Editor\u27s Notes, Chiba Medical Journal 90-1

Multiple sequence alignment of deduced amino acid sequences of 25 wheat annexin genes with rice annexin OsAnn2 (Os05g31760) obtained by ClustalW. (PDF 212 kb

<i>FZD7</i> regulates the size of metastases from MA-2 and MeWo Cells.

<p>A. Week1, week2, and week 3 metastases (arrows) from MA-2(shGFP) and MA-2(sh<i>FZD7</i>) cells were stained with the antibody against human vimentin (purple). B. Size distribution of metastases from MA-2(shGFP) and MA-2(sh<i>FZD7</i>) cells 1 week post injection. *: Student’s t test, <i>p</i> < 0.05. C. Size distribution of metastases from MA-2(shGFP) and MA-2(sh<i>FZD7</i>) cells 2 weeks post injection. *: Student’s t test, <i>p</i> < 0.05. D. Week1 and week2 metastases (arrows) from MeWo(shGFP) and MeWo(sh<i>FZD7</i>) cells were stained with the antibody against human vimentin as in A. E. Size distribution of metastases from MeWo(shGFP) and MeWo(sh<i>FZD7</i>) cells 1 week post injection. *: Mann-Whitney test, <i>p</i> < 0.05. F. Size distribution of metastases from MeWo(shGFP) and MeWo(sh<i>FZD7</i>) cells 2 weeks post injection. *: Mann-Whitney test, <i>p</i> < 0.05.</p

FZD7 promotes melanoma metastasis via activation of JNK.

<p>A. WNT3A treatment of MA-2(shGFP) and MA-2(sh<i>FZD7</i>) cells led to an increase in AXIN2 mRNA in both cell lines, but the level of AXIN2 mRNA was higher in the knockdown cells across all treatments. B. LiCl treatment of MA-2(shGFP) and MA-2(sh<i>FZD7</i>) cells led to an increase in AXIN2 mRNA in both cell lines, but the increase was more prominent in the knockdown cells. C. The cytosolic and nuclear fractions from LiCl-treated cells were probed with the anti-β-catenin antibody. Lamin and tubulin were used as the loading control for each fraction. D. MA-2(shGFP) and MA-2(sh<i>FZD7</i>) were treated with WNT5A and probed with antibodies against p-JNK and total JNK. GAPDH was used as a loading control. E. MeWo(shGFP) and MeWo(sh<i>FZD7</i>) were treated with WNT5A and probed with antibodies against p-JNK and total JNK. GAPDH was used as a loading control. F. The ratio between p-JNK and total JNK on western blots represented in D and E. G. qRT-PCR analyses to measure the over-expression of dnJNK in MA-2 cells. H. MA-2(EV) and MA-2(dnJNK) cells were injected intravenously into NSG mice. A significant reduction in metastasis formation was observed in dnJNK-overexpressing cells. *: Mann-Whitney test, <i>p</i> < 0.05.</p

<i>FZD7</i> knockdown reduces metastasis potential of multiple melanoma cell lines.

<p><i>FZD7</i> was knocked down by shRNA in MA-2 (A), WM266-4 (B), 451Lu-R (C) and MeWo (D) cell lines. The extent of knockdown was measured by qRT-PCR (left panels). The knockdown cell lines and the shGFP control were injected intravenously into NSG mice. The number of metastases formed in lung was counted (right panels). Student’s t test, *: <i>p</i> < 0.05; **: <i>p</i> < 0.01; ***: <i>p</i> < 0.001.</p

<i>FZD7</i> is required for cell proliferation in metastases from MA-2 cells.

