Fig 1: p16-overexpressing primary keratinocytes stimulate proliferation of naive cells through Wnt secretion.a Primary mouse keratinocytes infected with a p16-expressing or control lentivirus, co-stained for K14 (green), p16 (red) and BrdU (indicating S-phase, white), or for SA-βGal, 10 days after infection. b Relative mRNA levels of the indicated genes in the same keratinocytes, measured by qRT-PCR. n = 3 replicates. P < 0.05 for all genes. c Relative mRNA levels of the indicated Wnt-associated genes measured by qRT-PCR in control and p16-expressing keratinocytes. Values are also shown for the same cells treated with the β-catenin inhibitor XAV-939 (5 μM) for 24 h. n = 3 replicates. P < 0.05 for all comparisons indicated by lines: p16 versus control (dark blue versus dark grey, short lines), and p16 versus p16+XAV-939 (light blue versus dark blue, long lines). d Secreted Wnt3a protein levels in medium of control and p16-expressing cells, measured by ELISA. n = 3 replicates. ***P < 0.0001 e Fold increase over 4 days in numbers of primary keratinocytes cultured with conditioned media from p16-expressing (CM-p16) or from control (CM-Cont) keratinocytes, or with 10 ng/ml recombinant Wnt3a, in the absence or presence of XAV-939 (darker and lighter columns, respectively). n = 6 replicates. ***P < 0.0001. f Relative mRNA levels of the indicated Wnt-associated genes measured by qRT-PCR in keratinocytes treated for 24 h with conditioned media from p16-expressing or control cells, or with Wnt3a, in the presence or absence of XAV-939. n = 3 replicates. P < 0.05 for all comparisons indicated by lines: CM-p16 versus CM-Cont (dark blue versus dark grey, short lines), and CM-p16 versus CM-p16+XAV-939 (light blue versus dark blue, long lines). All graphs indicate mean across replicates ± S.E.M., t test. Scale bars—50 μm.
Fig 2: (A) Wnt‐3a concentration in the culture supernatant measured by ELISA in COL organoids cultured without additional growth factors. Supernatant was collected at medium exchange time points: Day 1 (24 h accumulation period), day 4 (72 h accumulation period), and day 7 (24 h accumulation period) (n = 3 per condition with 20 organoids per replicate). (B) Wnt‐3a concentration in supernatant measured by ELISA in response to varying trametinib concentrations (n = 3 per condition with 20 organoids per replicate). (C) Wnt‐3a concentration and (D) absolute Wnt‐3a levels in the culture supernatant measured by ELISA in COL organoids cultured without additional growth factors at varying organoid seeding densities. Supernatant was collected on day 4 (72 h accumulation period) and day 7 (24 h accumulation period). Seeding densities 3 and 50 were kept in 384‐well plate format with organoids growing within a 19 μL BME bed covered by 80 μL medium. Seeding density >200 was kept in 24‐well plate format with organoids growing in a 30 μL BME dome covered by 500 μL medium (n = 3 per condition). (E), (F), (G) Proliferation and morphological reorganization of untreated COL organoids over 8 days of culture without additional growth factors, analyzed in relation to different seeding densities. Scale bar: 100 μm.
Fig 3: (A) Representative image of untreated COL organoids cultured in COCM+GF (Noggin, R‐Spondin‐1, Wnt‐3a, EGF, CHIR‐99021, Y27632) over 8 days. Scale bar: 100 μm. (B) Organoid growth rates at different organoid seeding densities (n = 12,18,22,3,3; statistical analysis: Mann–Whitney test). (C) Metabolic activity of untreated organoids on day 8, measured by CellTiter‐Glo 3D assay, normalized to the number of organoids per well (n = 12,18,22,3,3; statistical analysis: Mann–Whitney test). (D) Dose response to trametinib at different organoid seeding densities measured on day 8 post plating. Each condition: n ≥ 3. IC50 values were: Seeding density 3: 1.2 nM; Seeding density 20: 3.1 nM; Seeding density 50: 27.9 nM. (E) Organoid growth rates at varying initial organoid diameters (n = 12 per condition with 20 organoids per replicate; statistical analysis: unpaired two‐tailed t‐test). (F) Dose response to trametinib based on initial organoid diameters assessed on day 8 post plating (n = 6 per condition with 20 organoids per replicate). IC50 values were: 50–100 μm: 3.6 nM; 100–150 μm: 6.9 nM; 150–200 μm: 18.9 nM. (G) Correlation between initial organoid volume and derived IC50 values from (F), with a goodness‐of‐fit of R 2 = 0.994. Error bars on the x‐axis represent the range of initial organoid volumes; error bars on the y‐axis indicate uncertainty in IC50 values obtained from dose response curves.
Supplier Page from R&D Systems, a Bio-Techne Brand for Wnt-3a ELISA
Available conjugates: Sizes Available: 1 Kit (for 5 Plates)