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

Article Title: N4-Acetylcytidine-Mediated CD2BP2-DT Drives YBX1 Phase Separation to Stabilize CDK1 and Promote Breast Cancer Progression.

doi: 10.1002/advs.202411834

Figure Lengend Snippet: Figure 3. CD2BP2-DT interacts with YBX1 to promote breast cancer cell proliferation. A) Schematic workflow of the ChIRP assay and MS used to identify CD2BP2-DT binding proteins. B) Silver-stained images of proteins pulled down by the CD2BP2-DT probe and control probe in ChIRP analysis. C) Overlap analysis was performed between MS detection (35–55 kDa) and catRAPID prediction (Z-score > 0.5), with results ranked according to MS scores. D) Ion chromatogram of a representative YBX1 peptide interacting with CD2BP2-DT, as identified by mass spectrometry. E) Biotin-labeled CD2BP2-DT RNA pull-down assay followed by Western blot analysis. F) The enrichment of CD2BP2-DT by anti-YBX1 in breast cancer cells was demonstrated through

Article Snippet: The membrane was blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against YBX1 (1:5000, Proteintech, 20339-1-AP), GAPDH (1:50 000, Proteintech 60004-1-Ig), CDK1Thr161 (1:200, ABclonal, AP0324), CDK1Tyr15 (1:200, Santa Cruz sc-136014), CDK1Thr14 (1:1000, ABclonal, AP1465), CDK1 (1:1000, Santa Cruz, sc-54), NAT10 (1:5000, Proteintech, 13365-1-AP) and Flag (1:10 000, Proteintech, 20543-1-AP).

Techniques: Binding Assay, Staining, Control, Mass Spectrometry, Labeling, Pull Down Assay, Western Blot

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

Article Title: N4-Acetylcytidine-Mediated CD2BP2-DT Drives YBX1 Phase Separation to Stabilize CDK1 and Promote Breast Cancer Progression.

doi: 10.1002/advs.202411834

Figure Lengend Snippet: Figure 4. CD2BP2-DT facilitates the interaction between YBX1 and CDK1 mRNA. A) Volcano plot of the DEGs after CD2BP2-DT knockdown in breast cancer cells. B) GO and KEGG enrichment analyses were conducted to investigate the functions and pathways associated with potential target genes of CD2BP2-DT. C) PPI networks of target genes were constructed using Cytoscape software. D) Hub genes were identified using the CytoHubba plugin in Cytoscape software. E) Western blotting and qRT-PCR analysis demonstrating the expression of CDK1 in breast cancer cells transfected with YBX1 overexpression or knockdown constructs (n = 3). F) qRT-PCR and Western blotting were used to analyze the effects of YBX1 knockdown or overexpression

Article Snippet: The membrane was blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against YBX1 (1:5000, Proteintech, 20339-1-AP), GAPDH (1:50 000, Proteintech 60004-1-Ig), CDK1Thr161 (1:200, ABclonal, AP0324), CDK1Tyr15 (1:200, Santa Cruz sc-136014), CDK1Thr14 (1:1000, ABclonal, AP1465), CDK1 (1:1000, Santa Cruz, sc-54), NAT10 (1:5000, Proteintech, 13365-1-AP) and Flag (1:10 000, Proteintech, 20543-1-AP).

Techniques: Knockdown, Construct, Software, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Over Expression

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

Article Title: N4-Acetylcytidine-Mediated CD2BP2-DT Drives YBX1 Phase Separation to Stabilize CDK1 and Promote Breast Cancer Progression.

doi: 10.1002/advs.202411834

Figure Lengend Snippet: Figure 5. YBX1 undergoes LLPS in breast cancer. A) The IDRs of YBX1 were predicted using the PONDR database. The bottom panel shows a schematic diagram of the YBX1 protein domains. B) Representative images of breast cancer cells transfected with YBX1-GFP (scale bars, 10 μm). C) Representative images of breast cancer cells transfected with YBX1-GFP and treated with 1.5% 1,6-hexanediol (scale bars, 10 μm). D) YBX1-GFP formed liquid droplets in vitro in a concentration-dependent manner (scale bars, 10 μm). E) 3D images of YBX1 forming droplet condensates in vivo and in vitro (scale bars, 10 μm, 1 μm). F) FRAP experiments were conducted to assess the recovery of fluorescence in YBX1-GFP droplets following photobleaching in breast cancer cells