<p>A. Immunostaining of metastases from MA-2(shGFP) or MA-2(sh<i>FZD7</i>) cells with the antibodies against phospho-histone H3 (dark red) and human vimentin (purple). Inset shows the high magnification of images pointed by black arrows. B. The percentage of pHH3+ metastases over the total number of metastases scored at week-1 (n = 6 for MA-2(shGFP) or MA-2(shFZD7) samples) or week-2 (n = 4 for MA-2(shGFP) and n = 6 for MA-2(shFZD7) samples) post injection. *: Student’s t test, <i>p</i> < 0.05. C. Subcutaneous tumor growth of MC-1(shGFP) (n = 13) and MC-1(sh<i>FZD7</i>) (n = 10) cells. **: Student’s t test, <i>p</i> < 0.01.</p

<i>FZD7</i> is required for tumor initiation of melanoma cells.

<p>A. Limiting dilution assays for MA-2(shGFP) and MA-2(shFZD7), or A375P and MA-2 cells. Tumor incidence was recorded 12 weeks after inoculation of the cells. B. Tumor-onset analyses on WM266-4(shGFP) or WM266-4(shFZD7) cells. The percent of tumor-free mice over time is shown. Total number of mice analyzed was 9 for WM266-4(GFP), 7 for WM266-4(shFZD7#3), and 9 for WM266-4(shFZD7#4) cells. Log-rank test, *: <i>p</i> < 0.05; **: <i>p</i> < 0.01. C. <i>FZD7</i> knockdown in MA-2 and WM266-4 cells led to reduced number of colonies in soft agar. Left: images of colonies in soft agar. Middle and right: the average number of colonies per field from control or FZD7-knockdown MA-2 or MeWo cells. D. Left: relative levels of <i>FZD7</i> mRNA in WM266-4 and WM115 cells measured by qRT-PCR. Right: limiting dilution assays for WM266-4 and WM115 cells.</p

Time course of FZD7 function during melanoma metastasis.

<p>Control or <i>FZD7</i>-knockdown MA-2 or MeWo cells were injected into NSG mice. Lungs were harvested 24 hrs (n = 5 for MA-2(shGFP), n = 8 for MA-2(shFZD7), n = 4 for MeWo(shGFP), n = 4 for MeWo(shFZD7)), 1 week (n = 6 for MA-2(shGFP), n = 6 for MA-2(shFZD7), n = 4 for MeWo(shGFP), n = 4 for MeWo(shFZD7), 2 weeks (n = 4 for MA-2(shGFP), n = 6 for MA-2(shFZD7)), or 3 weeks (n = 3 for MA-2(shGFP), n = 3 for MA-2 (shFZD7)) after injections. ~3–5 sections per lung were stained and metastases counted. No difference in the number of metastases was observed between control and knockdown at the 24 hr time point for both cell lines (A, C). For MA-2 cells, <i>FZD7</i> knockdown led to a reduction in metastases three weeks after injections (B). *: Mann-Whitney test, <i>p</i> < 0.05. For MeWo cells, <i>FZD7</i> knockdown led to a reduction in metastases one week after injections (D). *: Mann-Whitney test, <i>p</i> < 0.05.</p

<i>FZD7</i> expression correlates with melanoma malignancy.

<p>Expresson values of <i>FZD7</i> mRNA in tumor samples from highly metastatic derivatives and those from the poorly metastatic parental line. **: Mann-Whitney test, <i>p</i> < 0.01. (n = 11 for parental, n = 21 for metastatic derivatives).</p

<i>RIPK4</i> mRNA was down-regulated by fetal bovine serum from different sources and by human plasma.

A, B) The level of RIPK4 mRNA in HaCaT (A) or A431 (B) cells was reduced after treatment by FBE and FBS. Data for FBE treatment were pooled from three independent experiments.C. The level of RIPK4 mRNA in HaCaT cells was reduced after treatment of human plasma, similar to FBS. *: p < 0.05.</p

Similar down-regulation of <i>RIPK4</i> by FBS was observed when an alternative pair of primers was applied in the quantitative RT-PCR reaction.

Serum-starved A431 monolayer was treated with FBS or PBS for two hours and RNA was harvested. RIPK4 mRNA level was measured using an alternative pair of primers (see Materials and Methods). *: p (TIF)</p