Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 1: Immunohistochemical analyses of CDC25a and YBX1 proteins in different subtypes of lung adenocarcinoma. The expression of CDC25a and YBX1 in MIA was detected by IHC assay. MIA, minimally invasive adenocarcinoma; Lep, lepidic predominant adenocarcinoma; Aci, acinar predominant adenocarcinoma; Pap, papillary predominant adenocarcinoma; Mp, micropapillary predominant adenocarcinoma; Sci, solid predominant adenocarcinoma; Mu, mucinous predominant adenocarcinoma.100X magnification of the micrographs as insert in the figure.

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 2: CDC25a and YBX1 were highly expressed in lung adenocarcinoma cell lines. A. Co-localization of CDC25a and YBX1 in HLF, A549, H322 and Hcc827 cells by IF assay. Red: YBX1; Green: CDC25a. B,C,D. The expression of CDC25a and YBX1 proteins in total (W), nuclear (N) and cytoplasm (C) protein in lung adenocarcinoma cell lines and HLF cells detected by Western Blot. Expression of CDC25a and YBX1 were calculated relative to the Beta-actin or Histone H3 expression. The quantification of YBX1 and CDC25a was originated from all bands shown in the photograph. The date was repeated three times and presented as the mean±SD (*p<0.05, **p=<0.001 and # p>0.005).

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 3: The scattered/correlation blot between CDC25a and YBX1 expression and survival curves of CDC25a/YBX1 expression, IASLC/ATS/ERS classification and TNM stages in 116 patients with lung adenocarcinoma. A. A scattered/ correlation blot between CDC25a and YBX1 expression in 116 patients. Each number represents a patient. B. The overall 5-year survival rates in the patients with low CDC25a expression and high CDC25a expression were 79.7% and 45.5%, respectively (p=0.004). C. Survival curves of low and high expression of YBX1 was 74.9% and 40.5%, respectively (p=0.044). D. The 5-year survival rates of low, middle and high risk classification risk group was 75.0%, 61.3% and 42.1%, respectively (p=0.037). E. The 5-year survival rates of low TNM stage (I/ II) and high TNM stage (III/IV) was 65.0 and 23.7%, respectively (p<0.001).

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Expressing

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 4: YBX1 bound to CDC25a promoter region and positively regulated its transcriptional activation in lung adenocarcinoma cells. A. Three YBX1-binding possibility regions (indicated by the arrows) were checked in CDC25a promoter (GENE BANK: AJ242714.1). B. A 5’-flanking DNA fragment from position -841 to +336 (-841/+336, -235/+336, -183/+336, -72/+336) of human CDC25a gene was constructed into a promoter less luciferase expression vector pGL3. C. A549 cells were cotransfected with YBX1 and CDC25a promoter-driven luciferase plasmids for 48 hrs. The proteins were extracted, and luciferase activity was detected by luciferase reporter assay kit. pCDNA3.1-vector were negative control. Luciferase activity was detected.

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Activation Assay, Binding Assay, Construct, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay, Negative Control

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 5: YBX1 knockdown inhibited the expression of CDC25a and changed its downstream in G1/S checkpoint pathway. A. In accordance with luciferase activity, the ChIP assays were performed in HLF, A549 and H322 cells to confirm the endogenous of YBX1 binding to the CDC25a promoter (-352 to -153 bp) using an anti-YB-1 antibodyor normal rabbit IgG. B. YBX1 knockdown reduced endogenous YBX1 binding to the CDC25a promoter using ChIP assays in A549 and H322 cells. C. RT-PCR assay revealed si-YBX1 inhibited the mRNA expression of YBX1 and CDC25a in A549 and H322 cells. D. G1/S checkpoint pathway was detected by Western blot assay in A549 cells transfected with si-YBX1. E. Thesi-YBX1-transfected A549 and H322 cells stagnated in G0/ G1 phase by cell cycle analysis.

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Knockdown, Expressing, Luciferase, Activity Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Cell Cycle Assay

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 6: YBX1 knockdown inhibited the growth and migration of lung adenocarcinoma cells and induced apoptosis. A. Cell viability was analyzed by MTT in A549 and H322 cells transfected with si-YBX1 at 24 and 48h. The effect on cell viability was assessed as the percent cell viability compared with control-PBS group, which were arbitrarily assigned 100% viability. B. Colony formation assay of A549 and H322 cells transfected with si-YBX1 and its quantification. C. A549 and H322 cells were treated with si- YBX1 and the apoptosis was determined by a FACS analysis. D. Cleaved caspase-3/9 proteins in A549 and H322 cells were respectively analyzed by Western blot. E. Cell migration was analyzed by a wound-healing assay. A549 and H322 cells were seeded in 6-well plates and grown to full confluence. Migration rate was calculated as MR=[initial gap(0h)- terminal gap(48h)/ initial gap(0h)]100%. The date was repeated three times and presented as the mean±SD (*p<0.05,**p=<0.001 and# p>0.005).

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Knockdown, Migration, Transfection, Control, Colony Assay, Western Blot, Wound Healing Assay

Journal: Oncotarget

Article Title: YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

doi: 10.18632/oncotarget.10080

Figure Lengend Snippet: Figure 7: YBX1 knockdown inhibited tumor growth by down-regulating CDC25a expression. A. A photo of tumor grafts in nude mice injected with non-specific control siRNA (n=6) or si-YBX1 (n=6) 21 days latter and tumor volume of each group of nude mice was measured by V=(width2×length)/2; **P<0.01. B. The morphology of tumor xenografts in each nude mice after anatomy at 21 days of treatment was shown and tumor weight was analyzed by electronic scales; **P<0.01. C. H&E staining and D. Immunohistochemical analysis of YBX1, CDC25a, Ki 67 and cleaved caspase 3 protein expression in tumor samples.

Article Snippet: The overexpression vector of YBX1 (pcDNA3.1-YBX1) and control vector (pcDNA3.1-vector) plasmids were designed and synthesized by Cyagen (Cyagen Biosciences Inc., China).

