Fig 1: LRP8 depletion induces apoptosis. TNBC cells were transfected with one of the two siRNAs against LRP8 (#2 and #3: orange and red, respectively) or with a control siRNA (black). A, Percentage of sub‐G1 cells 144 h after siRNA treatment, in an analysis of cell cycle progression (Figure 4A). B, Annexin‐V‐positive cells 144 h after siRNA treatment. For the purposes of quantification (A, B), the data are expressed as the means ± SD from at least three independent experiments. P values determined with Student's t test (comparison with control siRNA) are indicated (**P < 0.01; ***P < 0.001). C, Western blot to evaluate caspase activity 144 h after LRP8 depletion. Antibodies recognizing the cleaved forms of caspase 7, caspase 8, and PARP were used. LRP8 depletion was verified with anti‐LRP8 antibodies. Actin was used as a loading control. The images shown are from a single experiment representative of three independent experiments performed
Fig 2: ApoER2 levels increase independently of the intracellular domain of ApoER2.A. Schematic of tagged ApoER2 constructs. Wild-type (WT) murine ApoER2 with both an N-terminal HA tag and a C-terminal myc tag. ΔICD is missing amino acids 731–841 and the myc tag. SP: Signal peptide. B. COS7 cells were co-transfected with either ApoER2 WT or ΔICD and either Fyn or empty vector. Lysates were collected in RIPA buffer and analyzed for HA, Fyn, and Tubulin. *p<0.05, **p<0.01 vs empty vector (Bonferroni’s Multiple Comparisons test). Data are mean +/− SD. N = 5.
Fig 3: ApoER2 is phosphorylated in vivo.A. Cortical (CTX) and hippocampal (HPC) lysates from wild-type mice were collected on postnatal days 1, 5, and 15, homogenized in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Fyn, and Tubulin. B. Levels of total ApoER2 were plotted from Western blots in A. #p<0.05 cortical P1 vs. P15; **p<0.01 hippocampal P1 vs. P5, ***p<0.001 hippocampal P1 vs. P15. (Bonferroni’s Multiple Comparisons Test). Data are mean +/− SD. N = 3. C. For the cortical samples, levels of total ApoER2, phosphorylated ApoER2, and total Fyn were plotted. D. For the cortical samples, a ratio of phosphorylated ApoER2 to total ApoER2 was calculated. *p<0.05 (Bonferroni’s Multiple Comparisons Test). Data are mean +/− SD. N = 5. E. Levels of total Fyn were plotted from Western blots in A. *p<0.05 (Bonferroni’s Multiple Comparisons Test). Data are mean +/− SD. N = 3.
Fig 4: Loss of IDOL impairs dendritic Spine morphogenesis and maturation.(A) Representative images showing immunofluorescence staining of dendrites from Mylipflox/flox hippocampal neurons transduced with either AAV-CamKII-GFP or AAV-CamKII-CRE-GFP. Neurons were transduced at DIV 5, and fixed and stained at DIV 19–20. Spine synapse, white arrow; Shaft synapse, red arrow; Green, GFP; Red, PSD95; Magenta, vGluT1. Scale bars, 10 µm. Quantification of spine density and spine-head width of neurons (including filopodia-like protrusions) is shown on the right (n = 10–12/group). **p<0.01 by Student’s t test. (B) Representative images showing immunofluorescence staining of dendrites from WT hippocampal neurons transduced with either Lenti-Cherry or Lenti-ApoER2. Neurons were transduced at DIV 10, and fixed and stained at DIV 19–20. Both groups were also transduced with a AAV-CamKII-GFP DIV15 to allow visualization of spines. Green, GFP; magenta, vGluT1. Scale bars, 10 µm. Quantification of spine density and spine-head width of neurons from (including filopodia-like protrusions, n = 10–12/group) is shown on the right. **p<0.01 by Student’s t test. (C) Representative images showing immunofluorescence staining of dendrites from Mylipflox/flox hippocampal neurons transduced with either AAV-CamKII-GFP or AAV-CamKII-CRE-GFP at DIV10. Control siRNA (1 µM) or ApoER2 siRNA (1 µM) were then added to the culture medium at DIV16 and applied again after 48 hr. Neurons were fixed and stained at DIV21. Green, GFP; magenta, vGluT1. Scale bars, 10 µm. Quantification of spine-head width of neurons (including filopodia-like protrusions, n = 10–12/ group) is shown on the right. (D) Representative images of primary CA1 apical dendrites (about 150 µM from the soma) in Golgi-impregnated slices from 5-weeks-old WT and IDOL-deficient mice. Scale bars, 5 µm; quantification of dendritic spine density along the dendrites of Golgi-impregnated CA1 hippocampal neurons obtained from WT (n = 9 total neurons, 3 animals) and IDOL-deficient mice (n = 10 total neurons, 3 animals) is shown on the right. Error bars represent SEM. **p<0.01 by Student’s t test. Primary neuron images presented in this figure are from ≥2 independent experiments, 5–8 randomly selected neurons are imaged for each experiment and used for statistical analysis.
Fig 5: IDOL suppresses dendritic spinogenesis through control of ApoER2.(A) mEPSC recordings from WT hippocampal neurons transduced with either AAV-hSyn-GFP, or AAV-hSyn-IDOL (right). Neurons were transduced at DIV 5 and recorded between DIV16-18. Quantification of mEPSC amplitude and frequency (n = 7–11) is shown on the right. Error bars represent SEM. **p<0.01 by Student’s t test. ns, not significant. (B) Representative images showing morphology of dendrites from WT hippocampal neurons incubated with control siRNA (1 µM) or ApoER2 siRNA (1 µM) from DIV5 to DIV16. Green, GFP; red, PSD95; magenta, vGluT1. Scale bars, 10 µm. Quantification of spine density is shown on the right (n = 9–12/group). **p<0.01 by Student’s t test. (C) Representative images showing morphology of dendrites from WT hippocampal neurons transduced with Lenti-Cherry, Lenti-ApoER2 or Lenti-VLDLR, as well as AAV-hSyn-GFP or AAV-hSyn-IDOL-GFP as labeled. Neurons were infected at DIV 5, and fixed and imaged at DIV 10. Scale bars, 10 µm. All data presented in Figure 2 are from ≥2 independent experiments. 5–8 randomly selected neurons are imaged for each experiment and used for statistical analysis. Quantification of spine density is shown on the right. **p<0.01 by Student’s t test.
Supplier Page from Abcam for Anti-ApoER2 antibody [EPR3326]