Journal: The Journal of Biological Chemistry

Article Title: Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs *

doi: 10.1074/jbc.M114.612648

Figure Lengend Snippet: 32D myeloid progenitor cell line expresses little or no endogenous FZDs but does express LRP5/6 co-receptors. A, the bar graph presents the FZD expression profile (FZD1–10) of 32D cells (hatched gray bars) and primary microglia cells (white bars) determined by qPCR (n ≥ 3). Error bars represent S.E. GAPDH was used as a housekeeping gene for normalization. By comparing FZD expression in primary microglia cells (previously published by Halleskog et al. (21)) and 32D cells, it becomes apparent that FZD levels in 32D cells are very low. B, WNT co-receptor profile in 32D cells determined by RT-PCR. LRP5 and LRP6 (but neither RYK nor ROR1/2) are expressed in 32D cells. The negative control consisted of 32D cell cDNA prepared without reverse transcriptase; mouse tail genomic DNA or cDNA from GL261 cells (RYK) was used as a positive control. C, shown are comparative PCR expression profiles of 32D and L929 cells, including WNTs, FZDs, LRPs, DKKs, and SFRPs. The table summarizes the average Ct values of two biological replicates. The following genes were not included on the PCR array: Wnt9b, Wnt10b, Fzd9, Fzd10, Ryk, Ror1, and Ror2. D, immunoblotting for HA-tagged FZDs in 32D cells expressing FZD2, FZD4, or FZD5. β-Actin was used as a loading control. The molecular masses of FZDs were determined with Bio-Rad software.

Article Snippet: The primer pairs used in this study were as follows: FZD 1 (Mm01320682_s1), FZD 2 (Mm02524776_s1), FZD 3 (Mm00445923_m1), FZD 4 (Mm03053556_s1), FZD 5 (Mm00445623_s1), FZD 6 (Mm00433383_m1), FZD 7 (Mm01255614_s1), FZD 8 (Mm00433419_s1), FZD 9 (Mm01206511_s1), FZD 10 (Mm00558396_s1), and GAPDH (4352339E) as an internal reference (all from Applied Biosystems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Western Blot, Software

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-101 inhibits cell migration and invasion in bladder cancer via targeting FZD4

doi: 10.3892/etm.2018.7084

Figure Lengend Snippet: T24 cells were transfected with NC siRNA or FZD4 siRNA. Following transfection, (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were used to examine the FZD4 mRNA and protein expression levels. (C) Wound healing and (D) Transwell assays were used to examine the migration and invasion capacities of T24 cells. Magnification for wound healing assay, ×40. Magnification for Transwell assay, ×200. **P<0.01 vs. NC siRNA. NC, negative control, siRNA, small interfering RNA; FZD4, frizzled class receptor 4.

Article Snippet: Cell transfection In brief, 2×10 5 T24 cells/well seeded in a 6-well plate were transfected with 100 nM miR-101 mimic (cat. no. HmiR0009-MR04), negative control miR (miR-NC; cat. no. CmiR0001-MR04), miR-101 inhibitor (cat. no. HmiR-AN0021-SN-10), NC inhibitor (cat. no. CmiR-AN0001-SN; all Guangzhou Fulengen Co., Ltd.), FZD4 small interfering (si)RNA (cat. no. SR305441) or NC siRNA (cat. no. SR30004; both OriGene Technologies, Inc., Rockville, MD, USA), 100 nM miR-101 mimic + 1 mg blank vector (cat. no. AMS10002) or 100 nM miR-101 mimic + 1 mg pc-DNA3.1-FZD4 expression plasmid (cat. no. AMS10116; both Hunan Nanhua Aishi Pulin Biotechnology; NanHua Bio-medicine Co., Ltd., Changsha, China) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Migration, Wound Healing Assay, Transwell Assay, Negative Control, Small Interfering RNA