Techniques: Knockdown, Expressing, Injection, Control, Staining, Immunohistochemical staining

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: High confidence TAM-induced protein stability hits.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques:

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: Pulse proteolysis with purified recombinant YBX1 +/− TAM. A) Pulse proteolysis of YBX1 +/− TAM in increasing concentration of urea. Red box depicting TAM-induced difference at 0.75 M Urea. B) Intact YBX1 and pulse proteolysis of YBX1 in the absence of TAM at low and high denaturant.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: Purification, Recombinant, Concentration Assay

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: SPROX data generated on YBX1 +/− TAM. The filled circles represent data generated on the YBX1 peptide RPQYSNPPVQGEVMEGADNQGAGEQGRPVR in the PAB-SPROX analysis of the purified recombinant YBX1 construct. The open circles represent data on the oxidized version of the same YBX1 peptide in the SILAC-SPROX analysis of the unpurified protein in the MCF-7 cell lysate. Both data sets are consistent with a TAM-induced stabilization of YBX1.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: Generated, Purification, Recombinant, Construct

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: STRING analysis of the 27 high-confidence TAM and NDT hits. The STRING analysis was conducted using medium confidence data from experiments. Also included in the STRING analysis were the MAPK1, AKT1 and ESR1 (see text). The red dashed line represents an experimentally observed direct interaction between ER and YBX1 that was recently reported in reference.34

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques:

Journal: Molecular cancer

Article Title: The m 6 A modification mediated-lncRNA POU6F2-AS1 reprograms fatty acid metabolism and facilitates the growth of colorectal cancer via upregulation of FASN.

doi: 10.1186/s12943-024-01962-8

Figure Lengend Snippet: Fig. 4 POU6F2-AS1 interacts with YBX1 to promote CRC cell growth and lipogenesis. (A) Schematic workflow of the biotinylated RNA pull-down assay and Mass spectrometry used for identification of POU6F2-AS1 binding proteins. (B) Venn diagram exhibiting the proteins pulled down by biotin-labeled POU6F2-AS1, and overlapping analysis with RBPDB and RBPmap database. (C) Representative YBX1 peptides binding with POU6F2-AS1 were detected by MS. (D) Western blotting of YBX1 pull downed by biotin-labeled POU6F2-AS1 was shown. (E) qRT–PCR analysis of POU6F2-AS1 enriched by YBX1 protein in SW480 cells was shown. (F) Colocalization analysis was assessed with the specific probes against POU6F2-AS1 (red) and the specific antibody against YBX1 (green). Nuclei are stained with DAPI (blue). (G) The potential interaction regions of POU6F2-AS1 with YBX1 were predicted by catRAPID website. (H) Schematic view of truncated fragments of POU6F2-AS1. (I) Western blotting analysis of YBX1 in samples pulled down by biotin-labeled full-length (F1, 2) or truncated POU6F2-AS1 (F3–F5). (J) Domain mapping of Flag-labeled full-length YBX1 (P4) or truncated YBX1 (P1–P3). (K) Western Blot analysis of Flag-tagged YBX1 constructs in SW480 cells transfected with each construct (P1: 1–129 aa, P2: 130–205 aa, P3: 206–324 aa). (L) RIP assays were performed by the antibodies against Flag, followed by POU6F2-AS1 qRT–PCR in SW480 cells. (M) CCK–8 assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. (N) Colony formation assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. (O) Oil Red O staining assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Immunoprecipitation of cross-linked protein/DNA was performed by incubating anti-YBX1 antibody (Proteintech, IL, USA) or normal rabbit IgG (Proteintech, IL, USA) with sheared chromatin overnight at 4 °C followed by incubation with Protein A/G Magnetic Beads for 2 h. Subsequently, cross-links of protein/ DNA were released with 5 M NaCl, and the DNA was purified using a PCR Clean Up Kit (Beyotime, Shanghai, China).

Techniques: Pull Down Assay, Mass Spectrometry, Binding Assay, Labeling, Western Blot, Quantitative RT-PCR, Staining, Construct, Transfection, CCK-8 Assay, Over Expression, Knockdown, Colony Assay

Journal: Molecular cancer

Article Title: The m 6 A modification mediated-lncRNA POU6F2-AS1 reprograms fatty acid metabolism and facilitates the growth of colorectal cancer via upregulation of FASN.

doi: 10.1186/s12943-024-01962-8

Figure Lengend Snippet: Fig. 5 POU6F2-AS1 tethers YBX1 to the FASN promoter to transcriptionally activate FASN and promotes CRC cell progression. (A) Venn diagram exhibit ing the downstream genes coregulated by POU6F2-AS1 and YBX1, and overlapping analysis with lipid metabolism-related genes (LMGs). (B) qRT–PCR and Western blotting showed that the reduction in FASN in HCT116 cells with transfected with POU6F2-AS1 siRNAs was reversed by YBX1 overexpression, whereas the increase in FASN in LoVo cells transfected with POU6F2-AS1 overexpression plasmid was reversed by YBX1 knockdown. (C) The YBX1-binding motif and sites enriched in the FASN promoter was predicted by JASPAR website. (D, E) Luciferase reporter assays showed that the luciferase activity driven by the − 447/-136 fragment of the FASN promoter region was significantly reduced in HCT116 cells with YBX1 knockdown but enhanced in LoVo cells with YBX1 overexpression. (F) Luciferase reporter assays for − 447/-136 fragment of the FASN promoter activity in SW480 cells with POU6F2-AS1 knockdown with or without overexpressing YBX1 (left), and in LoVo cells with POU6F2-AS1 overexpression with or without knocking down YBX1 (right). (G) The schematic diagram of truncated FASN promoter. (H) ChIP-qPCR analysis was performed to determine the binding affinity of YBX1 to nine FASN promoter regions in SW480 cells, showing that YBX1 bound to P2-4 regions in the FASN promoter. ChIP-qPCR with IgG was used as the negative control. (I) ChIP-qPCR analysis for the enrichment of YBX1 at the FASN promoter regions in SW480 cells with PO6F2-AS1 knockdown. (J) Representative images showing the expression of FASN in CRC tissues and paired ANTs detected by IHC. (K) CRC patient-derived organoid infected with POU6F2-AS1 knock down or FASN overexpression lentivirus. (L) CCK–8 assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. (M) Colony formation assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. (N) Oil Red O staining assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Immunoprecipitation of cross-linked protein/DNA was performed by incubating anti-YBX1 antibody (Proteintech, IL, USA) or normal rabbit IgG (Proteintech, IL, USA) with sheared chromatin overnight at 4 °C followed by incubation with Protein A/G Magnetic Beads for 2 h. Subsequently, cross-links of protein/ DNA were released with 5 M NaCl, and the DNA was purified using a PCR Clean Up Kit (Beyotime, Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Plasmid Preparation, Knockdown, Binding Assay, Luciferase, Activity Assay, ChIP-qPCR, Negative Control, Expressing, Derivative Assay, Infection, CCK-8 Assay, Colony Assay, Staining

