Journal: International Journal of Molecular Sciences

Article Title: Cadmium Disrupted ER Ca 2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

doi: 10.3390/ijms24065979

Figure Lengend Snippet: Effects of SERCA modulators on ER stress, autophagy, and apoptosis. ( A ) Effect of Cd 2+ , CDN1163, and TG on the expression of ER stress biomarkers BiP and PDI, autophagy biomarker p62, and apoptosis biomarker cleaved caspase-3 (CC-3) were detected by Western blotting. ( B – E ) Quantification of the relative protein levels was performed using the software Image J 1.54c. ( F , G ) Effect of Cd 2+ on the expression of PDI and CC-3 detected by Western blotting and quantification by Image J. NC—negative control. OE: SERCA2 overexpression. ( H ) MTT detection of cell viability in Cd 2+ -treated SERCA2 overexpressed mRTEC cells. Results are presented as mean ± SD ( n = 4 well cells/group). * indicates statistical significance between control and Cd 2+ treatment or between treatments. NS means no significant difference. p < 0.05, using one-way ANOVA followed by Duncan’s multiple range test.

Article Snippet: The staining procedure involved incubation of the cells or tissue sections with 3% normal goat serum in PBST to reduce non-specific staining, followed by overnight incubation at 4 °C with rabbit polyclonal antibodies against SERCA2 (1:50, Cell Signaling Technology, Shanghai, China), p-PLB (1:50, Abcam, Shanghai, China) KIM-1 (1:200, Thermo Fisher Scientific, Shanghai, China) antibodies.

Techniques: Expressing, Biomarker Discovery, Western Blot, Software, Negative Control, Over Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Cadmium Disrupted ER Ca 2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

doi: 10.3390/ijms24065979

Figure Lengend Snippet: Effects of R-467 on Cd 2+ -regulated SERCA2 and p-PLB expression in mice kidney cells and tissues. ( A ) Effects of R-467 on Cd 2+ -regulated SERCA2 and p-PLB expression in mRTEC cells were detected by Western blotting. ( B ) Quantification of the relative protein levels was performed using the software Image J 1.54c. Results are presented as mean ± SD ( n = 4 well cells/group). * Statistical significance between control and Cd 2+ treatment or between treatments. p < 0.05, using one-way ANOVA followed by Duncan’s multiple range test. Effects of R-467 on Cd 2+ -regulated SERCA2 and p-PLB expression in mRTEC cells ( C ) and mice kidney tissues ( D ) were detected by immunohistochemistry. After being treated by Cd 2+ for 24 h, with or without R-467 pretreatment, cells were fixed ( n = 4 well cells/group). After being exposed to Cd 2+ for 28 days, mice kidney tissues were collected ( n = 5 mice/group). The protein levels of SERCA2 and p-PLB in cells and tissues were investigated by immunofluorescence. SERCA2 (green), p-PLB (green), DAPI (blue). The red arrow refers to the glomerulus, and the yellow arrow refers to the renal proximal tubule.

Article Snippet: The staining procedure involved incubation of the cells or tissue sections with 3% normal goat serum in PBST to reduce non-specific staining, followed by overnight incubation at 4 °C with rabbit polyclonal antibodies against SERCA2 (1:50, Cell Signaling Technology, Shanghai, China), p-PLB (1:50, Abcam, Shanghai, China) KIM-1 (1:200, Thermo Fisher Scientific, Shanghai, China) antibodies.

Techniques: Expressing, Western Blot, Software, Control, Immunohistochemistry, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Cadmium Disrupted ER Ca 2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

doi: 10.3390/ijms24065979

Figure Lengend Snippet: Effects of Cd 2+ on SERCA2 stability in mRTEC cells. ( A , B ) After being treated with Cd 2+ (0–10 μM) for 24 h or treated with Cd 2+ (5 μM) or Cd (5 μM) + R-467 (1 μM) for 24 h, mRTEC cells were collected for RNA extraction, and the SERCA2 mRNA levels detected by RT-PCR. ( C ) Effects of MG132 and CQ on Cd 2+ -induced degradation of SERCA2. The mRTEC cells were pretreated with CQ (20 μM) for 1 h, followed by treatment with Cd 2+ (5 μM) for 24 h, or Cd 2+ (5 μM) for 18 h + MG132 (10 μM) for 6 h, and then the proteins were extracted for Western blotting analysis. ( E ) Effects of Cd 2+ on half-life of SERCA2 protein. Cells were treated with Cycloheximide (CHX) (10 μg/mL) for 3 and 6 h or pretreated with Cd 2+ (5 μM) for 24 h, and the protein was extracted for Western blotting analysis. ( D , F ) Quantification of the relative protein levels of SERCA2 was performed using the software Image J 1.54c. * indicates statistical significance between control and Cd 2+ treatment or between treatments. NS: no significant difference. ( n = 4 well cells/group) p < 0.05, using one-way ANOVA followed by Duncan’s multiple range test ( A , B , D ) and Student’s t -test ( F ).

Article Snippet: The staining procedure involved incubation of the cells or tissue sections with 3% normal goat serum in PBST to reduce non-specific staining, followed by overnight incubation at 4 °C with rabbit polyclonal antibodies against SERCA2 (1:50, Cell Signaling Technology, Shanghai, China), p-PLB (1:50, Abcam, Shanghai, China) KIM-1 (1:200, Thermo Fisher Scientific, Shanghai, China) antibodies.

Techniques: RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Control