Supplementary methods: soft agar assay, immunofluorescence, tumor cell proliferation assay, and isolation of tumor cells from tissues.
Guidelines on Breast-Feeding Appropriate for India?" -- concludes that babies can and should be given supplementary liquids or soft foods as young as 2 or 3 months to overcome the deficiencies in the milk of a malnourished mother.
Similarly, no significant differences were found between the two lines in their ability to grow independent of anchorage in soft agar (supplementary material Fig. S5), nor in their capacity to form mammospheres (supplementary material Fig. S6).
Here, in Supplementary Figure S1, by soft agar colony formation assay and wound healing assay, we also demonstrated that MALAT1 could promote colony formation and horizontal migration ability of LoVo and HCT116 cells.
While in suspension cultures, cells expressing HSF1WT were sensitive to metformin at all glucose concentrations, they only responded to metformin at normal and low glucose concentrations in soft agar (Supplementary Fig S7I).
After 3 weeks in culture, neither the 10A cont nor the 10A L2 cells proliferated in soft agar (Supplementary Figure 1), suggesting that LOXL2 expression alone cannot drive complete oncogenic transformation in the normal 10A cells.
No effect on cell growth/proliferation of the sNF96.2 cells was noted following transfection with the miR-29c mimics using a number of assays, including a live-cell imaging system IncuCyte, the MTS assay and growing the cells in 3-D culture using soft agar (Supplementary Figure S4).
Comparable results were obtained with the LATS-insensitive TAZS89A protein: overexpression of SOCS6 reduced TAZS89A levels and reduced soft agar growth (Supplementary Fig S7).
Silencing of P2Y2 receptor in 1E8 cells resulted in decreased tumour growth in nude mice compared with control cells, which is thought to be mainly due to decreased anchorage-independent growth as shown by soft agar assay (Supplementary Figure 2).
The CA dataset consisted of 203 characters randomly chosen from 7 publications of phylogenetic matrices for 499 extant and extinct vertebrate taxa that encompassed all regions of the hard and soft anatomy of vertebrates (Supplementary Table S2).
