Journal: Pflugers Archiv

Article Title: Soluble αKlotho downregulates Orai1-mediated store-operated Ca 2+ entry via PI3K-dependent signaling

doi: 10.1007/s00424-020-02510-1

Figure Lengend Snippet: A soluble form of αKlotho downregulates SOCE without affecting Orai1/STIM1. a Representative trace showing the effect of the Klotho family on SOCE. Flag-Orai1 and YFP-STIM1 co-transfected with Klotho isoforms: αKlotho (KLA), βKlotho (KLB), or γKlotho (KLG) in HEK293FT cells. Empty vector (pEF1 vector) used as transfection control. b Quantification of peak SOCE values is expressed as mean ± SEM ( n = 55–173 each group). c Immunoblotting showing transfection of Klotho isoforms and Flag-tagged Orai1 and YFP-tagged STIM1. d Quantitative real-time PCR for relative mRNA expression of Orais and STIMs in HEK293FT cells. e Representative trace of endogenous SOCE in HEK293FT cells transiently expressing full-length (KLFL) and secreted (KLΔTM) form of αKlotho. Empty vector (pEF1 vector) was used as a transfection control (Vector). f Summary of the SOCE in panel e ( n = 123–186 each group). g Effect of αKlotho (KLFL and KLΔTM) on endogenous Orai1 and STIM1 protein expression in HEK293FT cells. **Denotes p < 0.01. Data were analyzed by one-way ANOVA ( b left panel in d and f ) and t test (right panel in d)

Article Snippet: Primary antibodies were used following as: Orai1 (HPA016583, ATLAS antibodies, Stockholm, Sweden), STIM1 (11565-1-AP, ProteinTech Group Inc., Chicago, IL, USA), GFP (ab137687, Abcam, Cambridge, UK), αKlotho (clone KM2076, KAL-KO603, Cosmo Bio Co., Ltd., Tokyo, Japan), βKlotho (GTX45558, Gene Tex, Inc., Irvine, CA), γKlotho (AF5984-SP, R&D Systems, Minneapolis, MN, USA), Flag-HRP (A8592, Sigma-Aldrich, St. Louis, MO, USA), β-actin (ab6276, abcam, Cambridge, UK), p-Akt Ser407 (#9271), p-Akt Thr308 (#2965), and Akt (#9272) were provided from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Sequences for all the siRNAs

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Control

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 affected the PAN-induced podocyte detachment. (a) The detachment was determined in the podocytes at different doses (50,100,200,250 and 500 ng/ml) of rm-Angptl3 with or without PAN (50 μg/ml, 10 hrs) pretreatment for 24 hrs, it shown that rm-Angptl3 without PAN could not change the attached cells in normal podocytes significantly, while, in podocytes pretreated with PAN, rm-Angptl3 reduced the attached cells markedly, and the more the doses (100-500 ng/ml), the less the attached cells. (b) The rates of attached cells podocytes treated with rm-Angptl3 for different times after PAN pretreatment; the cells decreased from 9 h to 48 h, and the rate of 24 h was the lowest. (c) ELISA results showing weaker secretion of Angptl3 in the medium of Angptl3 siRNA group (lowered by 69.50%); (d) Western blot confirmed that Angptl3 was knocked down by siRNA successfully (reduced by 86.89 ± 3.58%). (e) There was no statistical difference between cell number in the Wt group and control siRNA group with or without PAN pretreatment. Cell number in the control siRNA group and Angptl3 siRNA group were almost the same, while, after PAN pretreatment, cell number in the Angptl3 siRNA group is more than in the control siRNA group. (Wt: wide type; Control siRNA: podocytes transfected with control siRNA; Angptl3 siRNA: podocytes transfected with Angptl3 siRNA. * P <0.05, ** P <0.01, *** P <0.0001) Angptl3, angiopoietin-like3; ELISA, enzyme-linked immunosorbent assay; PAN, puromycin aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Transfection, Small Interfering RNA

