Fig 1: shRNA knockdown of LGR5 and Msi-1 have opposite effects on cell growth in soft agar.A) and B): LIM1215 (A) and LIM1899 (B) cells were transduced with lentiviral particles containing shRNA to non target sequences (NT), to LGR5 (shLGR5) or to Musashi-1 (shMsi-1) and bulk selected in puromycin. Expression of LGR5 (left panels) and Musashi-1 (right panels) was assessed by qRT-PCR two weeks after transduction. Data are presented as gene expression relative to the parental cell lines. These results are representative of >5 separate experiments. C): Cells expressing shRNAs (shLGR5, shMsi-1 and NT) and parental cells were grown in antibiotic-free medium for three days then tested for their ability to form colonies in soft agar as described in Methods.. Data are presented as colony forming efficiency of test samples relative to control (untransfected) parental cells and are the average and sd of three separate experiments. D): Representative images of colonies in soft-agar plates stained with crystal violet. Images were acquired with a Nikon 90i with a DXM 1200C camera.
Fig 2: Effects of LGR5 modulation on cell-cell adhesion and migration.Lim1899 cells were transfected with vector controls (Cy3V and pTuneV), with Cy3- siLGR5 or with pTune/LGR5. Parental, transiently transfected cells (Tr) or stably transfected cell lines (St) were cultured under the following conditions: A) Cells were seeded in 30 µl droplets on a plastic surface, and the plate inverted to create hanging drops as described in Methods. Images were taken after 8 days by re-inverting the plastic support and imaging in bright field with a Nikon 90i microscope and a 10× lens. Digital images were acquired with a Photometrics CoolSnap digital camera. Spheroid volumes (left-hand graph) were calculated from these images using the modified ellipsoid formula. Spheroid sizes differed significantly between siLGR5 or M2LGR5 transfected cells and their counterparts transfected with empty vector (p = 0.0312 and p = 0.321, respectively, by the unpaired t-test). The cellularity of the spheroids (right hand graph) was assessed as described in Methods. In both graphs data represent the mean and standard deviation of 10 individual spheroids per cell line. B) Parental cells and cells stably transfected with pTune/LGR5 (clones 6-1) were plated at high density in 24-well plates. Wounds were scratched in the adherent monolyers and the wells were imaged every two days with a Nikon90i microscope using a 10× lens (upper panels). The photomicrograph on lower right shows a higher magnification of LGR5 6-1 wound at day 6 (20× lens). Insert: Rate of wound closure over 96 hr. C) Cells were seeded in Transwell inserts (8 mm pore size) and cultured for 4 days. Filters were fixed and stained with May-Grumwald/Giemsa. Cells on the upper side of the filters were removed, and filters mounted on glass slides. Cells present on the underside of the filters (migrating cells) were counted by light microscopy as described in Methods. The graph presents average and sd of three separate samples for each cell type. Tr and St denote transient and stable LGR5 transfectants. Significance levels were determined by the unpaired t-test. *** = p<0.001; ** = p<0.005.
Fig 3: Expression of junctional proteins in Lim 1899 cells with altered LGR5 expression.Cells transfected with empty vector, siRNA to LGR5 (siLGR5) or pTune/LGR5 (LGR5) were grown in chamber slides and prepared for immunofluorescence as described in Methods. Slides were stained with antibodies to E-cadherin (A), β-catenin (B) or Zo-1 (C) followed by Alexa 488 anti-mouse Ig (green channel in all samples). Cells were counterstained with rhodamin-phalloidin (red channel) and the nuclear stain DAPI (blue channel). Images are Z-stacks of sequential confocal images. For each antibody, the top panel shows the merged channels and the bottom panel the green channel only (grey scale).
Fig 4: Morphology and LGR5 reactivity of xenografts tumours.A) Haematoxilin and eosin staining of frozen sections from representative tumours generated from parental LIM1899, LIM1899 transfected with siRNA to LGR5 or LIM1899 transfected with pTune-LGR5 construct. Brightfield images were acquired on a Nikon 90i microscope with a 20× lens. B) Confocal images of frozen sections stained with antibodies to β-catenin (red), LGR5 (green) or the DNA stain DAPI (blue). All images are Z-stacks of confocal sections. For each set, the upper panel shows the three combined stains, the middle panel LGR5 (greyscale), and the bottom panel β-catenin (greyscale). Images were obtained on a Nikon C1 confocal microscope with a 60× oil lens. C) Specificity control for LGR5 staining: frozen sections were stained with antibody to β-catenin and normal rabbit serum, followed by Alexa 488 anti-rabbit Ig (green) and Alexa 546 anti-mouse Ig (red) and the nuclear stain DAPI (blue). The green channel (NRS) and red channel (β-catenin) are shown separately in greyscale. Images were obtained as in B).
Fig 5: Lgr5 and Sox9 are downregulated in epidermal tumours compared with normal skin. mRNA expression of Sox9, Lgr5 and Lgr6 in normal skin and epidermal tumours. Bars represent mean ± SEM. ** p < 0.05; *** p < 0.001. Normal skin tissues (normal skin, n = 3), basal cell carcinomas (BCC, n = 6), squamous cell carcinomas (SCC, n = 9).
Supplier Page from MilliporeSigma for Anti-LGR5 antibody produced in rabbit