Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Impact of low level of Calr in the Calr +/− mouse kidney on the expression of ER stress markers and EF-hand Ca2 + -binding proteins. ( A ): Illustration of the experimental design for immunohistology and Western blot. ( B , C ): Western blot analyses and immunohistostaining of the ER stress markers Grp78, Erp57, Canx, Chop, and p-eIF-2A showed no significant difference between the Calr +/+ and Calr +/− mouse kidneys; Actb was used as loading control. Below: Bar diagram represents the quantification of the Western blot results shown in ( B ) ( n = 4. *: p < 0.05). ( D , E ): Western blot analyses and immunohistostaining of some ER-hand Ca 2+ -binding proteins Pvalb, Calb1, Calm1, and S100A4 showed significant up-regulation in Calr +/− kidney compared to Calr +/+ . Below: Bar diagram represents the quantification of the Western blot results shown in ( D ) ( n = 4. *: p < 0.05, **: p < 0.01, ***: p < 0.001). The immunohistochemical staining of Grp78, Pvalb, Calm1, and Canx is shown in 40 wk old Calr +/− and Calr +/+ mouse kidney sections.

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Expressing, Binding Assay, Western Blot, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Calr expression underlays the control of calcium handling hormones and is important for Ca 2+ homeostasis. ( A ): Illustration of the experimental design for calcium measurement and immunoblotting. ( B ): MDCK cells were treated with PTH or VD3, or both, and Calr expression was monitored using Western blot analysis. Compared to other ER stress markers, the expression of Calr was significantly down-regulated upon PTH or VD3 treatment. ( C ): Primary cell isolated from mouse kidneys (Calr +/+ and Calr +/− ) were treated with PTH and the Calr expression was monitored using Western blot. Calr was significantly down-regulated upon PTH treatment. ( D ): The ratio of Fura-2 fluorescence emission intensity in response to 340 nm and 380 nm excitation (340/380) is proportional to intracellular (Ca 2+ ). Fura-2 340/380 emission ratios are plotted against measurement time. ( I ): Average of Fura-2 340/380 emission ratios of Calr +/+ kidney primary cells ( n = 20) and Calr +/− kidney primary cells are plotted against time. Fura-2 340/380 emission ratios of three representative tubular kidney primary cells from Calr +/+ or Calr +/− mice treated with ATP (5 µM) ( II ), Thapsigargin (1 µM) ( III ), or the ionophore A23187 (1 µM) ( IV ). ( E ): Categorization of differentially regulated proteins (2D gel analysis) was achieved by correlating GO identification numbers corresponding to cellular component and biological process with the regulated proteins. Values in figures presented the ratio distribution of proteins found in that respective category, (left) identified proteins categorized based upon their cellular component, (right) identified mitochondrial proteins categorized based upon their functional category. ( F ): Quantification of the proteomics data showed an up-regulation of protein sensing and transporting the calcium into the kidney cells. ( G ): Immunofluorescence staining of kidney tissue from Calr +/+ and Calr +/− with antibodies against Trpv5, Calb1, Ncx1, and Casr. ( H ): Expression alteration of proteins involved in the regulation of calcium, Western blot analyses confirming the up-regulation of Ncx1 and the down-regulation of Trpv5. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Expressing, Western Blot, Isolation, Fluorescence, Two-Dimensional Gel Electrophoresis, Functional Assay, Immunofluorescence, Staining

Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Down-regulation of mitochondrial proteins in Calr +/− mouse kidneys. ( A ): Western blot analysis of mitochondrial proteins: Vdac1, Cyc, and Cat from lysate of enriched mitochondria from Calr +/+ and Calr +/− kidney tissues. Quantification of protein expression is shown in bar diagram. ( B ): Immunofluorescence staining of Vdac1 and Cyc shows a clear decrease in the protein expression; micrograph shows the staining of Vdac1 using immunogold. ( C ): Quantification of proteomics data confirming the down-regulation of the investigated mitochondrial proteins. ( D ): Immunofluorescence staining of Cat coupled with ubiquitin shows enhanced expression in the glomerulus and nuclear translocation in the tubule cells of Calr +/− kidneys. ( E ): Quantification of the Cat expression using normalized spectral accounts, the expression of Cat is significantly down-regulated in Calr +/− mouse kidney. ( F ):Quantification of cytochrome c oxidase activity. Intact mitochondria were isolated for the quantification of cytochrome c oxidase activity. Comparison of the respiratory activity between Calr +/− and Calr +/+ kidneys revealed about a 50% decrease in mitochondrial activity in Calr +/− kidney cells. ** p <0.01, *** p <0.001).

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Translocation Assay, Activity Assay, Isolation

Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Severe mitochondrial damage and autophagy in Calr +/− mouse kidneys. ( A ): Representative electron micrographs for ultrastructural morphology of mitochondria from Calr +/− mouse kidney—( a , b ): distal convoluted tubule cells swelling mitochondria enclosed in membrane structures, some of the mitochondria are in advanced stages of autophagy; ( c – e ): damaged mitochondrial enclosed in multi-membrane structure undergoing autophagy, also shown are advanced stages where the mitochondria are almost completely eliminated; ( f ): a podocyte with damaged vacuolated mitochondria highlighted with red asterisks in Calr +/− mouse kidneys. ( B ): Western blot analysis of protein extract from Calr +/+ and Calr +/− kidney tissue showed down-regulation of Bcl-2 and up-regulation of Becn-1, indicating an activation of the autophagy. ( C ): Immunofluorescence staining of LC3 confirmed the initiation and formation of autophagosomes. ( D ): Western blot analysis with antibody against LC3 confirmed the shift toward LC3-II in Calr +/− kidney, as evidenced by the ratio LC3-II/LC3-I calculation. ( E ): Proteomic analysis revealed an up-regulation of the Atg3, an important player in autophagy in Calr +/− kidneys. Results are given as means ± SD of the relative intensity in the case of Western blot analysis, or of the normalized spectral accounts in the case of proteomic data **: p <0.01, ***: p <0.001).

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Western Blot, Activation Assay, Immunofluorescence, Staining

Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Chronic increased cytosolic calcium level results in activation of PKC and NF-κB pathway in Calr +/− kidney. ( A ): Activity assay demonstrated significant activation of PKC in Calr +/− mouse kidney because of alteration in cytosolic calcium concentration. ( B ): Western blot analysis of protein extracts from Calr +/+ and Calr +/− kidneys showed an activation of NF-κB pathway as evidenced by up-regulation of p65 in heterozygous kidney and down-regulation of the pathway inhibitor IkB. ( C , D ): The nuclear translocation of p65 confirmed the activation of NF-κB pathway, the nuclear protein Lamin A/C was used as control. ( E ): Western blot analysis of iNos was performed for kidney lysates of Calr +/− and Calr +/+ mice. Actb was used as loading control. Bar diagram representing the quantification of the MM and DM of iNos. Western blot results are shown in ( B ) ( n = 4 *, p < 0.05, **: p < 0.01, ***: p < 0.001). MM: monomer, DM: dimer. ( F ): Immunohistochemical staining of iNos shows no significant change in expression pattern of protein in Calr +/− compared to Calr +/+ . ( G ): Proteomic data showed an up-regulation of IL6st the interleukin-6 receptor, revealing an increased inflammation upon NF-κB pathway activation in Calr +/− mouse kidney.