Article Snippet: The membrane was blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against YBX1 (1:5000, Proteintech, 20339-1-AP), GAPDH (1:50 000, Proteintech 60004-1-Ig), CDK1Thr161 (1:200, ABclonal, AP0324), CDK1Tyr15 (1:200, Santa Cruz sc-136014), CDK1Thr14 (1:1000, ABclonal, AP1465), CDK1 (1:1000, Santa Cruz, sc-54), NAT10 (1:5000, Proteintech, 13365-1-AP) and Flag (1:10 000, Proteintech, 20543-1-AP).

Techniques: Transfection, In Vitro, Concentration Assay, In Vivo

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

Article Title: N4-Acetylcytidine-Mediated CD2BP2-DT Drives YBX1 Phase Separation to Stabilize CDK1 and Promote Breast Cancer Progression.

doi: 10.1002/advs.202411834

Figure Lengend Snippet: Figure 7. CD2BP2-DT-mediated YBX1 phase separation promotes the stability of CDK1 mRNA and CD2BP2-DT/CDK1 axis promotes proliferation of breast cancer cells. A) Representative images from in vitro phase separation experiments involving purified YBX1-WT, YBX1-Mut, and FusIDR proteins complexed with Cy3-tagged CDK1 mRNA (scale bars, 10 μm). B) ActD assay to detect the degradation rate of CDK1 mRNA in breast cancer cells transfected with YBX1-WT, YBX1-Mut, or FusIDR (n = 3). C) The expression levels of CDK1 in breast cancer cells transfected with YBX1-WT, YBX1-Mut, or FusIDR were assessed using qRT-PCR (n = 3). D, E) CCK-8 (D) and colony formation (E) assays were used to analyze the effects of CDK1 overexpression

Article Snippet: The membrane was blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against YBX1 (1:5000, Proteintech, 20339-1-AP), GAPDH (1:50 000, Proteintech 60004-1-Ig), CDK1Thr161 (1:200, ABclonal, AP0324), CDK1Tyr15 (1:200, Santa Cruz sc-136014), CDK1Thr14 (1:1000, ABclonal, AP1465), CDK1 (1:1000, Santa Cruz, sc-54), NAT10 (1:5000, Proteintech, 13365-1-AP) and Flag (1:10 000, Proteintech, 20543-1-AP).

Techniques: In Vitro, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Over Expression

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A. YB-1 mRNA levels compared to KRAS copy number status in invasive breast carcinoma samples in the TCGA dataset. Values for YB-1 are shown as RPKMs. B. Kaplan-Meier curves of overall survival (OS) for the TCGA cohort, with respect to KRAS copy number (N=206 for tumours with amplified KRAS or a gain of function KRAS gene, and N=522 for tumours with diploid KRAS ). C. YB-1 mRNA levels compared to KRAS copy number status in invasive breast carcinoma samples in the METABRIC dataset. Values for YB-1 are shown as RPKMs. D. HIF1A (left panel) CAIX (middle panel) and G3BP1 (right panel) mRNA expression according to KRAS copy number status in invasive breast carcinomas in TCGA dataset. Values for HIF1A, CAIX and G3BP1 are shown as RPKMs. E. Scatter plot of YB-1 and G3BP1 mRNA expression in amplified- KRAS invasive breast carcinomas. F YB-1 (left panel) HIF1A (middle panel) and G3BP1 (right panel) mRNA expression according to AKT1 copy number status in invasive breast carcinomas in METABRIC dataset. Values for YB-1 , HIF1A and CAIX are shown as RPKMs. G. Scatter plot of YBX1 and G3BP1 mRNA expression in amplified- AKT1 invasive breast carcinomas. P-values in panels (A, C-D) were determined by Student’s t-test, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Amplification, Expressing

