Figure 6. Cell death is increased in Slc7a5‐null embryos, and exogenous ER stress induction in wild‐type embryos upregulates Chac1 and Trib3 .

• A, B
  Western blot of individual E9.5 wild‐type (n = 4) and Slc7a5‐null (n = 4) embryo lysates immunoblotted using an antibody against phospho‐GCN2 (Thr899) and total GCN2 (loading control), ***P = 0.0001; or against phospho‐eIF2α (Ser51) and total eIFα (loading control), *P = 0.0482, band intensities measured with FIJI and analysed with unpaired t‐test (see original source data).
• C–F
  Phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wild type (C, c1) (n = 2 embryos, 0/14 sections) and Slc7a5‐null (D, d1) (n = 2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wild type (E–e2) (n = 4 embryos, 0/62 sections) and Slc7a5‐null (F–f2) (n = 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C–e1, D–f1) 100 µm and in (e2 and f2) 25 µm. Arrowheads indicate ventral midline.
• G
  An increase in Trib3 protein was detected by Western blot in Slc7a5‐null embryos; each lane represents an E9.5 embryo lysate (wild type n = 3, Slc7a5‐null, n = 4) with α‐tubulin loading control, unpaired t‐test *P = 0.0153 (see original source data).
• H–L
  TUNEL assay to detect apoptotic cells in wild type (n = 3 embryos, 32 sections) or Slc7a5‐null (n = 5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H–K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL‐positive cells within spinal cord sections was performed as stated in Materials and Methods. Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t‐test, P = 0.250, F‐test to compare variances, P = 0.0003 (see original source data).

Data information: All error bars indicate SEM.Source data are available online for this figure.