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Activation Assay, Activity Assay, Concentration Assay, Western Blot, Translocation Assay, Immunohistochemical staining, Staining, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway

doi: 10.2147/DMSO.S380053

Figure Lengend Snippet: FST overexpression decreased lipid accumulation in FFA-treated LO2 cells. ( A and B ) The efficiency of FST overexpression in LO2 cells was demonstrated by RT-qPCR and Western blotting. ( C and D ) Oil red O staining and analysis result of control cells (EGFP) and FST-overexpressing LO2 cells treated with 1.0 mM FFA for 24 h (400×, scale bar=50 µm). ( E – G ) The protein levels and analysis results of ACC1, FASN, SREBP1, ChREBP, Akt, Akt-Thr308, Akt-Ser473, p-mTOR, and mTOR in the control cells and FST-overexpressing LO2 cells treated with 1 mM FFA for 24h. β-actin was used as a loading control.

Article Snippet: The primary antibodies were β-actin (#AC026) and ChREBP (#A7360) from Abclonal Technology; SREBP1 (#ab28481) from Abcam Technology; mTOR (#T55306) and p-mTOR (#T55996) from Abmart Technology; ACC1 (#4190), FASN (#3180), Akt-Thr308 (#13038), Akt-Ser473 (#4060), and Akt (#9272) from Cell Signaling Technology; and FST (#AF669) from R&D Technology.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway

doi: 10.2147/DMSO.S380053

Figure Lengend Snippet: FST knockdown aggravated lipid accumulation in LO2 cells and this effect was inhibited by rapamycin. ( A and B ) The efficiency of FST knockdown in LO2 cells was demonstrated by RT-qPCR and Western blotting. ( C and D ) Oil Red O staining and analysis results of FFA-treated negative control shRNA (NCsh) cells, FFA-treated FST-knockdown (FSTsh) cells, and FFA- and rapamycin-treated FSTsh (FSTsh+RAPA) cells (400×, scale bar: 50 µm). ( E and F ) The protein levels and analysis results of Akt, Akt-Thr308, Akt-Ser473, p-mTOR, and mTOR in FFA-treated NCsh cells and FFA-treated FSTsh cells. ( G and H ) The protein levels and analysis results of ACC1, FASN, SREBP1, ChREBP, and mTOR in NCsh, FSTsh, FSTsh+RAPA cells. β-actin was used as a loading control.

Article Snippet: The primary antibodies were β-actin (#AC026) and ChREBP (#A7360) from Abclonal Technology; SREBP1 (#ab28481) from Abcam Technology; mTOR (#T55306) and p-mTOR (#T55996) from Abmart Technology; ACC1 (#4190), FASN (#3180), Akt-Thr308 (#13038), Akt-Ser473 (#4060), and Akt (#9272) from Cell Signaling Technology; and FST (#AF669) from R&D Technology.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Negative Control, shRNA

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway

doi: 10.2147/DMSO.S380053

Figure Lengend Snippet: FST overexpression inhibited lipid synthesis and Akt/mTOR pathway in HFD mice. ( A and B ) The protein expression and analysis results of ACC1, FASN, SREBP1, ChREBP, Akt, Akt-Thr308, Akt-Ser473, p-mTOR, and mTOR in the control (treated with an AAV vector encoding GFP) and FST-overexpressing (treated with an AAV vector encoding FST288 and FST315) HFD mice. β-actin was used as a loading control. ( C and D ) GTT and ITT results in the control and FST-overexpressing HFD mice. ( E and F ) AUC for GTT and ITT. ( G ) Immunohistochemical staining of hepatic FST in the control and FST-overexpressing HFD mice (400×, scale bar: 50 µm).

Article Snippet: The primary antibodies were β-actin (#AC026) and ChREBP (#A7360) from Abclonal Technology; SREBP1 (#ab28481) from Abcam Technology; mTOR (#T55306) and p-mTOR (#T55996) from Abmart Technology; ACC1 (#4190), FASN (#3180), Akt-Thr308 (#13038), Akt-Ser473 (#4060), and Akt (#9272) from Cell Signaling Technology; and FST (#AF669) from R&D Technology.

Techniques: Over Expression, Expressing, Plasmid Preparation, Immunohistochemical staining, Staining