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

Journal: PLoS ONE

Article Title: SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

doi: 10.1371/journal.pone.0027451

Figure Lengend Snippet: A) Primary airway epithelial cells were cultured on collagen-coated 24-well plates. Replication-deficient lentivirus each carrying 5 different SERCA2 target shRNA (pLKO.1-CMV-tGFP-ATP2A2) (SERCA2A-1-5) were transduced at different MOI according to manufacturer's instructions and SERCA2 knock down was tested using real time RTPCR. The data shown are mean±SEM (n = 3) *p<0.05, significant difference between cells treated with SERCA2 shRNA and control. B) Primary airway epithelial cells were cultured on collagen-coated 6-well plates and transduced with SERCA2 shRNA (pLKO.1-CMV-tGFP-ATP2A2) or pLKO.1-CMVtGFP-TurboGFP control. Forty-eight hour post transduction cell lysates were prepared and SERCA2 Western blot was performed. C) Airway epithelial cell phenotype with and GFP expression by control and SERCA2 shRNA transduced cells. Cultured cells retained the morphology of airway epithelial cells (Brightfield light microscopy of untransduced, A & B, transduced C & E) and expressed GFP (Fluorescence microscopy of transduced D & F, matching area to the Brightfield in C & E). D) Flow cytometry of control, SERCA2 shRNA (pLKO.1-CMV-tGFP-ATP2A2) or pLKO.1-CMVtGFP-TurboGFP control cells with 7-AAD a cell death marker.

Article Snippet: Western blots were performed as previously described in detail and the membranes were probed with rabbit polyclonal antibodies against SERCA2 (Affinity Bioreagents, Golden, CO) at 1∶1,000 dilution for each, overnight at 4°C.

Techniques: Cell Culture, shRNA, Reverse Transcription Polymerase Chain Reaction, Transduction, Western Blot, Expressing, Light Microscopy, Fluorescence, Microscopy, Flow Cytometry, Marker

Journal: PLoS ONE

Article Title: SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

doi: 10.1371/journal.pone.0027451

Figure Lengend Snippet: Cells were cultured on collagen-coated snapwells at air-liquid interface (ALI) or on collagen-coated 6-well plates. (A) Primary airway epithelial cells were cultured at ALI and exposed to 500 or 1000 ppb ozone. At the end of exposure (4 h), lysates were prepared and SERCA2 protein was analyzed using Western blot. A representative blot from cells of one donor is shown. (B) Cells transduced with lentiviral GFP control or lentiviral GFP-SERCA2 shRNA were cultured on collagen-coated snapwells at air-liquid interface (ALI) and exposed to 0 (white column) or 200 (black column) ppb ozone as described in legend to . Apical media was collected and analyzed. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb control and # indicates significant difference from 200 ppb exposed lentiviral GFP transduced cells, and $ indicates significant difference from 0 ppb exposed lentiviral GFP-SERCA2 shRNA transduced cells p<0.05. C) Primary airway epithelial cells cultured on 6-well plates were transduced with Ad.GFP or Ad.SERCA2. Exposure to ozone, 0 (white column) or 200 (black column) ppb for 18 h was carried out 48 h post transduction. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb and # indicates significant difference from 200 ppb ozone-exposed Ad.GFP transduced cells p<0.05.

Article Snippet: Western blots were performed as previously described in detail and the membranes were probed with rabbit polyclonal antibodies against SERCA2 (Affinity Bioreagents, Golden, CO) at 1∶1,000 dilution for each, overnight at 4°C.

Techniques: Cell Culture, Western Blot, Transduction, shRNA

Journal: PLoS ONE

Article Title: SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

doi: 10.1371/journal.pone.0027451

Figure Lengend Snippet: NF-κB luciferase reporter-expressing 16HBEo- cells were cultured on collagen-coated 6-well plates and transfected with either control siRNA or SERCA2 siRNA. Forty-eight hour post transfection cells were treated with ‘Cytomix’ containing TNFα (20 ng/ml), IL-1β (10 ng/ml), IFNγ (10 ng/ml) and LPS (50 ng/ml) with or without thapsigargin (thaps; 2 µM)). (A) Cell lysates were prepared 24 h after treatment and luciferase activity was measured as described. The data shown are mean±SEM (n = 6). The image is representative of four independent experiments. * Indicates significant difference from untreated siControl transfected cells, # indicates significant difference from untreated siSERCA2 transfected cells, $ indicates significant difference from cytomix-treated siControl transfected cells, and $$ indicates significant difference from cytomix+thaps-treated siControl-transfected cells p<0.05. (B) Cells were cultured and treated as described above and supernatant media was collected and analyzed for IL-8. The data shown are mean±SEM (n = 3). The image is representative of two independent experiments. * Indicates significant difference from untreated siControl transfected cells, # indicates significant difference from untreated siSERCA2 transfected cells, $ indicates significant difference from cytomix-treated siControl transfected cells and $$ indicates significant difference from cytomix+thaps-treated siControl-transfected cells p<0.05.

Article Snippet: Western blots were performed as previously described in detail and the membranes were probed with rabbit polyclonal antibodies against SERCA2 (Affinity Bioreagents, Golden, CO) at 1∶1,000 dilution for each, overnight at 4°C.

Techniques: Luciferase, Expressing, Cell Culture, Transfection, Activity Assay

Journal: PLoS ONE

Article Title: SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

doi: 10.1371/journal.pone.0027451

Figure Lengend Snippet: NF-κB luciferase reporter-expressing 16HBEo- cells were cultured on collagen-coated 6-well plates and transfected with either control siRNA or SERCA2 siRNA. Forty-eight hour post transfection cells were exposed to ozone (0 or 500 ppb). (A) Cell lysates were prepared after treatment and luciferase activity was measured as described. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb siControl transfected cells, # indicates significant difference from 500 ppb siControl transfected cells p<0.05. (B) Cells were cultured and treated as described above and supernatant media was collected and analyzed for IL-8. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb siControl transfected cells and # indicates significant difference from 500 ppb siControl transfected cells.

Article Snippet: Western blots were performed as previously described in detail and the membranes were probed with rabbit polyclonal antibodies against SERCA2 (Affinity Bioreagents, Golden, CO) at 1∶1,000 dilution for each, overnight at 4°C.

Techniques: Luciferase, Expressing, Cell Culture, Transfection, Activity Assay

Journal: PLoS ONE

Article Title: SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

doi: 10.1371/journal.pone.0027451

Figure Lengend Snippet: 16HBEo- cell lines stably transfected with sense (16HBE-S, non-CF) and antisense (16HBE-AS, CF) CFTR oligonucleotide were cultured on 6-well plates as described in the . On the 2 nd day of plating they were transduced with Ad.GFP or Ad. SERCA2 as described in the . Cells were exposed to cytomix after 24 h incubation. Supernatant media was collected for IL-8 assay (A) and nuclear lysates were prepared for total p65 assay by ELISA. The data shown are mean±SEM (n = 6). * Indicates significant difference from untreated control cells, # indicates significant difference from cytomix treated non-CF and ! indicates significant difference from cytomix treated non-CF and CF cells p<0.05.

Article Snippet: Western blots were performed as previously described in detail and the membranes were probed with rabbit polyclonal antibodies against SERCA2 (Affinity Bioreagents, Golden, CO) at 1∶1,000 dilution for each, overnight at 4°C.

Techniques: Stable Transfection, Transfection, Cell Culture, Transduction, Incubation, Enzyme-linked Immunosorbent Assay