Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 1. SPRY4 positively correlated with hAMSC adipogenic differentiation capacity. (a) Oil red O staining gradually increased from 0 to 12 days. (b) Quantification of Oil red O staining for evaluating the efficiency of hAMSC adipogenesis. (c) qRT-PCR detected the mRNA levels of SPRY4, PPARG, CEBPA and FABP4 mRNA levels during adipogenic differentiation (0 d, 3 d, 6 d, 9 d and12 d). (d) Western blotting showed the protein levels of SPRY4, C/EBPα and FABP4 during adipogenic differentiation (0 d, 3 d, 6 d, 9 d and 12 d), and quantitative analysis of the intensity of protein expression in the indicated groups. GAPDH was used as the control for normalization in the and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

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

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 2. Downregulation of SPRY4 impaired hAMSC adipogenic differentiation in vitro. SPRY4 was silenced in hAMSCs using two independent siRNAs (siSPRY4-1 and siSPRY4-2). Knockdown efficiency was verified by qRT-PCR at mRNA level (a) and western blotting at protein level (b). (c) qRT-PCR detected PPARG, CEBPA, FABP4 and LPL on day 6 of adipogenic differentiation. (d) Western blotting detected PPARγ, C/EBPα, FABP4and LPL on day 6 of adipogenic differentiation, and quantitative analysis of the intensity of protein expression in the indicated groups. (e, f) Oil red O staining and quantification were performed on day 10 of

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

Techniques: In Vitro, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Staining

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 3. Ectopic expression of SPRY4 promoted hAMSC adipogenic differentiation in vitro. hAMSCs was infected with specific lentivirus to overexpress SPRY4. The efficiency of ectopic expression was verified by qRT-PCR at mRNA level (a) and western blotting at protein level (b). (c) qRT-PCR detected PPARG, CEBPA, FABP4 and LPL on day 6 of adipogenic differentiation. (d) Western blotting detected PPARγ, C/EBPα, FABP4 and LPL on day 6 of adipogenic differentiation, and quantitative analysis of the intensity of protein expression in the indicated groups. (e, f) Oil red O staining and quantification were performed on day 10 of adipogenic differentiation to evaluate the efficiency of hAMSC adipogenesis after ectopic expression of SPRY4. GAPDH was used as the control for normalization in the qRT-PCR and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

Techniques: Expressing, In Vitro, Infection, Quantitative RT-PCR, Western Blot, Staining, Control

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 4. SPRY4 accelerated hAMSC heterotopic lipid formation in vivo. hAMSCs were infected with lentivirus (sh-NC, sh-siSPRY4-1, sh-siSPRY4-2, LV-NC, or LV-SPRY4), and then incubated in adipogenic medium for 4 days before being transplanted subcutaneously in vivo, which was detected after 8 weeks. (a, b) Heterotopic lipid formation was detected by HE staining, and quantitative analysis of relative number of adipocytes. (c, d) Immunofluorescence detected perilipin 1-positive cell numbers for representing the efficiency of hAMSC ectopic fat formation (n = 6, scale bar = 50 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

Techniques: In Vivo, Infection, Incubation, Staining, Immunofluorescence

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 6. SPRY4 functioned by activating the MEK–ERK1/2 pathway. SPRY4 was overexpressed in hAMSCs, and then the cells were treated with DMSO or 10 μM U0126 during adipogenic induction. (a) qRT-PCR detected PPARG, CEBPA, FABP4 and LPL on day 6 of adipogenic differentiation. (b) Western blotting detected PPARγ, C/EBPα, FABP4 and LPL as well as SPRY4, T-ERK and p-ERK on day 6 of adipogenic differentiation, and quantitative analysis of the intensity of protein expression in the indicated groups. (c, d) Oil red O staining and quantification were performed on day 10 of adipogenic differentiation to evaluate the efficiency of hAMSC adipogenesis after different treatments. GAPDH was used as the control for normalization in the qRT-PCR and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

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

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 1. SPRY4 positively correlated with hAMSC adipogenic differentiation capacity. (a) Oil red O staining gradually increased from 0 to 12 days. (b) Quantification of Oil red O staining for evaluating the efficiency of hAMSC adipogenesis. (c) qRT-PCR detected the mRNA levels of SPRY4, PPARG, CEBPA and FABP4 mRNA levels during adipogenic differentiation (0 d, 3 d, 6 d, 9 d and12 d). (d) Western blotting showed the protein levels of SPRY4, C/EBPα and FABP4 during adipogenic differentiation (0 d, 3 d, 6 d, 9 d and 12 d), and quantitative analysis of the intensity of protein expression in the indicated groups. GAPDH was used as the control for normalization in the and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