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A. Western blots of MDA-MB-231 cells transduced with shYB-1(#1307), shYB-1(#1309) or sh Y B-1(#948) or a control Scr vector, showing YB-1 protein levels (relative to GAPDH). B. Representative pictures of bioluminescence signals in mice injected SQ with MDA-MB-231 cells transduced with shYB-1(#1307), shYB-1(#1309), shYB-1(#948) or shScr. Dot plots show the bioluminescence exhibited by these tumours 34 days post-transplant. C. Representative pictures of bioluminescence signals obtained in mice 34 days after being injected intravenously with MDA-MB-231 cells transduced with shYB-1 or shScr vectors. Dot plot shows the measured bioluminescence exhibited by the progeny of these cells generated in vivo . D. Representative photomicrographs of H&E- or YB-1-stained sections of lungs of mice injected intravenously with MDA-MB-231 cells transduced with shYB-1 or shScr vectors. Dot plot shows the number of metastasis per mouse (2 lungs). P-values in panels (B-D) were determined by Student’s t-test, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Western Blot, Transduction, Plasmid Preparation, Injection, Generated, In Vivo, Staining

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A. Cell cycle analysis of shYB-1 - or shScr -transduced MDA-MB-231 cells. B. Western blots showing NRF2, HIF1α, G3BP1, CAIX and YB-1 levels (relative to GRB2) of MDA-MB-231 cells transduced with shYB-1 or shScr vectors grown in ultra-low attachment plates for 24 hours. C. Western blots showing HIF1α and YB-1 levels (relative to GAPDH) in shYB-1 and shScr -transduced MDA-MB-231 cells maintained in vitro for up to 24 hours in 1% O 2 . D-E. Representative images of CAIX ( D )-, G3BP ( E )-stained sections of tumours produced from shYB-1 - or shScr -transduced MDA-MB-231 cells. Scale bar, 100 μm. Bar graph shows measured levels of CAIX or G3BP (staining intensity). F. Dot plots of tumor sizes obtained in mice injected SQ with T47D cells transduced with GFP-control and KRAS G12D and assessed 30 days post-transplant. G. Representative views of YB-1 levels in the IHC-stained in vivo progeny of GFP-control and KRAS G12D -transduced T47D cells. Bar graph shows quantification of YB-1 expression.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Cell Cycle Assay, Western Blot, Transduction, In Vitro, Staining, Produced, Injection, In Vivo, Expressing

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A Representative views of YB-1 immunostaining of normal human mammary tissue (left) and 8-week tumours derived from KRAS G12D -transduced normal mammary cells isolated from the same matching 3 different donors (right). Scale bar, 50 μm. B Western blots showing YB-1 levels (relative to H3) in human BCs, LPs, LCs and SCs isolated from 3 normal donors. Subsets were sorted according to their surface EPCAM and CD49f levels (top panel). C Representative FACS profile of a 4 week xenograft of inducible KRAS G12D -transduced human BCs obtained from mice maintained on doxycyline-supplemented water (Dox) for 2 or 4 weeks post-transplant. D KRAS mRNA levels measured in 4 week xenografts of inducible KRAS G12D -transduced cells obtained from mice maintained on Dox for 0, 2, or 4 weeks, as shown. E Representative views of YB-1 immunostaining of 4 week tumours derived from inducible KRAS G12D -transduced cells in mice maintained on Dox for 2 or 4 weeks (N=3 donors). Scale bar, 50 μm.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Immunostaining, Derivative Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A Representative photos of bioluminescence signals measured in mice injected SQ 7 weeks earlier with human mammary cells transduced with lenti-viral vectors encoding Luc-YFP alone or in combination with KRAS G12D -, YB-1-, KRAS G12D +YB-1-, or myrAKT1 . BCs and LPs from 3 donors were pooled separately before transduction. Graph plot shows changes in bioluminescence activity over time. B Representative FACS plots of human (CD298/EPCAM) + and mCherry (myrAKT1) + or YFP (KRAS G12D ) + cells present in dissociated tumours generated from human mammary cells transduced with KRAS G12D or myrAKT1 . C Representative images of SMA-, CK14-, CK8-18- and p63-stained sections of myrAKT1 -derived tumours initiated from either BCs or LPs. Scale bar, 100 μm. D Representative views of YB-1 immunostaining of 18-week primary myrAKT1 -derived tumours generated from normal mammary cells from 3 different donors (#1-3). Bar graph shows a comparison of YB-1 staining intensity in KRAS G12D - or myrAKT1 -derived tumours. N = 10 ( KRAS G12D ) or 6 ( myrAKT1 ) tumours. E Dot plot of the bioluminescence measured in mice injected SQ with myrAKT1 +inducible YB-1-transduced human mammary cells and given water with or without doxycyline. N = 3 donors.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Injection, Transduction, Activity Assay, Generated, Staining, Derivative Assay, Immunostaining