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 1. YBX1 interacts with HBZ. (A) MS-MS experiments were performed to identify unique protein interactions. Numbers indicate the total spectral counts, or numbers of unique peptides, found in a subset of proteins with HBZ and APH-2 pulldown. (B) HEK293T cells were transfected with empty, HBZ, or APH-2 S-tagged expression vectors. Proteins were purified by S-tag affinity 24 h after transfection. Pulldowns were examined by immunoblot analysis using antibodies to YBX1 and S-tag. (C) PBL-HTLV-1 (HTLV-1 newly immortalized), SLB-1 (HTLV-1 transformed), and (D) ATL-ED or TL-Om1 (ATL-derived) T-cell lines were immunoprecipitated with a control rabbit antibody or HBZ rabbit antisera. Immunoprecipitated proteins were examined by immunoblot analysis using antibodies to YBX1 and HBZ. 10% of IP was included as a direct load.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Tandem Mass Spectroscopy, Transfection, Expressing, Western Blot, Transformation Assay, Derivative Assay, Immunoprecipitation, Control

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 2. YBX1 influences viral transcription. (A–C) HEK293T cells were co-transfected with (A) pcDNA3.1(+) empty, HBZ, and increasing amounts of myc-tagged YBX1 expression vectors; (B) pcDNA3.1(+) empty, Tax, and increasing amounts of myc-tagged YBX1 expression vectors; or (C) pcDNA3.1(+) empty, Tax, HBZ, and/or myc-tagged YBX1 expression vectors with an HTLV-1 LTR-firefly luciferase construct and tk-renilla luciferase construct (internal control). At 48 h post- transfection, cells were collected for luciferase assays to measure relative LTR transactivation (left panels). The empty vector was set at 1. Total cell lysates were examined by immunoblot analysis using antibodies to YBX1, myc (YBX1), HBZ, Tax, and β-actin (right panels). HEK293T cells (D,E) were transduced with lentiviral vectors expressing shRNA directed against YBX1. After a brief puromycin selection, the cells were co-transfected with (D) an HTLV-1 LTR-firefly luciferase construct, a tk-renilla luciferase construct (internal control), and (E) pcDNA3.1(+) empty, Tax, and/or HBZ expression vectors. At 48 h post-transfection, cells were collected for luciferase assays to measure relative LTR transactivation (left panels). Empty vector was set at 1. Total cell lysates were examined by immunoblot analysis using antibodies to YBX1, HBZ, Tax, and β-actin (right panels). (F) Jurkat cells were transduced with lentiviral vectors expressing shRNA directed against YBX1. After a brief puromycin selection, the cells were co-transfected with an HTLV-1 LTR-firefly luciferase construct, a tk-renilla luciferase construct (internal control), pcDNA3.1(+) empty, Tax, and/or HBZ expression vectors. 48 h post-transfection, cells were collected for a luciferase assay to measure relative LTR transactivation (left panel). The empty vector was set at 1. Total cell lysates were examined by immunoblot analysis using antibodies to YBX1 and β-actin (right panel). Graphs represent data generated from duplicate samples, and error bars represent the standard deviation (SD). The data are representative of at least three experimental repeats. Statistical significance was determined using a Student’s t-test; * p ≤0.05, ** p ≤0.01, **** p ≤0.0001.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Transfection, Expressing, Luciferase, Construct, Control, Plasmid Preparation, Western Blot, Transduction, shRNA, Selection, Generated, Standard Deviation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 3. YBX1 interacts with Tax. (A) PBL-HTLV-1 and (B) SLB-1 cell lines were immunoprecipitated with a control rabbit IgG, Tax, or HBZ antibody. IPs were examined by immunoblot analysis using antibodies to YBX1, Tax, and Hbz. 10% of IP was included as a direct load. (C) JPX-M cells (subclone of Jurkat cells with a metallothionein promoter-driven Tax mutant expression plasmid) were treated with 15 µM cadmium chloride (CdCl2) for 24 h to produce Tax protein. Uninduced and induced JPX-M cells and Jurkat cells were immunoprecipitated with a control rabbit IgG or YBX1 antibody. IPs were examined by immunoblot analysis using antibodies to YBX1 and Tax. 10% of IP was included as a direct load.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Immunoprecipitation, Control, Western Blot, Mutagenesis, Expressing, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 4. YBX1 activates sense and antisense viral transcription. (A) HEK293T cells were co- transfected with an HTLV-1 proviral clone and a YBX1 expression plasmid, as indicated. Protein levels were examined 48 h post-transfection by immunoblot analysis using antibodies to HBZ, myc (YBX1), and β-actin. (B) Quantitative RT-PCR for hbz and gapdh was performed on mRNA isolated from cells in (A). The total hbz mRNA level was determined using the ∆∆Ct method, normalized to relative gapdh levels, and the HTLV-1 provirus alone was set at 1. (C) HEK293T scramble and shYBX1 cells were transfected with an HTLV-1 proviral clone for 48 h. Protein levels were examined by immunoblot analysis using antibodies to Tax, YBX1, and β-actin. Tax protein intensity relative to β-actin without YBX1 knockdown was set at 1. (D) Quantitative RT-PCR for hbz, tax, and gapdh was performed on mRNA isolated from cells in (C). Total hbz and tax mRNA levels were determined using the ∆∆Ct method, normalized to relative gapdh levels, and control cells (scramble) were set at 1. (E) HTLV-1 gene expression was quantified from cells in (C) through the detection of the p19 Gag protein in the culture supernatant using ELISA. (F) SLB-1 and (G) ATL-ED transformed T-cell lines were transduced with lentiviral vectors expressing shRNA directed against YBX1. After a brief puromycin selection, cells were collected and analyzed by immunoblot using antibodies to Hbz, Tax, YBX1, and β-actin. Tax and Hbz protein intensity relative to β-actin in each scramble condition was set at 1. All graphs represent data generated from duplicate samples, and error bars represent the standard deviation (SD). The data are representative of at least three experimental repeats. Statistical significance was determined using Student’s t-test: * p ≤0.05, ** p ≤0.01, **** p ≤0.0001.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Isolation, Knockdown, Control, Gene Expression, Enzyme-linked Immunosorbent Assay, Transformation Assay, Transduction, shRNA, Selection, Generated, Standard Deviation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 5. YBX1 associates with the 5′ and 3′ viral LTRs. ChIP assays were performed on cross- linked chromatin from PBL-HTLV-1, SLB-1, TL-Om1, and ATL-ED cells using either IgG or YBX1 antibodies. Retained DNA was amplified using (A) 5′ LTR or (B) 3′ LTR-specific primers and qPCR. Fold enrichment for each cell line was calculated relative to the IgG sample. Each ChIP experiment was repeated twice in duplicate. Error bars represent the standard deviation of the sample means. Statistical significance was determined using Student’s t-test: * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Standard Deviation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 6. Mutation of the proposed YBX1 binding site does not affect viral transcription. (A) The proposed YBX1 binding site was mutated in the LTR-1-luciferase expression vector through site- directed mutagenesis (shown as bold, underlined text). An alignment of the YBX1 mutation (YBX1m) region and the wt region from the HTLV-1 LTR is shown. (B) HEK293T cells were co-transfected with pcDNA3.1(+) empty, YBX1, wt HTLV-1 LTR-firefly luciferase, or YBX1m HTLV-1 LTR-firefly luciferase, and tk-renilla luciferase (internal control) constructs, as indicated. At 48 h post-transfection, cells were collected for luciferase assays to measure relative LTR transactivation. The empty vector was set at 1. (C) HEK293T cells were co-transfected with pcDNA3.1(+) empty, YBX1, HBZ, Tax, HTLV-1 LTR-firefly luciferase, YBX1m LTR-firefly luciferase, and tk-renilla luciferase (internal control) constructs, as indicated. At 48 h post-transfection, cells were collected for luciferase assays to measure relative LTR transactivation (top panel). The empty vector was set at 1. Total cell lysates were examined by immunoblot analysis using antibodies to YBX1, HBZ, Tax, and β-actin (bottom panel). (D) HEK293T scramble and shYBX1 cells were co-transfected with pcDNA3.1(+) empty, Tax, HBZ, HTLV-1 LTR-firefly luciferase, YBX1m LTR-firefly luciferase, and tk-renilla luciferase (internal control) constructs, as indicated. At 48 h post-transfection, cells were collected for luciferase assays to measure relative LTR transactivation. The empty vector was set at 1. Total cell lysates were examined by immunoblot analysis using antibodies to YBX1 and β-actin (right panel). Graphs represent data generated from duplicate samples, and error bars represent the standard deviation (SD). The data are representative of at least three experimental repeats.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Mutagenesis, Binding Assay, Luciferase, Expressing, Plasmid Preparation, Transfection, Control, Construct, Western Blot, Generated, Standard Deviation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 7. YBX1 enhances Tax binding, and HBZ represses YBX1 transcriptional activation. (A) SLB-1 cells were transduced with lentiviral vectors expressing shRNA directed against YBX1. After a brief puromycin selection, ChIP assays were performed on cross-linked chromatin using either IgG or Tax antibodies. Retained DNA was amplified using 5′ LTR-specific primers and qPCR. Fold enrichment was calculated relative to the IgG sample. Each ChIP experiment was repeated twice in duplicate. (B) HEK293T cells were co-transfected with pcDNA3.1(+) empty, YBX1, wt.HTLV-1 proviral clone, a HTLV-1 proviral clone that lacks HBZ protein (HTLV-1.∆HBZ), HTLV-1 LTR-firefly luciferase, and tk-renilla luciferase (internal control) constructs, as indicated. At 48 h post-transfection, cells were collected for luciferase assays to measure relative LTR transactivation. The empty vector was set at 1. (C) Total hbz mRNA levels from HTLV-1.∆HBZ cells in (B) were determined using the ∆∆Ct method, normalized to relative gapdh levels. HTLV-1.∆HBZ provirus alone was set at 1. (D) HEK293T scramble and shYBX1 cells were co-transfected with wt.HTLV-1 or HTLV-1.∆HBZ proviral clones, as indicated. After 48 h, total cell lysates were examined by immunoblot analysis using antibodies to YBX1, Tax, and β-actin. Tax protein intensity relative to β-actin in each scramble condition was set at 1. Graphs represent data generated from duplicate samples (5 samples for B), and error bars represent the standard deviation (SD). The data are representative of at least three experimental repeats. Statistical significance was determined using Student’s t-test: ** p ≤0.01, *** p ≤0.001.

