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. 2021 Aug 6;22(9):e52247. doi: 10.15252/embr.202052247

Figure EV3. FoxO1 and PDK4 are target genes of miR‐183 and miR‐96 in skeletal muscle, related to Fig 3 .

Figure EV3

  • A
    Relative mRNA expression of FoxO1 (left) and PDK4 (right) in C2C12 cells treated with Ago‐183/96 or Ant‐183/96 (n = 3 for FoxO1 analysis; n = 5 for PDK4 analysis).
  • B, C
    Western blot analysis of FoxO1 (B) and PDK4 (C) in C2C12 cells treated with Ago‐183/96 or Ant‐183/96.
  • D
    The activity of the reporter containing the 3′UTR of FoxO1 or PDK4 was determined in HEK 293T cells transfected with Ago‐183/96, or Ant‐183/96 as indicated (n = 6 for FoxO1 3′UTR; n = 5 for PDK4 3′UTR).
  • E
    The relative expression of miR‐183 and miR‐96 in C2C12 cells transfected with miR‐183 mimics (left) or miR‐96 mimics (right) as indicated (n = 3).
  • F
    The activity of the reporter containing the 3′UTR of FoxO1 (left) and PDK4 (right) was determined in HEK 293T cells transfected with mimics for miR‐183 (183 mimics) or miR‐96 (96 mimics) (n = 6 for FoxO1 3′UTR; n = 5 for PDK4 3′UTR).
  • G
    The activity of the reporter containing the 3′UTR of ATGL was determined in HEK 293T cells transfected with Ago‐183/96, or Ant‐183/96 as indicated (n = 6).
  • H
    The activity of the reporter containing the 3′UTR of ATGL was determined in HEK 293T cells transfected with mimics for miR‐183 (183 mimics) or miR‐96 (96 mimics) (n = 6).
  • I
    Relative mRNA expression of ATGL in C2C12 cells treated with Ago‐183/96 or Ant‐183/96 (n = 3).
  • J
    Sequence alignment of miR‐182 and its target site in the 3′UTR of ATGL from various species.
  • K
    Relative mRNA expression of ATGL in C2C12 cells treated with Ago‐182 or Ant‐182 (left) (n = 3). Western blot analysis of ATGL in C2C12 cells treated with Ago‐182 or Ant‐182 (right).
  • L
    Relative mRNA expression of miR‐182 (left) and primary miR‐183 cluster (right) in C2C12 cells treated with Ant‐183/96 (n = 3).
  • M
    Relative mRNA expression of miR‐183, miR‐96, miR‐182, FoxO1, PDK4, ATGL, HSL, PGC1α, and PLIN5 in Cas9‐mediated miR‐183 and miR‐96 knockout C2C12 cells (n = 3).

Data information: Means ± SEM are shown for all panels. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student’s t‐test). All experiments were performed at least three times, and representative data are shown.Source data are available online for this figure.