fgf bp1  (R&D Systems)

Journal: Cell Death & Disease

Article Title: Inhibitory effect of the novel tyrosine kinase inhibitor DCC-2036 on triple-negative breast cancer stem cells through AXL-KLF5 positive feedback loop

doi: 10.1038/s41419-022-05185-x

Figure Lengend Snippet: A DCC-2036 inhibited the expression of KLF5, as well as its downstream targeted genes Nanog and FGF-BP1 in TNBC cells. The cells were incubated with DCC-2036 at indicated concentrations for 48 h, and the protein expression was examined by Western blotting. B KLF5 or vector plasmids were transfected into MDA-MB-231 and HS-578T cells for 24 h. The cells were then treated with either DCC-2036 (5 μM) or DMSO for 48 h, and the expression of KLF5 was detected by WB. C , D Ectopic expression of KLF5 partially but significantly rescued the mammosphere formation reduction induced by DCC-2036. MDA-MB-231 cells and HS-578T cells were transfected with KLF5 or vector plasmid, followed by DCC-2036 (0 or 5 μM) treatment for 48 h before they were plated onto 24-well ultra-low attachment plates (5000 cells each well). The number of mammospheres were counted 7 days after seeding and shown in bar charts. Scale bars, 100 μm. D The histogram represents the mean value of three experiments. The statistical significance was determined by Student’s t-t est. *** P < 0.001. E DCC-2036 did not reduce the mRNA level of KLF5 in MDA-MB-231 cells but downregulated the mRNA level of Nanog. The cells were treated with DCC-2036 (0 or 5 μM) for 48 h and the mRNA levels were measured by q RT-PCR. The statistical significance was determined by Student’s t -test, *** P < 0.001. F WB showed that DCC-2036 could promote the degradation of the KLF5 protein in MDA-MB-231 cells and HS-578T cells, which were treated with DCC-2036 (5 μM) or DCC-2036 (0 μM) for 12 h, followed by cycloheximide (CHX, 100 μg/ml) for 0.5, 1, or 2 h. G The proteasome inhibitor MG132 mitigated the DCC-2036-induced KLF5 decrease in MDA-MB-231 cells and HS-578T cells. The cells were treated with DCC-2036 (5 μM) or DCC-2036 (0 μM) for 44 h, followed by MG132 (20 μM) for 4 h and then for the Western blot assay. H Treated with 10 μM DCC-2036 for 24 h, KLF5 ubiquitination was assessed by an anti-KLF5 antibody in the presence of MG132 after ubiquitin were transfected into MDA-MB-231 cells.

Article Snippet: The following antibodies were used for immunoblotting: phospho-AXL (Y702) #5724, phospho-AKT (S473) #4060, AKT #4691,phospho-GSK3β (S9) #5558, GSK3β #12456, β-actin #4970, purchased from Cell Signaling Technology (Beverley, MA); ALDH1A1 #15910-1-AP, OCT4 #11263-1-AP, KLF4 #11880-1-AP, SOX2 #11064-1-AP, KLF5 #21017-1-AP, NANOG #14295-1-AP, AXL #13196-1-AP, MET #25869-1-AP, FGF-BP1 #25006-1-AP purchased from Proteintech (Wuhan, Hubei, China); NF-κB (P65) #ab16502, purchased from Abcam; FGF-BP1 #AF1593 purchased from R&D; phospho-KLF5 (S303) #AF7042, phospho-AXL (Tyr702) # AF8523 purchased from Affinity (Changzhou, Jiangsu, China).

Techniques: Expressing, Incubation, Western Blot, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: International Journal of Biological Sciences

Article Title: Histone Deacetylase Inhibitors (HDACi) Promote KLF5 Ubiquitination and Degradation in Basal-like Breast Cancer

doi: 10.7150/ijbs.65322

Figure Lengend Snippet: Both HDACi and HDAC1 knockdown downregulated the expression of KLF5 protein and its downstream targets genes. A, HDACi inhibited KLF5-mediated downstream target gene (FGF-BP1, Cyclin D1, and p21 expression changes. HCC1937 and HCC1806 cells were treated with HDACi for 48 h at the indicated concentrations. The protein expression was measured by Western blotting. B, HDAC1 knockdown by three different siRNAs in HCC1937 and HCC1806 cells for 48 h; cell lysates were collected for western blotting, which indicated that KLF5 and its downstream target gene (FGF-BP1 and Cyclin D1) expression were blocked. C, HDAC1 knockdown-induced reduction in KLF5 expression in BLBC was inhibited by proteasome inhibitor. HDAC1 was knocked down by three different siRNAs in HCC1937 and HCC1806 cells for 24 h. Subsequently, MG132 (3 µM) was used to treat the cells for 24 h. Cell lysates were collected for Western blotting.

Article Snippet: The anti-human FGF-BP1 (MAB1593) antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Histone Deacetylase Inhibitors (HDACi) Promote KLF5 Ubiquitination and Degradation in Basal-like Breast Cancer

doi: 10.7150/ijbs.65322

Figure Lengend Snippet: HDACi in combination with BRD4i inhibit the KLF5 expression and function and the survival of BLBC cells. A, HDACi were combined with compound 870 to regulate the expression of KLF5 and its downstream target genes (FGF-BP1, Cyclin D1, and p21). HCC1937 and HCC1806 cells were treated with HDACi and compound 870 for 24 h at the indicated concentrations. The protein expression was measured by Western blotting, and cell viability was assessed after 24 h by the SRB assay. B, KLF5 knockdown decreased the cytotoxicity of HDACi in BLBC cell lines. KLF5 was knocked down by two different siRNAs in HCC1937 and HCC1806 cells and cell lysates were collected for Western blotting. The cells were treated with HDACi for 48 h to measure cell viability by the SRB assay.

Article Snippet: The anti-human FGF-BP1 (MAB1593) antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Sulforhodamine B Assay, Knockdown