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques: Binding Assay, Activation Assay, Transduction, Expressing, shRNA, Selection, Transfection, Luciferase, Control, Construct, Plasmid Preparation, Clone Assay, Western Blot, Generated, Standard Deviation

Journal: International journal of molecular sciences

Article Title: The Pleiotropic Effects of YBX1 on HTLV-1 Transcription.

doi: 10.3390/ijms241713119

Figure Lengend Snippet: Figure 8. Model depicting YBX1 effects on HTLV-1 transcription. YBX1 associates with both the 5′

Article Snippet: Lysates were rocked overnight at 4 ◦C with 2 μg of the following antibodies: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA), Tax [80], HBZ [14], or YBX1 (Bethyl Laboratories, Montgomery, TX, USA; A303-231A).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: SSAT RNA-interacting proteins and knockdown effects. (A) Chimeric RNA. Bait RNA was created using the first 170 bases of the 5′ end of the SSAT ORF and the last 181 bases of the 3′ end linked by a streptomycin-binding aptamer (red) that allowed the bait to bind to a Sepharose-streptomycin column. (B) Proteins interacting with SSAT RNA. HEK293T cell cytoplasmic proteins were incubated in the column, unbound proteins were washed away, and bound proteins were eluted with streptomycin-containing buffer. Eluted proteins were separated by SDS-PAGE and stained with Sypro ruby. No proteins were detected in the control column eluate, but multiple proteins were eluted from the SSAT-RNA column and then identified by mass spectroscopy (Table 1). Proteins and approximate gel positions are labeled: ENO1 (enolase 1), YBX1 (Y box protein 1), CSDA (DNA-binding protein A), SSB (La protein), DHX9 (ATP-dependent RNA helicase A), and NCL (nucleolin). (C) siRNA knockdowns. Western blots demonstrate individual siRNA knockdown of RNA-interacting proteins. (D) Knockdown effects on SSAT translation. Expression of recombinant SSAT was assayed by anti-His tag Western blotting with actin as the loading control. Lanes 1 to 6 are from cells treated with the indicated siRNA. Lane 7 is a negative control from cells treated with nontargeting siRNA. Lane 8 is a positive control with proteins from cells exposed to DENSPM to induce SSAT translation. Only nucleolin knockdown significantly increased expression of recombinant SSAT (lane 5).

