(A) Western blot following RNA-pull down showing Lnc34a interaction with PHB2, Dnmt3a and HDAC1 in CCSC1 (left) and CCSC2 (right) sphere cells. RNA-pull down was performed using CCSC lysates with biotin-labeled Lnc34a, antisense and tRNA. Actin was used for input control. (B) RNA immunoprecipitation (RIP) showing Lnc34a interaction with PHB2, Dnmt3a and HDAC1 in CCSC1 (left) and CCSC2 (right) sphere cells. (C) RIP showing PHB2 knockdown disrupts Lnc34a interaction with Dnmt3a, but has no effect on Lnc34a interaction with HDAC1. (D) RIP showing Dnmt3a knockdown does not affect Lnc34a interaction with PHB2 or HDAC1. (E) RIP showing HDAC1 knockdown has limited effect on Lnc34a interaction with PHB2 or Dnmt3a. (F) Mapping PHB2 and HDAC1 interaction domains on Lnc34a. Upper panel, schematic illustration of full-length Lnc34a and the truncated fragments for RNA put-down. Lower panel, Western blot of PHB2 and HDAC1 from RNA put-down of the fragments. (G) EMSA showing Lnc34a/PHB2 (left) and Lnc34a/HDAC1 (right) interactions. (H) RT-qPCR of miR-34a levels after expressing full-length or truncated fragments of Lnc34a. (I) In vitro interaction assay binding of the truncated fragment (267–560 bp) to the DNA containing the miR-34a promoter sequence. (J) Schematic illustration of Lnc34a interaction with PHB2, Dnmt3a and HDAC1. (K, L, M) RT-qPCR showing knockdown of Dnmt3a (K), HDAC1 (L), and PHB2 (M) increased miR-34a expression in sphere cells. (N, O) RT-qPCR showing treatments with HDAC inhibitor SAHA (N) or TSA (O) increased miR-34a expression in sphere cells. Error bars denote s.d. of triplicates. ***p<0.001. p-value was calculated based on Student’s t-test.