Journal: Nature Communications

Article Title: Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

doi: 10.1038/s41467-023-43716-y

Figure Lengend Snippet: a Dependency map of Rb from the Cancer Dependency Map Project . b Percentage of proliferating cells in wild-type and tKO MCF-10A cells treated with either DMSO or palbociclib (1 µM). Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05; ** p ≤ 0.001). c Heatmap of single-cell traces for CDK4/6 and CDK2 activities and Cdt1 degron levels in tKO MCF-10A cells treated with palbociclib (1 µM). During 25‒48 h after drug treatment, cells activating CDK2 (>1) for over 2 h were classified as proliferating cells. d Percentage of S-phase cells in tKO MCF-10A cells without and with expression of a DHFR-p27 construct. Data are shown as mean ± SD ( n = 4 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). e Average traces of CDK2 activity and p53 and p21 levels in MCF-7 cells treated with palbociclib (1 µM). Data are shown as mean ± 95% CI (proliferation: n = 447 cells; quiescence: n = 3582 cells). f , g Accumulative percentage of S-phase cells in tKO ( f ) or wild-type ( g ) MCF-10A cells. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the one-way ANOVA test (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). h Percentage of S-phase cells in palbociclib-resistant MCF-10A cells exposed to different concentrations of mitogens and NCS as indicated for 24 h. Data are shown as mean ± SD ( n = 4 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05).

Article Snippet: Anti-p21 (#sc-271610; 1:300 for immunoblotting in PC-12 and OP-9 cells) was ordered from Santa Cruz Biotechnology.

Techniques: Two Tailed Test, Expressing, Construct, Activity Assay

Journal: Nature Communications

Article Title: Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

doi: 10.1038/s41467-023-43716-y

Figure Lengend Snippet: a Percentage of S-phase cells in PLB-985, PC-12, and OP-9 cells after differentiation for the indicated time. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (PC-12 cells) or one-way ANOVA test (PLB-985 and OP-9 cells) (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). b Percentage of neurite bearing PC-12 cells before and after one-day differentiation. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). c Percentage of high-PPARγ expressing OP-9 cells. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the one-way ANOVA test (* p ≤ 0.05; ** p ≤ 0.001). d Immunoblot showing Rb, c-Myc, p21, p27, and GAPDH expression before and after differentiation. FPR1, SYN1, and PPARγ are cell differentiation markers. e Percentage of S-phase OP-9 cells expressing a doxycycline-inducible c-Myc construct. After 6 days of differentiation, cells were treated with palbociclib (1 µM) and EdU (10 µM) + DMSO or doxycycline (5 µM) for 72 h. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). f Schematic diagram illustrating CDK4/6-independent cell-cycle entry by multiple steps: (1) reduction in Rb-protein levels, (2) c-Myc-mediated amplification of E2F activity, and (3) inhibition of CDK2 activity by Cip/Kip.

Article Snippet: Anti-p21 (#sc-271610; 1:300 for immunoblotting in PC-12 and OP-9 cells) was ordered from Santa Cruz Biotechnology.

Techniques: Two Tailed Test, Expressing, Western Blot, Cell Differentiation, Construct, Amplification, Activity Assay, Inhibition

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Slc25a5 is increased in adipogenic differentiation. RNA-seq data were obtained from OP9 cells and ASCs undergoing adipogenic differentiation and those that were not. A Upregulated and downregulated gene expression profiles were selected. B Venn diagram illustrating the upregulated genes in both OP9 and ACSs during adipogenic differentiation. C Ten GO terms representing the most upregulated genes during adipogenic differentiation. D , E Expression profiles of the Slc25 gene family during adipogenic differentiation. F The relative mRNA level of Slc25a5 was measured with RT–qPCR. The values are mean ± SEM ( n = 3). ** P < 0.01; *** P < 0.001 versus controls

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Inhibiting Slc25a5 prevents adipogenic differentiation. Before adipogenic differentiation, OP9 cells were transfected with Slc25a5 siRNA, and 24 h later, the cells were incubated with 1 mM rosiglitazone to induce adipogenic differentiation. A RT–qPCR verified the efficiency of Slc25a5 knockdown in OP9 cells. B Oil Red O staining; scale bar is 200 μm. C Cellular TG levels were measured. D RT–qPCR was performed to assess the expression of the adipogenic factors PPARγ, FABP4, FASN, and C/EBPα. E Western blot of selected subunits of the OXPHOS complexes. F Cellular levels of ATP. Values are the mean ± SEM ( n = 3). * P < 0.05; ** P < 0.01 versus control

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Transfection, Incubation, Quantitative RT-PCR, Knockdown, Staining, Expressing, Western Blot, Control

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Transcriptomics analysis implicates ERK pathway as a target of Slc25a5 . After adipogenic differentiation, OP9 cells with or without Slc25a5 knockdown were collected, and a transcriptome analysis was performed. A PCA score plot showing clear clusters in each group. B Scatterplot of DEGs between the control and Slc25a5 -knockdown cells (fold change ≥ 1.5, and P ≤ 0.06). C Heat map of the DEGs between the control and Slc25a5 -knockdown cells. D GO analysis of downregulated DEGs. E Protein levels of ERK, p-ERK, P38, p-P38, JNK, p-JNK, and PPARγ were measured by western blot analysis. The values are the mean ± SEM ( n = 3). * P < 0.05 versus control

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knockdown, Control, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: ERK inhibitor reverses Slc25a5 knockdown-induced adipogenesis inhibition. Before adipogenic differentiation was induced, OP9 cells were transfected with Slc25a5 siRNA, and 24 h later, these cells were incubated with 1 mM rosiglitazone and PD98059 (50 μM). A Cellular triglyceride (TG) levels were measured. B Oil Red O staining was performed after adipogenic differentiation, and the results were visualized by light microscopy. The scale bar is 200 µm. C RT–qPCR was performed to assess the expression of the adipogenic factors PPARγ , FABP4 , FASN , and C/EBPα after adipogenic differentiation. D Protein levels of ERK, p-ERK, and PPARγ were measured by western blot analysis. The values are the mean ± SEM ( n = 3). a versus b represents differences between groups; b versus b represents no differences between groups

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knockdown, Inhibition, Transfection, Incubation, Staining, Light Microscopy, Quantitative RT-PCR, Expressing, Western Blot