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Knockdown, Binding Assay, Incubation, SDS Page, Staining, Control, Mass Spectrometry, Labeling, Western Blot, Expressing, Recombinant, Negative Control, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: SSAT RNA binding proteins identified by Gel-LCMS a

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: RNA Binding Assay, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: Nucleolin-SSAT RNA interaction in situ. Protein-RNA coprecipitation was detected using RT-PCR. Lanes 1 to 4 are products of RT-PCRs. Lanes 5 to 8 are negative controls omitting reverse transcriptase. Lanes 1 and 5 are positive controls with the PCR target being whole-cell lysate. Lanes 2 and 6 are negative controls with the PCR target being sham immunoprecipitate from protein G-coated beads without bound antibody (No Ab). Lanes 3 and 7 are negative controls with the PCR target being immunoprecipitate using antiactin monoclonal antibody. Lanes 4 and 8 used immunoprecipitate from beads with antinucleolin monoclonal antibody as the PCR target. The data show SSAT RNA coprecipitates with nucleolin.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: In Situ, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: Glycine/arginine-rich nucleolin domain is required for SSAT mRNA binding. (A) Nucleolin schematic with amino acid numbers: N-term, N-terminal region; RRM1 to RRM4, “RNA recognition motif” domains 1 to 4; GAR, glycine/arginine-rich domain. (B) Western blots using an anti-His tag monoclonal antibody confirmed expression of nucleolin fragments by cells transiently transfected with plasmids containing the indicated constructs (native nucleolin or fragments not detected). (C) Anti-His tag Western blots of SSAT-RNA column eluates from transiently transfected cell lysates show that the binding does not occur without the GAR domain.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Construct

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: DENSPM and spermine effects on recombinant nucleolin peptide 1234GAR. HEK293T cells were transiently transfected to overexpress His-tagged 1234GAR (HEK293T-1234GAR); anti-His antibody was used to detect the peptide. (A) 2D blots were prepared as for Fig. 2: control HEK293T NCL-1234GAR cells are on the left, and cells exposed to 10 mM DENSPM for 12 h are on the right. Red arrows indicate a peptide isoform present in controls but absent in cells treated with DENSPM. (B) 2D blots of peptides that bound to SSAT RNA columns; peptides from controls are on the left, and those from cells exposed to 10 mM DENSPM for 12 h are on the right. 1234GAR peptides bind to the column and are markedly reduced in cells exposed to DENSPM. (C) One-dimensional (1D) Western blots show 1234GAR peptide autocatalysis in cells exposed to 2 mM spermine for 12 h.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Recombinant, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: Exogenous spermine reduces 55-kDa nucleolin isoform. 2D Western blots in panels A, B, and C used IPG strips (pI 3 to 6) for the first separation, SDS-PAGE for the second, and monoclonal antinucleolin antibody for the blots. Red arrows point to the position of the 55-kDa isoform that binds to the SSAT-RNA column and is missing or reduced in cells exposed to spermine. (A) HEK293T cells grown under control conditions. (B) HEK293T cells exposed to 2 mM spermine for 12 h. (C) Proteins eluted from a SSAT-RNA interaction column (high intensity of the 55-kDa spot results from column enrichment). (D) Western blots of cells exposed to 0, 1, and 2 mM spermine for 12 h. Upper and lower blots are from the same 3 cultures: upper panels show dose-dependent spermine-induced degradation of the 55-kDa nucleolin isoform, and lower panels show corresponding spermine-induced increases in the SSAT protein. (E) Polyamine-mediated nucleolin degradation by 48 h of spermine exposure. (F) An Alamar blue assay was used to test viability of HEK293T cells grown for 48 h in the indicated spermine concentrations. Aminoguanidine (1 mM) was included in all cultures to inhibit generation of reactive oxygen species by amine oxidase activity of serum in the media.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Western Blot, SDS Page, Control, Alamar Blue Assay, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: Characterization of 55-kDa nucleolin isoform by mass spectroscopy. The nucleolin isoform interacting with SSAT RNA was separated by 2DE, trypsin digested, and analyzed by mass spectroscopy. The tandem mass spectrometry (MS/MS) profile of each the four identified peptides (panels A to D) and their representative sequence is shown on the right of each panel. The location of each peptide is shown in panel E.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: SSAT RNA bases essential for nucleolin binding and translation repression. (A) Chimeric RNA-bait molecules. Top schematic, labeled 1-516, is the original chimera used to isolate nucleolin RNA binding proteins. Graphics and labels of others indicate nucleotides omitted in other bait molecules. (B) Nucleotides essential for nucleolin binding. Individual columns were prepared using the seven bait molecules plus a control with nonspecific RNA. HEK293T cell cytoplasmic lysate was incubated with the columns, and bound proteins were eluted and then concentrated for Western blot analysis using an antinucleolin monoclonal antibody. “C+” is a positive control using whole HEK293T lysate. “C-” is a negative control from the nonspecific RNA column. Lanes 3 to 9 are from columns prepared with the indicated bait molecules. All the columns bound nucleolin except those with baits lacking nucleotides 1 to 117. Since the 50–486 and 50–417 columns did bind nucleolin, binding depends on nucleotides within the 50-to-118 range. (C) Nucleotides essential for SSAT translational repression. HEK293T cells were transiently transfected with plasmids containing the complete SSAT ORF (ORF) and mutants using constructs lacking ORF nucleotides 52 to 117 (Δ52-117) or 4 to 45 (Δ4-45). Anti-His antibody was used for the blots, with antiactin for the loading control. Lanes 1 and 3 show that translation of the full SSAT ORF occurs only when the cells are exposed to spermine. Lanes 2 and 4 show that the Δ52-117 and Δ4-45 mutants are translated without spermine and thus both regions are required for translational control even if nucleolin binds to only one.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Binding Assay, Labeling, RNA Binding Assay, Control, Incubation, Western Blot, Positive Control, Negative Control, Transfection, Construct

