Supplementary Figure 4: HDAC screening and analysis of the role of HDAC3 in OSKM reprogramming.

(a) RT-qPCR analysis showing the knockdown efficiency of Hdac members in MEFs. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each) relative to shLuc control. (b) Immunoblotting with the indicated antibodies using lysates from MEFs transduced with OSKM and FLAG-tagged HDAC3, HDAC3-YF, and HDAC3-YF-KA on day 7 in serum+Vc. (c) Co-immunoprecipitations of FLAG-tagged NCoR/SMRT and HA-tagged HDAC3 in lysates from HEK293T cells. (d) Phase contrast and Oct4-GFP⁺ cells in OSKM reprogramming in serum+Vc at the indicated days. Scale bar, 100 μm. (e) Time course measurement of the appearance of Oct4-GFP⁺ colonies in MEFs transduced with OSKM and HDAC3-YF or empty vector in serum+Vc. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test. (f) Representative flow cytometry plots showing the appearance of Oct4-GFP⁺ cells in MEFs transduced with OSKM and HDAC3-YF or empty vector in KSR. (g) Measurements of cell numbers during a reprogramming time course in serum+Vc. Cells were transduced with OSKM and HDAC3-YF or empty vector. Data are the mean ± s.e.m. (n=3 biological replicates with 2 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test. (h) RT-qPCR detection of the indicated pluripotency genes in MEFs transduced with OSKM and HDAC3-YF or empty vector on day 13 in serum+Vc. MEFs and E14 ESCs are included as comparisons. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each), relative to OSKM and empty vector, P value was calculated using two-tailed unpaired Student’s t-test, * indicates P value <0.05. (i) RT-qPCR analysis showing the expression of the indicated MEF-enriched genes on day 5 in MEFs transduced with OSKM and HDAC3-YF or empty vector in serum+Vc (n=3 biological replicates with 3 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test. (j) Heatmap of all genes passing quality control from the single-cell RT-qPCR analysis organized into categories according to the type of gene; each column is a single cell. Data were calculated as the ACx (relative expression). (k) Principal component gene loading of the single-cell RT-qPCR data. (l, m) Violin plots of single-cell RT-qPCR expression of pluripotency genes (panel l) or the somatic gene S100a4 (panel m). The red bar represents the mean, and samples sizes are indicated in panel j.