Skip to main content
. 2020 Feb 18;21(3):e48692. doi: 10.15252/embr.201948692

Figure 6. Loss of Abca12 results in altered lipid raft levels and changes in F‐actin and CDC42 activity.

Figure 6

  • A
    Detection of lipid rafts in 8‐week‐old islets with recombinant cholera toxin subunit B (CT‐B) conjugated to Alexa Fluor 488 (green). Islets are co‐stained with DAPI (blue) (scale bar = 50 μm.).
  • B
    FACS profiling of dispersed islets from 12‐week‐old mice stained with insulin and CT‐B.
  • C
    Confocal images of rafts (using CT‐B‐Alexa Fluor 647) in MIN6 cells transfected with Fluorescein‐488‐labelled siRNAScr or Dy547‐labelled siRNAABCA12 (scale bar = 10 μm).
  • D
    Quantitation by confocal microscopy of the effect of ABCA12 deficiency on CT‐B binding to MIN6 cells (mean ± SEM, *P = < 0.05, Student's t‐test, n = 4 biological replicates, 50–100 cells for each).
  • E
    Quantitation by flow cytometry of the effect of ABCA12 deficiency on CT‐B binding to MIN6 cells (mean ± SEM, n = 3 biological replicates, *P = < 0.05, Student's t‐test).
  • F
    The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 (incubation for 5 or 10 min as indicated, mean ± SEM, n = 4 (biological replicates), *P = < 0.05, Student's t‐test).
  • G
    Western blot of the abundance of CDC42 in ABCA12‐deficient MIN6 cells.
  • H
    The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells (BDK, 100 ng/ml, 4 min; *P = < 0.05, Student's t‐test, significance versus ABCA12‐deficient BDK‐activated cells are indicated, n = 4 biological replicates, mean ± SEM).
  • I, J
    (I) Confocal images of F‐actin in ABCA12‐deficient MIN6 cells (scale bar = 20 μm) and (J) quantification of abundance showing the effect of ABCA12 deficiency (mean ± SEM, *P = < 0.05, Student's t‐test, n = 4 biological replicates, 50–100 cells for each).
  • K
    F‐actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 (mean ± SEM, n = 3 biological replicates, *P = < 0.05, Student's t‐test).
  • L, M
    (L) Representative image of F‐actin using LifeAct staining of purified islets isolated from Abca12 tm1d versus control mice (scale bar = 10 μm) and (M) quantitation of fluorescent signals from these samples (n = 3 biological replicates, mean ± SEM, ***P = < 0.001, ****P = < 0.0001 Student's t‐test).
  • N
    Co‐localisation between ABCA12 (red) and CDC42 (green) in MIN6 cells (scale bar = 10 μm).
  • O
    Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 cells with ABCA12 deficiency (mean ± SEM, n = 4 biological replicates, *P = < 0.05, Student's t‐test).
  • P
    The effect of overexpression of constitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12 deficiency. Treatments are defined in the table below.
  • Q
    Effects of Jasplakinolide on F‐actin levels (green) in siRNA‐treated cells (scale bar = 10 μm).
  • R
    Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose (mean ± SEM, n = 4 biological replicates, *P = < 0.05, **P = < 0.01, significance relative to untreated cells, Student's t‐test).