Journal: bioRxiv

Article Title: YB-1 IS REQUIRED FOR THE GENESIS AND METASTATIC CAPACITY OF HUMAN BREAST CANCER

doi: 10.1101/372524

Figure Lengend Snippet: A Representative views of G3BP1, NRF2, CAIX and HIF1α immunostaining of normal human mammary tissue (left) and 8-week tumours derived from KRAS G12D -transduced normal mammary cells isolated from the same matching 3 different donors (right). Scale bar, 50 μm. Bar graph shows quantification of G3BP1, NRF2, HIF1α and CAIX expression. B Representative photos of the bioluminescence signals obtained in mice injected SQ with KRAS G12D + shYB-1 - or KRAS G12D + shScr -transduced primary human mammary cells 2 weeks earlier. Dot plot showing the bioluminescence activity of tumours derived from BCs (blue) and LPs (red). C-F Representative images of YB-1 ( C )-, HIF1α ( D )-, CAIX ( E )- and VEGF ( F )-stained sections from different BC- or LP-derived tumours arising from KRAS G12D + shYB-1 - or KRAS G12D + shScr -transduced cells. Scale bar, 100 μm. Bar graphs shows quantification of YB-1 ( C )-, HIF1α ( D )-, CAIX ( E )- and VEGF ( F )-intensities in tumours derived from KRAS G12D + shYB-1 - or KRAS G12D + shScr -transduced cells. N = 8. P-values in panels (A-E) were determined by Student’s t-test, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Membranes were then blotted overnight at 4°C with appropriate primary antibodies, such as anti-ACTIN (Santa Cruz, sc-1615, 1/10,000), anti-H3 (Cell Signaling Technology, 12648, 1/10,000), anti-RAS (Cell Signaling Technologies, 3339, 1/1,000), anti-YB-1 (Cell Signaling Technology, 4202, 1/1,000) and anti-HIF1a (Cayman; 10006421; 1/1000).

Techniques: Immunostaining, Derivative Assay, Isolation, Expressing, Injection, Activity Assay, Staining

Journal: Molecular Metabolism

Article Title: White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

doi: 10.1016/j.molmet.2020.101137

Figure Lengend Snippet: YBX1 abundance acutely increased in thermogenic adipose tissue upon cold exposure in mice. (A) Representative YBX1 and AKT immunoblots in various fat depots from mice kept at 5 °C for 0, 3, 8, and 24 s, 1 week, or 3 weeks. Quantification of the band intensities (n = 4 replicates per time point). Values are mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 analyzed by one-way ANOVA. (B) Immunoblots of YBX1 and AKT in various fat depots from mice kept at 29 °C for 3 weeks. Quantification of the band intensities (n = 3 replicates per time point). Values are mean ± SEM, ∗p < 0.05 analyzed by one-way ANOVA. (C) Histological analysis of scWAT from mice kept at either thermoneutrality (29 °C) or cold (5 °C) for 24 h. scWAT was stained with hematoxylin and eosin or YBX1 fluorescent antibody (red) together with DAPI (blue). Tissue autofluorescence is shown in white. Representative images (from n = 3 mice per group) are shown. Scale bars 200 μm and magnified scale bars 50 μm. (D) Ybx1 mRNA expression in the isolated mature adipocyte fraction (MAF) and stromal-vascular fraction (SVF) from scWAT of mice exposed to thermoneutrality (29 °C) or cold (5 °C) for 24 h (n = 5 replicates per condition). Values are mean ± SEM. ∗∗∗p < 0.001 analyzed by 2-way ANOVA with Bonferroni's multiple comparisons test.

