Fig 1: Tox4 suppression impedes intermediate reprogramming stages. (A) Immunofluorescence analysis for TOX4/NANOG in ESCs grown in S/L and MEFs, showing expression and nuclear localization in both cell types. Representative images of all lines examined for TOX4 (green), NANOG (red) and DAPI (blue, nuclei counterstaining) are shown. Scale bars: 20 µm. (B) Western blot for TOX4 (Sigma) and GAPDH in MEFs and ESCs. (C) Quantification of TOX4 western blot analysis using GAPDH as a loading control. Results are shown as the mean of technical duplicates (n=1). (D) Schematic of siRNA-mediated somatic Tox4 knockdown at the start of reprogramming to iPSCs. Indicated genes were targeted at D0 by siRNA transfection of STEMCCA MEFs after subsequent DOX induction of reprogramming. (E) The number of AP+ colonies at D12 of reprogramming in S/L+AA. Results for control, Oct4, Tox4, C2Orf88, Ubr4 and Ube2a siRNA are shown as mean±s.d. (n=2 or 3 with biological duplicates in total). Results for Bex2, Tcl1a, Bcor, Zhx3 and Alkbh1 siRNA are shown as the mean±s.d. (n=2 with two biological replicates in total). *P<0.05, **P<0.01 (one-way ANOVA with Dunnett's multiple comparisons test compared to control). (F) The number of AP+ colonies at D12 of reprogramming in S/L+AA. Counts were normalized to counts in control conditions. Results are shown as the normalized mean±s.d. (n=1 with biological duplicates in total). Squares, triangles and circles represent one independent experiment each.
Fig 2: siRNA screen for modulators of reprogramming to iPSCs identifies Tox4 as a novel modulator of reprogramming. (A) Schematic of targeted siRNA approach for modulators of reprogramming to iPSCs. Target genes were targeted every other day by siRNA transfection of STEMCCA MEFs induced to reprogram. ‘STEMCCA’ MEFs allow for a DOX-inducible expression of Oct4, Sox2, Klf4 and Myc resulting in the generation of iPSCs. (B) The number of AP+ colonies at D14 or 15 of reprogramming in S/L with no AA. Colony counts were normalized to colony counts in control conditions. Results are shown as the mean±s.d. (n=3 with two biological replicates in total). *P<0.05; **P<0.01 (one-way ANOVA with Dunnett's multiple comparisons test compared to control). (C) Western blot analysis for TOX4 (Sigma antibody) and actin after 6 days and 9 days of STEMCCA MEFs reprogramming and transfection of Tox4 or control siRNAs every other day. (D) The number of AP+ colonies at D11 or 12 of reprogramming in S/L+AA. Colony counts were normalized to colony counts in control conditions. Results are shown as the mean±s.d. (n=3 with two biological replicates in total). **P<0.01 (one-way ANOVA with Dunnett's multiple comparisons test compared to control). (E) The number of AP+ colonies at D11 or 12 of reprogramming in KSR+AA. Colony counts were normalized to colony counts in control conditions. Results are shown as the mean±s.d. (n=3 with two biological replicates in total). **P<0.01, ***P<0.001 (one-way ANOVA with Dunnett's multiple comparisons test compared to control). Squares, triangles and circles represent one independent experiment each.
Supplier Page from MilliporeSigma for In Vitro Toxicology Assay Kit, Neutral Red based