File S1 - Pemt Deficiency Ameliorates Endoplasmic Reticulum Stress in Diabetic Nephropathy

Figure S1. A schematic diagram of the strategy used to disrupt the mouse Pemt gene. a. Schematic drawings of the Pemt targeting vector. b. The targeted recombination in ES cells (lines 135, 144, 152 and 162) was confirmed by the Southern blot analyses of genomic DNA digested with EcoRI, and the expected sizes of wild-type and Pemt gene-targeted bands using a 5′-probe, 3′-probe and NEO-probe are shown, respectively. Figure S2. Oxidant fluorescence microtopography using hydroethidine in streptozotocin (STZ)-treated diabetic Pemt+/+ and Pemt−/− C57BL/6JJcl mice. The Pemt+/+ and Pemt−/− mice were treated with citrate buffer (CON) or STZ. a–d. The Pemt+/+ (STZ) mice demonstrated a prominent increase in hydroethidine fluorescence, and Pemt deficiency markedly reduced the renal cell-derived superoxide. Bars = 300 μm (a–d). Figure S3. The intraglomerular macrophage infiltration in streptozotocin (STZ)-treated diabetic Pemt+/+ and Pemt−/− C57BL/6JJcl mice. Pemt+/+ and Pemt−/− mice were treated with citrate buffer or STZ. a–d. Immunoperoxidase staining for F4/80, e. The number of F4/80 positive cells/glomerulus. The number of glomerular F4/80-positive cells was significantly reduced in Pemt−/− (STZ) mice compared with Pemt+/+ (STZ) mice. Bars = 20 μm (a–d). ##PPemt+/+ (CON). **PPemt+/+ (STZ). Figure S4. The interstitial macrophage infiltration in streptozotocin (STZ)-treated diabetic Pemt+/+ and Pemt−/− C57BL/6JJcl mice. Pemt+/+ and Pemt−/− mice were treated with citrate buffer or STZ. a–d. Immunoperoxidase staining for F4/80, e. The number of F4/80 positive cells in the interstitium per mm2. The number of interstitial F4/80-positive cells was significantly reduced in Pemt−/− (STZ) mice compared with Pemt+/+ (STZ) mice. Bars = 50 μm (a–d). ##PPemt+/+ (CON). **PPemt+/+ (STZ). Figure S5. The results of the densitometric analyses of the Western blots of renal cortex tissues from Pemt+/+ and Pemt−/− mice treated with citrate buffer or streptozotocin (STZ), which appeared in Figure 6. ##PPemt+/+ (CON). **PPemt+/+ (STZ). Figure S6. Endoplasmic reticulum (ER) stress-related markers in mProx24 cells treated with MISSION shRNA lentivirus transduction particles for Pemt (shRNA-Pemt) and Non-Target shRNA control lentivirus transduction particles (shRNA-CON). The mProx24 renal tubule cells were treated with normal glucose (NG), high glucose (HG), an osmotic control using mannitol (Mn), DMSO, tunicamycin or thapsigargin. **PCip1, p27Kip1 and p-Akt in mProx24 cells treated with MISSION shRNA lentivirus transduction particles for Pemt (shRNA-Pemt) and Non-Target shRNA control lentivirus transduction particles (shRNA-CON). The mProx24 cells were treated with normal glucose (NG), high glucose (HG), an osmotic control using mannitol (Mn), DMSO, tunicamycin or thapsigargin. The treatments with tunicamycin and thapsigargin upregulated cyclin D1 and downregulated p27Kip1 and p-Akt. shRNA-Pemt led to prominent recovery and increased the expression of p-Akt. However, the anti-proliferative activities of tunicamycin and thapsigargin were not reversed by the treatment with shRNA-Pemt, as revealed by the CellTiter96 Aqueous One Solution Cell Proliferation Assay. Figure S8. The results of the densitometric analyses of the Western blot analyses of the levels of cyclin D1, p21Cip1, p27Kip1 and p-Akt in mProx24 cells treated with MISSION shRNA lentivirus transduction particles for Pemt (shRNA-Pemt) and Non-Target shRNA control lentivirus transduction particles (shRNA-CON). **PPemt (shRNA-Pemt) and Non-Target shRNA control lentivirus transduction particles (shRNA-CON). The treatments with tunicamycin and thapsigargin increased the levels of cleaved caspases 3 and 7, while the treatment with shRNA-Pemt reduces the levels of cleaved caspases 3 and 7. **P (PDF)