Article Snippet: The slides were then incubated overnight at 4 °C with anti-YBX1 antibody (#A303-231A, 1:1000, Bethyl Laboratories), followed by 45 min of incubation at RT with secondary antibody Alexa Fluor 568 (1:500, Life Technologies).

Techniques: Western Blot, Staining, Expressing, Isolation

Journal: Molecular Metabolism

Article Title: White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

doi: 10.1016/j.molmet.2020.101137

Figure Lengend Snippet: YBX1 was necessary for the expression of thermogenic genes in adipocytes. Brown WT-1 preadipocytes and C3H/10T1/2 mesenchymal stem cells were transfected with Ybx1 -specific (si Ybx1 , light bars) or negative control siRNA (siCon, dark bars) and induced to differentiate (d0). (A) The efficiency of Ybx1 knockdown (KD) in WT-1 cells is shown at the mRNA level at different days of differentiation following siRNA transfection. (B) The efficiency of Ybx1 knockdown (KD) in WT-1 cells is shown at the protein level by Western blotting on different days of differentiation following siRNA transfection. The blot quantification is on the right. YBX1 was normalized to vinculin (VCL) (C) Effects of Ybx1 KD on the expression of key thermogenic genes ( Ppargc1a , Elovl3 , Prdm16 , Cidea , and Ucp1 ) across differentiation. (D) The effect of Ybx1 KD on UCP1 protein expression in WT-1 cells is shown by Western blotting, and the quantification is on the right. UCP1 was normalized to vinculin (VCL) (E) The efficiency of Ybx1 KD in C3H/10T1/2 mesenchymal cells is shown at mRNA levels on different days of differentiation following siRNA transfection. (F) The efficiency of Ybx1 KD in C3H/10T1/2 cells is shown at the protein level by Western blotting on different days of differentiation following siRNA transfection. The blot quantification is on the right. YBX1 was normalized to vinculin (VCL). (G) Effects of Ybx1 KD on the expression of key thermogenic genes ( Ppargc1a , Elovl3 , Prdm16 , Cidea , and Ucp1 ) across differentiation. (H) The effects of Y bx1 KD on UCP1 protein expression in C3H/10T1/2 cells is shown by Western blotting, and the blot quantification is on the right. UCP1 was normalized to vinculin (VCL). For all of the panels in this figure with qPCR data, n = 4 biological replicates were used and the values represent means ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 analyzed by 2-way ANOVA with Bonferroni's multiple comparisons test. For all of the panels in this figure with Western blotting data, n = 3 biological replicates were used and the values represent means ± SEM. ∗p < 0.05 analyzed by the unpaired t-test.

Article Snippet: The slides were then incubated overnight at 4 °C with anti-YBX1 antibody (#A303-231A, 1:1000, Bethyl Laboratories), followed by 45 min of incubation at RT with secondary antibody Alexa Fluor 568 (1:500, Life Technologies).