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: SSAT RNA stem-loop and translational repression. (A) Mfold-calculated RNA secondary structure of first 140 bp of the SSAT ORF. Bases deleted in the Δ52-117 construct are marked in red; blue indicates the Δ4-45 construct. (B) Transiently transfected HEK293T cells. Transfecting plasmids contained the full SSAT ORF (ORF) or mutants with deletions that impair formation of a stem-loop by bases 2 to 76. The Δ4-75 mutant lacks the entire stem-loop, Δ4-30 lacks the first half, and Δ31-75 lacks the second half. Anti-His Western blots show that protein expression by the wild type is dependent on spermine exposure (lanes 1 and 5), but all mutants express protein without spermine (lanes 2, 3, and 4). (C) Fusion of the SSAT ORF stem-loop with a reporter. HEK293T cells were transiently transfected with plasmids producing RNA for the His-tagged reporter protein eGFP or RNA with the SSAT 1–75 stem-loop appended in frame to the 5′ end of eGFP (Loop eGFP). Anti-His Western blots show that Loop eGFP expression is repressed (lane 2) compared to that of eGFP (lane 1), and repression is partially relieved by exposure to 2 mM spermine (lane 3). (D) 5′ UTR and SSAT ORF stem-loop repression. To determine whether the 5′ORF stem-loop represses translation when the full 5′ UTR is present, HEK293T cells were transiently transfected with plasmids containing the 5′ UTR of SSAT RNA and the full coding region (5′UTR-ORF), the 5′ UTR with Δ4-30 ORF (5′UTR-Δ4-30), and the 5′UTR with Δ49-114 ORF (5′UTR-Δ49-114). Expression of 5′UTR-ORF required spermine exposure (lanes 1 and 4) while the mutants did not. Expression of 5′UTR-Δ49-114 (lane 3), which doesn't form the stem-loop and lacks the nucleolin binding region, was greater than that of 5′UTR-Δ4-30 (lane 2), which also doesn't form the stem-loop but does have the nucleolin binding region; the same relative relationship can be seen in panel B, although it is not as marked.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Construct, Transfection, Mutagenesis, Western Blot, Expressing, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: SSAT expression is elevated in mutants lacking uORFs. Red dots show locations of uORF starting codons. HEK293T cells were transiently transfected with plasmids having SSAT RNA 5′uORF plus the full coding region (5′UTR-ORF), mutations eliminating uORF starting codons plus the full coding region (ΔuORF-ORF), mutations eliminating the uORFs plus coding region deletions preventing 5′ORF stem-loop formation (ΔuORF-Δ4-3 ORF), or mutations eliminating the uORFs plus coding region deletions preventing 5′ ORF stem-loop formation and nucleolin binding (ΔuORF-Δ49-114ORF). (A) uORFs affect translation. Lanes 1 and 2 show that elimination of uORFs does not allow SSAT expression without spermine, but lanes 5 and 6 show that uORF elimination enhances spermine-induced expression. Lanes 3 and 4 show that elimination of both the uORFs and stem-loop allows greater expression, and further elimination of the nucleolin binding site enhances expression further. (B) uORF translational control is not affected by spermine. SSAT expression was compared among three constructs that require spermine for translation: 5′UTR-ORF, ΔuORF-ORF, and ORF. Since spermine-induced SSAT translation of ORF was markedly less than that for 5′UTR ORF but the difference was eliminated when the start codons of the uORFs were eliminated, that indicates that the translation repression by uORFs is independent of the presence of spermine. The slight translation leakage of ΔuORF-ORF without spermine supports this interpretation.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Expressing, Transfection, Binding Assay, Control, Construct

Journal: Molecular and Cellular Biology

Article Title: Polyamine-Regulated Translation of Spermidine/Spermine- N 1 -Acetyltransferase

doi: 10.1128/MCB.06444-11

Figure Lengend Snippet: Model of the SSAT translational control mechanism. (A) SSAT translational repression in the presence of low levels of polyamines. Success of ribosome scanning to the correct initiation codon is reduced in the presence of uORFs. Translation therefore depends on either leaky scanning or reinitiation of ribosome scanning 3′ to the uORFs. Those ribosome subunits escaping uORF control are then blocked by the strong stem-loop at the initiation site, which is stabilized by nucleolin. (B) Polyamine-regulated translational derepression of SSAT. An isoform of nucleolin blocks translation by binding to and stabilizing the stem-loop at the extreme 5′ end of the ORF. Increased polyamines cause autocatalysis of this nucleolin isoform, thereby reducing the stability of the stem-loop and allowing ribosomal subunits that escaped the uORF control to initiate translation. This increase in expression acetylates the polyamines and lowers their concentration, which reduces nucleolin degradation and thereby reestablishes translation repression. Together, these provide a negative feedback mechanism to regulate cellular polyamine concentrations.

Article Snippet: The following antibodies from Santa Cruz Biotechnologies were used for other proteins: nucleolin monoclonal (SC-8031), actin monoclonal (SC-47778), SSB (SC-80655), CSDA (SC-21318), YBX1 (SC-18057), and ENO1 (SC-15343).

Techniques: Control, Binding Assay, Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: Y-box binding protein 1/cyclin A1 axis specifically promotes cell cycle progression at G 2 /M phase in ovarian cancer

doi: 10.1038/s41598-024-72174-9

Figure Lengend Snippet: Effects of YBX1 siRNA on the expression levels of YBX1, cyclin A1, cyclin A2, cyclin B1, cyclin D1, cyclin E1, CDC6, and CDC20 mRNAs in ovarian cancer cell lines. ( A ) The eight selected genes are listed. Correlations of YBX1 with other genes are presented according to Spearman scores based on gene ontology analysis (TCGA database, n = 316) of HGSC, and the chromosomal locations of the 8 genes are indicated. The right panel of the illustrated model shows the sites of action of 7 cell cycle-related genes in the cell cycle. ( B ) RT-qPCR analysis showing mRNA expression levels of cell cycle-related genes in ovarian cancer cells. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. * p < 0.05; ** p < 0.01. Error bars indicate standard deviations for 3 independent experiments. ( C ) RT-qPCR analysis showing mRNA expression levels of 7 genes in ovarian cancer cell lines following transfection with YBX1 siRNA#1 for 3 days. Expression levels were expressed as ratios relative to the expression in the corresponding negative control based on 3 independent assays. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. * p < 0.05; ** p < 0.01. Error bars indicate standard deviations for 3 independent experiments. A heatmap plot of cell cycle-related genes from the comparison of the YBX1 siRNA#1-transfection group and the control group was presented. High expression levels in the YBX1 siRNA#1-transfection group are indicated in red, and low expression levels are indicated in blue. ( D ) JHOC-5 cells were transfected with luciferase reporter constructs containing the 1166 bp 5′-flanking region of the cyclin A1 gene with YBX1 siRNA#1 for 2 days. The firefly luciferase activity was normalized to the Renilla luciferase activity. Student’s t-tests were used to compare differences between YBX1 siRNA#1-transfection groups and the control group. ** p < 0.01. Error bars indicate standard deviations for 3 independent experiments. (E) Schematic representation of potential YBX1 binding sites (Black boxes) and primer locations (arrows) used for ChIP assay in the promoter region of Cyclin A1 gene. (F) The YBX1 binding to the cyclin A1 promoter by ChIP assay in JHOC-5 cells.