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

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 3. Ectopic expression of SPRY4 promoted hAMSC adipogenic differentiation in vitro. hAMSCs was infected with specific lentivirus to overexpress SPRY4. The efficiency of ectopic expression was verified by qRT-PCR at mRNA level (a) and western blotting at protein level (b). (c) qRT-PCR detected PPARG, CEBPA, FABP4 and LPL on day 6 of adipogenic differentiation. (d) Western blotting detected PPARγ, C/EBPα, FABP4 and LPL on day 6 of adipogenic differentiation, and quantitative analysis of the intensity of protein expression in the indicated groups. (e, f) Oil red O staining and quantification were performed on day 10 of adipogenic differentiation to evaluate the efficiency of hAMSC adipogenesis after ectopic expression of SPRY4. GAPDH was used as the control for normalization in the qRT-PCR and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

Techniques: Expressing, In Vitro, Infection, Quantitative RT-PCR, Western Blot, Staining, Control

Journal: Adipocyte

Article Title: SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

doi: 10.1080/21623945.2022.2123097

Figure Lengend Snippet: Figure 6. SPRY4 functioned by activating the MEK–ERK1/2 pathway. SPRY4 was overexpressed in hAMSCs, and then the cells were treated with DMSO or 10 μM U0126 during adipogenic induction. (a) qRT-PCR detected PPARG, CEBPA, FABP4 and LPL on day 6 of adipogenic differentiation. (b) Western blotting detected PPARγ, C/EBPα, FABP4 and LPL as well as SPRY4, T-ERK and p-ERK on day 6 of adipogenic differentiation, and quantitative analysis of the intensity of protein expression in the indicated groups. (c, d) Oil red O staining and quantification were performed on day 10 of adipogenic differentiation to evaluate the efficiency of hAMSC adipogenesis after different treatments. GAPDH was used as the control for normalization in the qRT-PCR and western blotting. All data are the mean ± SD (n = 3 independent experiments with three biological repetitive tests, scale bar = 100 μm).

Article Snippet: Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China).

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

Journal: The Journal of Clinical Investigation

Article Title: Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer

doi: 10.1172/JCI145035

Figure Lengend Snippet: (A) Genomic distribution of H3K27ac, H3K4me1, and H3K4me3 peaks in A2780-P and A2780-R cells. (B) Plot of the average level of H3K27ac, H3K4me1, and H3K4me3 peaks centered at the TSS in A2780-P or A2780-R cells. (C) Venn diagram showing the number and overlaps of enhancers and promoters between A2780-P and A2780-R cells overlapped by MAnorm (https://github.com/shao-lab/MAnorm). Common peaks merged. (D and E) Distribution of histone markers surrounding the summit of H3K27ac peaks, in gained or lost enhancer regions (D) and promoter regions (E). Black borders, A2780-P; dark red borders, A2780-R. Only one of the duplicates was plotted, as consistent results were observed in the other replicate. Each row represents 1 peak centered at the midpoint between two 2-kb flanking regions. (F) Scatterplot of the correlation between H3K27ac mark intensity and fold change of corresponding gene expressions. The horizontal axis illustrates the differences between resistant and parental cell lines, measured in counts per million mapped reads (CPM). Only genes of at least 4-fold change and H3K27ac changes of 500 CPM are shown. (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis enriched by downregulated genes in A2780-R (relative to A2780-P) showed that the MAPK pathway was significantly enriched. NES, normalized enrichment score. (H) Relative mRNA levels of ERK transcriptional targets in A2780-P/R cells (left) and OVCAR5-P/R cells (right). Results are represented as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test. (I) ChIP-Seq profiles show the ChIP-Seq signal (y axis, reads per million) for H3K27ac, H3K4me1, and H3K4me3 at genomic loci of DUSP6, ETV4, and SPRY4. (J) The results showed relative mRNA level of DUSP6 in 9 commercial cell lines and 7 patient-derived cells. Results are represented as mean ± SD of 4 independent experiments.

Article Snippet: The primary antibodies used were as follows: ERK1/2 (1:2000; 4696S), p-ERK1/2 (1:2000; 4370S), MEK (1:1000; 9126S), p-MEK (1:1000; 9154S), β-actin (1:2000; 8456S), and SPRY2 (1:1000; 14954S) were purchased from Cell Signaling Technology; DUSP6 (1:1000, pa5-15552; or 1:1000, ab76310) was obtained from Invitrogen or Abcam and ETV5 (1:1000; ab102010) was from Abcam; and SPRY4 (1:2000; 22765-1-AP) was from Proteintech.

Techniques: ChIP-sequencing, Derivative Assay