Techniques: Expressing, Transfection, Negative Control, Knockdown, Western Blot

Journal: Molecular Metabolism

Article Title: White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

doi: 10.1016/j.molmet.2020.101137

Figure Lengend Snippet: YBX1 potentiated the thermogenic capacity of C3H/10T1/2 mesenchymal stem cells. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA (gRNA) directed to the promoter regions of Pparg2 (P) or Ybx1 (Y) as indicated. (A – B) mRNA expression levels of Ybx1 and Pparg2 post-transfection. (C) Gene expression analysis of thermogenic markers Ucp1 , Cidea , and Elovl3 . Data are based on n = 3 biological replicates. Two-way ANOVA; overall effects of the overexpression and differentiation day are shown for selected groups of interest (P vs EV; P vs P + Y). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (D – E) The effects of Y bx1 overexpression on the levels of YBX1 (D) and UCP1 (E) proteins in C3H/10T1/2 cells is shown by Western blotting and the quantification is added. UCP1 was normalized to vinculin (VCL). Four biological replicates were used, and the values are mean ± SEM. ∗p < 0.05 analyzed by unpaired t-test was applied for statistical analysis. (F) Oxygen consumption rates (OCR) in mature C3H/10T1/2-CRISPRa-SAM cells following injection with isoproterenol (ISO), FCCP, and rotenone/antimycin A (Rot/Anti). A representative experiment is shown. (G) AUC quantification of ISO-induced OCR is illustrated for n = 4 independent experiments. Data are shown as mean ± SEM. ∗p < 0.05 analyzed by paired t-test.

Article Snippet: The slides were then incubated overnight at 4 °C with anti-YBX1 antibody (#A303-231A, 1:1000, Bethyl Laboratories), followed by 45 min of incubation at RT with secondary antibody Alexa Fluor 568 (1:500, Life Technologies).

Techniques: Transfection, Plasmid Preparation, Expressing, Gene Expression, Over Expression, Western Blot, Injection

Journal: Molecular Metabolism

Article Title: White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

doi: 10.1016/j.molmet.2020.101137

Figure Lengend Snippet: YBX1 induced early transcriptional changes to promote browning in C3H/10T1/2 mesenchymal stem cells. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA directed to the promoter region of Pparg2 (P) or Pparg2 and Ybx1 (PY) as indicated. (A – C) Volcano plots displaying transcripts significantly regulated by induction of Pparg2 expression (A) or combined induction of Pparg2 and Ybx1 expression compared to EV-transfected control cells (B) and a set of genes upregulated specifically by Ybx1 (C) . (D) Heat map of fold changes in relative abundance of the set of genes significantly regulated by Pparg2 and Ybx1 co-induction across days of differentiation. (E) Gene set enrichment analysis (GSEA) across different days of differentiation upon Ybx1 over-expression; GO terms with FDR < 0.1 were considered significantly enriched and the top 10 GO terms with the lowest FDR for each contrast are shown in the dot plot. The extended list is provided in Table S6 . NES stands for normalized enrichment score for GSEA. Data represent the relative abundance based on counts per million (CPM) and 0.1 false discovery rate (FDR) cut-off.

Article Snippet: The slides were then incubated overnight at 4 °C with anti-YBX1 antibody (#A303-231A, 1:1000, Bethyl Laboratories), followed by 45 min of incubation at RT with secondary antibody Alexa Fluor 568 (1:500, Life Technologies).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Over Expression

Journal: Molecular Metabolism

Article Title: White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

doi: 10.1016/j.molmet.2020.101137

Figure Lengend Snippet: JMJD1c contributed to YBX1-induced browning. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA directed to the promoter region of Ybx1 as indicated. All of the transfections included the vector delivering a single-guide RNA directed to the promoter region of Pparg2. Furthermore, siRNA targeting Jmjd1c was used for knockdown. (A) Overexpression of Ybx1 in combination with siRNA targeting Jmj1dc . Transfection was performed two days pre-confluency (d2), the cells were harvested at d0, and gene expression levels of Ybx1 and Jmjd1c were assessed. (B–C) On day 6 of differentiation, the cells were harvested after 6 h of 10 μM NE stimulation or not, and the gene expression levels of Ucp1 , Elovl3 , Ppargc1a , and Cidea (B) , and gene expression levels of Fasn , Fabp4 , and Adipoq (C) were assessed. Data are based on n = 4 biological replicates and the values are the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 analyzed by 2-way ANOVA; overall effects of overexpression and knockdown are shown.

Article Snippet: The slides were then incubated overnight at 4 °C with anti-YBX1 antibody (#A303-231A, 1:1000, Bethyl Laboratories), followed by 45 min of incubation at RT with secondary antibody Alexa Fluor 568 (1:500, Life Technologies).

Techniques: Transfection, Plasmid Preparation, Knockdown, Over Expression, Gene Expression