Article Snippet: Antibodies against p-YBX1 (cat. no. #2900), cyclin A2 (cat. no. #67955), cyclin B1 (cat. no. #4138), cyclin D1 (cat. no. #55506), cyclin E (cat. no. #4129), and CDC6 (cat. no. #3387), PKCα (cat. no. #59754), p-PKCα/β II (cat. no. #9375), AKT (cat. no. #9272) p-AKT (cat. no. #4060), and CREB (cat. no. #9197) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Negative Control, Comparison, Luciferase, Construct, Activity Assay, Binding Assay

Journal: Scientific Reports

Article Title: Y-box binding protein 1/cyclin A1 axis specifically promotes cell cycle progression at G 2 /M phase in ovarian cancer

doi: 10.1038/s41598-024-72174-9

Figure Lengend Snippet: Effects of cyclin A1 knockdown by cyclin A1 siRNA on cell growth, and the expression levels of YBX1 and cell cycle-related genes in HGSC and OCCC tumors. ( A ) Cell growth of ovarian cancer cell lines on days 1, 3, and 5 after transfection with cyclin A1 siRNA. Student’s t-tests were used to compare differences between control- and cyclin A1 siRNA-transfected cells. * p < 0.05; ** p < 0.01. Error bars indicate standard deviations for 3 independent experiments. ( B – D ) Percentages of cells in G 0 /G 1 ( B ), S ( C ), and G 2 /M ( D ) phases following transfection with cyclin A1 siRNA for 3 days. Percentages were determined by gating the FACS plot. Student’s t-tests were used to compare differences between control- and cyclin A1 siRNA-transfected cells. * p < 0.05; ** p < 0.01. Error bars indicate standard deviations for 3 independent experiments. ( E ) Expression levels of YBX1 and cyclin A1, as assessed by RT-qPCR analysis in HGSC (n = 50) and OCCC tumors (n = 51). The statistical significance was determined using the Mann–Whitney U tests. ( F , G ) Correlation of YBX1 mRNA expression levels with the mRNA expression of cyclin A1 in HGSC (n = 50) ( F ) and OCCC tumors (n = 51) ( G ). The statistical significance of the correlations was determined using Pearson’s correlation coefficients and p values.

Article Snippet: Antibodies against p-YBX1 (cat. no. #2900), cyclin A2 (cat. no. #67955), cyclin B1 (cat. no. #4138), cyclin D1 (cat. no. #55506), cyclin E (cat. no. #4129), and CDC6 (cat. no. #3387), PKCα (cat. no. #59754), p-PKCα/β II (cat. no. #9375), AKT (cat. no. #9272) p-AKT (cat. no. #4060), and CREB (cat. no. #9197) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Expressing, Transfection, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 3. Effect of RNA demethylase ALKBH5 on ferroptosis levels in High-glucose Hypoxia-Reoxygenation (HH/R) model. A) Schematic diagram illustrating the experimental protocol for treating H9c2 and Neonatal Mouse Ventricular Cardiomyocytes (NMVCs) cells. The process includes hypoxia for 4 h, reoxygenation, synchronization for 12 h, and treatment with lentivirus (LV) carrying siRNAs against G3BP1, ALKBH5, and YBX1, followed by collection of cardiomyocytes at various time – points (0, 24, 48, 72 h). B) The ultrastructural changes of cells were detected by TEM. C,D) Bar graphs depicting cell viability percentages and LDH release levels in H9c2 and NMVCs cells across different experimental groups. E) Dot blot was used to detect the overall level of m6A in NMVCs cell model at 200 and 400 ng mRNA levels. F,G) Bar graphs presenting the levels of Fe2+ and p53N/T protein levels in H9c2 and NMVCs cells for different experimental groups. H) The changes of GPX4 mRNA in each group of cells were detected by RT-PCR. I,J) western blot was used to detect the changes of SLC7A11 and GPX4 protein in each group of cells. The data was expressed as means ± SD. n = 6 for Figure B–D, F,G. n = 3 for Figure H–J. Data were tested for normality via the Shapiro–Wilk test and confirmed to be normally distributed. For multiple group comparisons, one-way ANOVA with Tukey’s post-hoc test was used; for two-group comparisons, Student’s t-test was applied. The scale bars in Figure B represent 20 μm. **p < 0.01, *p < 0.05. ##p < 0.01, #p < 0.05. @@p < 0.01, @p < 0.05. &&p < 0.01, &p < 0.05.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Dot Blot, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 4. ALKBH5 can regulate G3BP1 through RNA methylation. A) Target molecules regulated by ALKBH5 were detected by RNA-seq and MeRIP sequencing. B) volcano map of RNA-seq sequencing. C) KEEG analysis of differential pathways after RNA-seq sequencing. D) peak downregulation by ALKBH5-siRNA versus NC, MeRIP sequencing. E) ALKBH5-siRNA versus NC, peak upregulation by MeRIP sequencing. F) KEEG analysis of differential pathways after MeRIP sequencing. G) RNA-seq and MeRIP sequencing were used to analyze differentially expressed genes. H) Cluster map of differentially expressed genes selected by RNA-seq and MeRIP combined sequencing. I) Nucleic acid sequence of G3BP1 molecule. J) m6A modification sites of G3BP1 predicted by SRAMP website. K) Flowchart of SELECT- m6A kit to detect the level of m6A methylation sites. L,M) SELECT- m6A kit was used to detect the levels of 142 and 173 m6A sites in each group. The data was expressed as means ± SD. n = 3. Data were tested for normality using the Shapiro–Wilk test and confirmed normally distributed. Two-group comparisons performed via Student’s t-test. **p < 0.01, ##p < 0.01. t-test was used.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Methylation, RNA Sequencing, Sequencing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 5. Role of G3BP1 3′UTR region and m6A modification site in cardiomyocyte ferroptosis. A) Schematic diagrams illustrating experimental setups in H9c2 and NMVCs cells, including treatments with LV-Ctrl, LV-ALKBH5, 3′-UTR mut, and 3′-UTR WT, Mut WT, Mut 142, Mut 173, siRNA-Ctrl and siRNA-ALKBH5 and manipulations to study RNA degradation and ferroptosis. LV-Ctrl Group: Transfection with lentiviral control vector (MOI = 50, 48 h). LV-ALKBH5 Group: Lentiviral overexpression of ALKBH5 (MOI = 50, 48 h). 3′UTR WT Group: Wild-type G3BP1 3′UTR luciferase reporter plasmid (2 μg, 24 h). 3′UTR Mut Groups: Site-specific mutations at m6A sites 142 (Mut 142) and 173 (Mut 173) (2 μg, 24 h). siRNA-Ctrl/siRNA-ALKBH5 Groups: Transfection with control or ALKBH5-specific siRNA (50 nm, 72 h). B,D,K) Determination of the rate of G3BP1 mRNA degradation in H9c2 cells. Actinomycin D treatment (5 μg mL−1) to block transcription, G3BP1 mRNA levels measured by RT-PCR at 0, 2, 4, 6 h post-treatment. C,J) Dual-luciferase assay was used to detect the fluorescence intensity of the reporter gene in H9c2 cells. Firefly/Renilla luciferase activity measured 48 h post-transfection. Normalized to Renilla luciferase for transfection efficiency. E–I,L–P) The kits were used to detect the levels of p53N/T, SLC7A11 mRNA, GSH, ROS and Fe2+ in each group. The data was expressed as means ± SD. n = 6. Normality was assessed using the Shapiro–Wilk test. For comparisons between two groups, Student’s t-test was used. For multiple groups, one-way ANOVA with Tukey’s post-hoc test was applied. **p < 0.01. ##p < 0.01.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Control, Plasmid Preparation, Over Expression, Luciferase, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 6. Effect of ALKBH8 on G3BP1-YBX1-p53 axis. A) BioGRID and IntAct database were used to search the proteins interacting with the human G3BP1 protein and make a Venn diagram. B,D,E) western blot was used to detect the changes of G3BP1 and YBX1 protein in cells and myocardial tissue. C) IF was used to detect the expression of G3BP1 protein in rat myocardial tissue. E) IF was used to detect the expression of knockdown G3BP1 protein in each group of cells. G–I) The kits were used to detect cell viability, LDH, and Fe2+ levels. J) Ultrastructural changes of the wash bar were detected by

