Fig. 1

Inducible depletion of SPT16 triggers reduced pluripotency in ES cells. A Schematic of auxin-inducible degron (AID) and V5-tagged SPT16 protein. NTD = N-terminal domain, DD = dimerization domain, MD = middle domain, CTD = C-terminal domain, AID = minimal auxin-inducible degron tag, V5 = 3xV5 epitope tag. B Western blot showing depletion of SPT16 after 0, 3, 6, 24, and 48 h treatments with IAA (+) or vehicle control (EtOH, −). 40 µg total protein loaded per lane. Top: V5 antibody (for tagged SPT16) and bottom: β-actin antibody. Representative blot shown from SPT16-V5-AID clone 1; additional blots can be found in Additional File 1: Fig. S1. “M” denotes a molecular weight marker lane. C Timecourse of IAA or EtOH treatment for 6, 12, 24, or 48 h to deplete SPT16 showing morphological changes following alkaline phosphatase staining. Images are representative of plate-wide morphological changes. Alkaline phosphatase staining is quantified in Additional File 1: Fig. S1E. D. Timecourse of IAA or EtOH treatment for 12, 24, or 48 h to deplete SPT16, followed by immunocytochemistry showing a progressive reduction in the expression of OCT4. Images are representative of plate-wide immunofluorescence changes. Left panels are DAPI-stained (blue), while right panels show OCT4 immunofluorescence (green). OCT4 immunofluorescence is quantified in Additional File 1: Fig. S1F