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 affected apoptosis in PAN-induced podocyte injury model. (a) Flow cytometry analysis of the rate of apoptosis in the cultured podocytes with different treatments. No significant differences in the number of apoptotic cells were observed among control, rm-Angptl3, control siRNA and Angptl3 siRNA groups. After PAN pretreatment, the rate of apoptosis was significantly increased in the rm-Angptl3 group compared to the control group (34.80±7.9 vs. 1.89±0.92 3%, P <0.0001; n=6). And in comparison to the control siRNA group, apoptosis rate in the podocytes treated with Angptl3 siRNA was a little lower (9.29±1.27vs. 17.42±2.61%, P <0.05; n=6). (b, c) This finding was confirmed using TUNEL assay for apoptosis in cultured podocytes. In PAN-induced podocyte injury model, TUNEL positive cells in rm-Angptl3-treating group are significantly more than that without rm-Angptl3 treating (56±9 vs. 8±10 %, P <0.0001; n=6), and TUNEL positive cells in the Angptl3 siRNA group are significantly less than that in control siRNA group (32±4 vs. 54±9 %, P <0.05; n=6) (magnification ×400). (Control: podocytes treated without rm-ANGPTL3; Rm-ANGPTL3: podocytes treated with rm-ANGPTL3. *P<0.05, ***P<0.0001). Angptl3, angiopoietin-like3; PAN, puromycin aminonucleoside; siRNA, small interfering RNA; TUNEL. deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Flow Cytometry, Cell Culture, Control, Comparison, TUNEL Assay, Small Interfering RNA, End Labeling

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 influenced the F-actin rearrangement in podocytes. (a) Phalloidin fluorescence labelling of F-actin showed podocyte stress fibers. Confocal microscopy showed no difference in the F-actin staining pattern between control group and Angptl3 siRNA group. However, F-actin rearrangement in Angptl3 group, characterized by the formation of lamellipodia (arrowhead) and increase in cell spikes (arrow). In PAN induced podocyte injury model,cells became retracted and F-actin seemed partial disrupted,moreover, F-actin almost completely disrupted in Angptl3 protein group. However, disruption did not occur in Angptl3 knockdown group. (b) The percentage of stress fibers also showed that Angptl3 protein could significantly aggravate the effect of PAN on decreaseing podocyte stress fibers while Angptl3 siRNA could prevent PAN from decreasing podocyte stress fibers. (c) Quantitative analysis showed the MFI of F-actin in different groups under confocal microscopy. After PAN treatment, MFI value in the Angptl3 siRNA group is higher than in the control siRNA group. (Scale bar, 5 μm * P <0.05, ** P <0.01. n=100). Angptl3, angiopoietin-like3; MFI, mean fluorescence intensity; PAN, puromycin-aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Fluorescence, Confocal Microscopy, Staining, Control, Disruption, Knockdown, Small Interfering RNA

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 orchestrated integrin α3β1, ILK and p53, rather than caspase 3 in podocytes on treatment with PAN. (a) Western blots were performed on the expression of integrin α3, total integrin β1, phospho-integrinβ1, and ILK in podocytes with different treatment, it was showed that, there was no significant difference of in the observed molecular expression in podocytes without PAN pretreatment, while, after treatment with PAN, expression of integrin α3 and total integrin β1 became lower (ILK and phospho- integrin β1, higher) in the rm-Angptl3 group than that in the control group, and Angptl3 siRNA group showed higher expression of integrin α3, total integrin β1 (ILK, lower) than in control siRNA group, while, phospho- integrinβ1 in the two group had no statistical difference. (b, c, d, e) Relative levels of integrin α3, total integrin β1, phospho- integrinβ1 and ILK. (f, g) Western blots suggesting that there was rarely no activated caspase 3 in any observed groups; with PAN pretreatment, p53 protein level was markedly increased in rm-Angptl3 group with comparison to the control group, while, p53 protein level in Angptl3 siRNA group was a little lower than that in the control siRNA group, no p53 protein was detected in any other groups. (*P<0.05, **P<0.01, ***P<0.0001 n=6). Angptl3, angiopoietin-like3; ILK, integrin-linked kinase; PAN, puromycin aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Western Blot, Expressing, Control, Comparison, Small Interfering RNA