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Knockdown

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 7. Interaction mechanism between G3BP1 and YBX1 and between YBX1 and p53. A) 3D structure of the interaction between G3BP1 and YBX1 protein. B) The binding domain between G3BP1 and YBX1. C) The experimental procedure of CO-IP to detect the interaction between G3BP1 protein and YBX1 protein. D) Construct truncated fragments targeting different domains of YBX1 protein. E) Construct truncated fragments targeting different domains of G3BP1 protein. F) Construction of truncates targeting different domains of p53 protein. G,H) CO-IP was used to detect the YBX1 domain that bound to G3BP1 protein. I,J) CO-IP was used to detect the G3BP1 domain that bound to YBX1 protein. K,L) CO-IP was used to detect the YBX1 protein domain that interacted with p53 protein. M,N) CO-IP was used to detect the p53 protein domain interacting with YBX1 protein.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Construct

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 8. Interaction mechanism between G3BP1 and p53 and the effect of G3BP1 on the distribution of YBX1 and p53 in the nucleus and cytoplasm. A,B) Predicted 3D structure and binding domains of YBX1-p53 interaction. C,D) Co-IP analysis of G3BP1 domains (NTF2/RRM/RGG) and p53 domains (N/M/C) in H9c2 cells transfected with HA-G3BP1/FLAG-p53 plasmids (48 h). E) ChIP-qPCR for p53 binding to p21 (positive control) and SLC7A11 promoters in siG3BP1 versus siCtrl cells (72 h post-transfection). F) CO-IP was used to detect the binding of YBX1, p53, and G3BP1 proteins in the cytoplasm and nucleus from HH/R-treated cells (6 h hypoxia/24 h reoxygenation). G–J) western blot was used to detect the expression of G3BP1, YBX1, and p53 proteins in the cytoplasm and nucleus. The data was expressed as means ± SD. n = 3. Normality was confirmed using the Shapiro–Wilk test. Two-group comparisons were analyzed by Student’s t-test. **p < 0.01, ##p < 0.01.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, ChIP-qPCR, Positive Control, Western Blot, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: ALKBH5-Mediated M 6 A Demethylation of G3BP1 Attenuates Ferroptosis Via Cytoplasmic Retention of YBX1/p53 in Diabetic Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202507254

Figure Lengend Snippet: Figure 10. Pattern diagram of ALKBH5 regulating G3BP1 through m6A modification affecting cardiomyocyte ferroptosis. In the process of diabetic myocardial ischemia-reperfusion, the protein level of G3BP1 is decreased, and the amount of binding p53 and YBX1 proteins is decreased. After p53 translocation into the nucleus, it binds to the promoter region of SLC7A11 gene, the transcription level of SLC7A11 is decreased, the expression of GSH and GPX4 is decreased, and then the level of ROS-mediated ferroptosis is increased. ALKBH5 promoted the expression of G3BP1 by regulating the 142 and 173 m6A methylation sites on G3BP1 mRNA. Subsequently, G3BP1 binds to the CTD domain of YBX1 through the NTF2 domain and the NLS domain of p53 through the RGG and RRM domains, thereby inhibiting p53 and YBX1 nuclear translocation. p53 and YBX1 can synergistically enter the nucleus to aggravate myocardial cell injury. Therefore, overexpression of G3BP1 can reduce the nuclear translocation of p53 by capturing YBX1 and p53, which increases the transcription level of SLC7A11 and the expression of GSH and GPX4 and subsequently inhibits ROS-mediated ferroptosis of cardiomyocytes.

Article Snippet: Rabbit anti-rat ALKBH5 (Cat No. 16837-1-AP), G3BP1 (Cat No. 13057-2-AP), GPX4 (Cat No. 30388-1-AP), YBX1 (Cat No. 20339-1-AP), GAPDH (Cat No. 60004-1-Ig) α-tubulin (Cat No. 11224-1-AP) primary antibodies, Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (Cat No. SA00009-2), and fluorescein (FITC)conjugated AffiniPure goat anti-mouse IgG (H+L) secondary antibodies (Cat No. SA00003-1) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Translocation Assay, Expressing, Methylation, Over Expression