: 1:5000; IF: 1:100 Thermo Fisher Scientific MA1-13042 Mouse IgG anti-human VIM IF: 1:100 Merck MAB3400 Goat IgG anti-mouse HRP WB: 1:10,000 Dianova 115-035-003 Goat IgG anti-rabbit HRP WB: 1:3000 Cell Signaling Technology 7074 Goat IgM anti-mouse HRP WB: 1:4000 Thermo Fisher Scientific 62-6820 Donkey IgG anti-goat Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11055 Goat IgG anti-mouse Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11001 1 3

Techniques: Marker, Immunofluorescence, Staining

Journal: Molecular neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity.

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Fig. 6 Proposed mechanisms for APOE and its impact on aging markers for regulating NSC plasticity. Observed effects on PTEN, ATG7, FGF2, and SIRT1 may reflect the impact of APOE on RTK signaling including FGF2 as key signaling molecule, SIRT1 as transcrip- tional regulator, and PTEN and ATG7 as target genes. RTKs: receptor tyrosine kinases; JAKs: Janus kinases; AKT: protein kinases B; gray: Golgi apparatus; green arrows: APOE signaling; violet arrows: aging markers signaling via JAKs, RTKs, and Notch

Article Snippet: 1 3 Mouse IgG anti-human ACTB WB: 1:40,000 Merck A5441 Mouse IgG anti-human APOE WB: 1:500; IF: 1:500 Bio-Techne NB110-60531 Mouse IgG anti-human ATG7 WB: 1:1000; IF: 1:100 Bio-Techne MAB6608 Mouse IgG anti-human COL1A1 IF: 1:100 Merck C2456 Rabbit IgG anti-human CDH1 IF: 1:100 Abcam Ab40772 Mouse IgG anti-human FGF2 WB: 1:100 Santa Cruz sc-136255 Mouse IgG anti-human FGF2 IF: 1:100 Bio-Techne NBP1-47749 Rabbit IgG anti-human MSI1 IF: 1:100 Merck AB5977 Mouse IgG anti-human NES IF: 1:100 Santa Cruz Sc-23927 Rabbit IgG anti-human NEUROG3 IF: 1:100 Abcam Ab38548 Mouse IgG anti-human OCT4 (POU5F1) IF: 1:100 Santa Cruz Sc-5279 Mouse IgG anti-human PAX6 IF: 1:100 Santa Cruz Sc-53108 Goat IgG anti-human PRRX1 IF: 1:100 Bio-Techne NBP1-06067 Rabbit IgG anti-human p21m (CDKN1A) WB: 1:1000; IF: 1:800 Cell Signaling Technology 2947 Mouse IgG anti-human p16 (CDKN2A) IF: 1:100 Origene TA500036 Mouse IgM anti-human PTEN WB: 1:100; IF: 1:200 Thermo Fisher Scientific MA5-12278 Mouse IgG anti-human SIRT1 WB: 1:1000; IF: 1:1000 Bio-Techne NBP1-51641 Goat IgG anti-human SOX2 IF: 1:100 Santa Cruz Sc17320 Mouse IgG anti-human SOX17 IF: 1:100 Santa Cruz Sc-130295 Mouse IgM anti-human SSEA1 IF: 1:100 Santa Cruz Sc-21702 Mouse IgG anti-human STAT3 WB: 1:5000; IF: 1:100 Thermo Fisher Scientific MA1-13042 Mouse IgG anti-human VIM IF: 1:100 Merck MAB3400 Goat IgG anti-mouse HRP WB: 1:10,000 Dianova 115-035-003 Goat IgG anti-rabbit HRP WB: 1:3000 Cell Signaling Technology 7074 Goat IgM anti-mouse HRP WB: 1:4000 Thermo Fisher Scientific 62-6820 Donkey IgG anti-goat Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11055 Goat IgG anti-mouse Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11001 1 3

Techniques: