Journal: Oxidative Medicine and Cellular Longevity

Article Title: Oxalate Activates Autophagy to Induce Ferroptosis of Renal Tubular Epithelial Cells and Participates in the Formation of Kidney Stones

doi: 10.1155/2021/6630343

Figure Lengend Snippet: Oxalate induced ferroptosis in HK-2 cells. The concentration of oxalate was 2 mmol/L, and the intervention time was 24 hours. We compared the Fe 2+ content of the NC group and the Ox group after 24 hours of intervention (a). The mitochondrial damage of the two groups under a 10 K field of a projection electron microscope (b). The fluorescence intensity of the two groups under a 200× field of the automatic microscope; the stronger the fluorescence intensity, the higher the level of GPX4 (c). The bands and histograms of western blotting represent the relative levels of ACSL4, TFR1, FTL, GPX4, and xCT proteins in the two groups relative to GAPDH (d). Data are presented as the means ± SEM from three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the NC group.

Article Snippet: After transfer and blocking, the proteins on the membrane were incubated with primary antibodies recognizing GPX4 (14432-1-AP, 1 : 1000, Proteintech), acyl-CoA synthetase long-chain family member 4 (ACSL4) (22401-1-AP, 1 : 1000, Proteintech), solute carrier family 7 member 11 (xCT) (26864-1-AP, 1 : 1000, Proteintech), transferrin receptor (TFR1/CD71) (10084-2-AP, 1 : 1000, Proteintech), Ferritin Light Chain (FTL) (10727-1-AP, 1 : 1000, Proteintech), BECN1 (11306-1-AP, 1 : 2000, Proteintech), microtubule-associated protein 1 light chain 3 alpha (LC3) (14600-1-AP, 1 : 2000, Proteintech), NCOA4 (DF4255, 1 : 1000, Affbiotech, Jiangsu, China), sequestosome 1 (P62) (18420-1-AP, 1 : 2000, Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1 : 50000, Proteintech) at 4°C for 12 hours.

Techniques: Concentration Assay, Microscopy, Fluorescence, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Oxalate Activates Autophagy to Induce Ferroptosis of Renal Tubular Epithelial Cells and Participates in the Formation of Kidney Stones

doi: 10.1155/2021/6630343

Figure Lengend Snippet: Knockdown of the expression of BECN1 in HK-2 cells can reduce the ferroptosis of oxalate-induced cells. The concentration of oxalate was 2 mmol/L, and the intervention time was 24 hours. Among the four groups of cells, we compared the levels of BECN1 protein relative to GADPH levels (a), the MDA content (b), the GSH content (c), the Fe 2+ content (d), the ROS levels (e), the mitochondrial membrane potential (g), the GPX4 protein levels (h), the BECN1 and GPX4 mRNA levels (relative to GADPH expression) (f), and the levels of P62, LC3 II, ACSL4, TFR1, TFH1, GPX4, and xCT proteins relative to GADPH levels (i). The fluorescence images were all taken under a 200× field of view under an automatic microscope. Data are presented as the means ± SEM from three independent experiments. # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 versus the ctrl Ox group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the ctrl NC group. ns: not significant.

Article Snippet: After transfer and blocking, the proteins on the membrane were incubated with primary antibodies recognizing GPX4 (14432-1-AP, 1 : 1000, Proteintech), acyl-CoA synthetase long-chain family member 4 (ACSL4) (22401-1-AP, 1 : 1000, Proteintech), solute carrier family 7 member 11 (xCT) (26864-1-AP, 1 : 1000, Proteintech), transferrin receptor (TFR1/CD71) (10084-2-AP, 1 : 1000, Proteintech), Ferritin Light Chain (FTL) (10727-1-AP, 1 : 1000, Proteintech), BECN1 (11306-1-AP, 1 : 2000, Proteintech), microtubule-associated protein 1 light chain 3 alpha (LC3) (14600-1-AP, 1 : 2000, Proteintech), NCOA4 (DF4255, 1 : 1000, Affbiotech, Jiangsu, China), sequestosome 1 (P62) (18420-1-AP, 1 : 2000, Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1 : 50000, Proteintech) at 4°C for 12 hours.

Techniques: Expressing, Concentration Assay, Fluorescence, Microscopy

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Oxalate Activates Autophagy to Induce Ferroptosis of Renal Tubular Epithelial Cells and Participates in the Formation of Kidney Stones

doi: 10.1155/2021/6630343

Figure Lengend Snippet: Overexpression of BECN1 in HK-2 cells can exacerbate the ferroptosis of cells induced by oxalate. The concentration of oxalate was 2 mmol/L, and the intervention time was 24 hours. Among the four groups of cells, we compared the levels of BECN1 protein relative to GADPH levels (a), the MDA content (b), the GSH content (c), the Fe 2+ content (d), the ROS levels (e), the mitochondrial membrane potential (g), GPX4 protein level of the ctrl Ox and Ox+BECN1 groups (h), and the expression of BECN1 and GPX4 mRNA in the four groups of cells relative to that of GAPDH (f). We compared the levels of P62, LC3 II, ACSL4, TFR1, TFH1, GPX4, and xCT proteins in the four groups of cells relative to the GAPDH levels (i). The fluorescence images were all taken under a 200× field of view under an automatic microscope. Data are presented as the means ± SEM from three independent experiments. # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 versus the ctrl Ox group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the ctrl NC group. ns: not significant.

Article Snippet: After transfer and blocking, the proteins on the membrane were incubated with primary antibodies recognizing GPX4 (14432-1-AP, 1 : 1000, Proteintech), acyl-CoA synthetase long-chain family member 4 (ACSL4) (22401-1-AP, 1 : 1000, Proteintech), solute carrier family 7 member 11 (xCT) (26864-1-AP, 1 : 1000, Proteintech), transferrin receptor (TFR1/CD71) (10084-2-AP, 1 : 1000, Proteintech), Ferritin Light Chain (FTL) (10727-1-AP, 1 : 1000, Proteintech), BECN1 (11306-1-AP, 1 : 2000, Proteintech), microtubule-associated protein 1 light chain 3 alpha (LC3) (14600-1-AP, 1 : 2000, Proteintech), NCOA4 (DF4255, 1 : 1000, Affbiotech, Jiangsu, China), sequestosome 1 (P62) (18420-1-AP, 1 : 2000, Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1 : 50000, Proteintech) at 4°C for 12 hours.

Techniques: Over Expression, Concentration Assay, Expressing, Fluorescence, Microscopy

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Oxalate Activates Autophagy to Induce Ferroptosis of Renal Tubular Epithelial Cells and Participates in the Formation of Kidney Stones

doi: 10.1155/2021/6630343

Figure Lengend Snippet: NCOA4 might be a key factor for oxalate induction of excessive autophagy in HK-2 cells to cause ferroptosis of cells. The concentration of oxalate was 2 mmol/L, and the intervention time was 24 hours. Among the six groups of cells, we compared the levels of BECN1 and NCOA4 proteins relative to GADPH levels (a), the MDA content (b), the GSH content (c), the Fe 2+ content (d), the ROS levels (e) of the two groups Ox+BECN1 and Ox+BECN1+NCOA4-shRNA, the mitochondrial membrane potential level (g), the protein levels of GPX4 (h), the expression of BECN1 , GPX4 , and NCOA4 mRNA in the six groups of cells relative to that of GAPDH (f), and the protein levels of P62, LC3 II, ACSL4, TFR1, TFH1, GPX4, and xTC in the six groups of cells (i). The fluorescence images were all taken under a 200× field of view under an automatic microscope. Data are presented as the means ± SEM from three independent experiments. ^^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001, and ^^^^ P < 0.0001 versus the Ox+BECN1 group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the NC+BECN1 group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 versus the ctrl NC group. ns: not significant.

Article Snippet: After transfer and blocking, the proteins on the membrane were incubated with primary antibodies recognizing GPX4 (14432-1-AP, 1 : 1000, Proteintech), acyl-CoA synthetase long-chain family member 4 (ACSL4) (22401-1-AP, 1 : 1000, Proteintech), solute carrier family 7 member 11 (xCT) (26864-1-AP, 1 : 1000, Proteintech), transferrin receptor (TFR1/CD71) (10084-2-AP, 1 : 1000, Proteintech), Ferritin Light Chain (FTL) (10727-1-AP, 1 : 1000, Proteintech), BECN1 (11306-1-AP, 1 : 2000, Proteintech), microtubule-associated protein 1 light chain 3 alpha (LC3) (14600-1-AP, 1 : 2000, Proteintech), NCOA4 (DF4255, 1 : 1000, Affbiotech, Jiangsu, China), sequestosome 1 (P62) (18420-1-AP, 1 : 2000, Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1 : 50000, Proteintech) at 4°C for 12 hours.

Techniques: Concentration Assay, shRNA, Expressing, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: SEC23B Loss-of-Function Suppresses Hepcidin Expression by Impairing Glycosylation Pathway in Human Hepatic Cells

doi: 10.3390/ijms23031304

Figure Lengend Snippet: N-linked glycosylation is required for proper BMP/SMADs signaling . ( A ) Left panel. Representative immunoblots of TFR2, HFE, HJV, and TMPRSS6 proteins in total cells lysate of HuH7 sh-74 and sh-CTR cells. GAPDH is the loading control. Right panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01; Student’s t -test). ( B ) Left panel . Representative immunoblots of TFR2, HFE, and TMPRSS6 in HuH7 control cells treated with DMSO (vehicle) or increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). GAPDH is the loading control. Right panel . Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, °° p < 0.01 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle, §§ p < 0.01 0.5 ug/mL tunicamycin-treated cells vs. vehicle; * p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle; ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). ( C ) Upper panel . Representative immunoblots of pSMAD1/5/8 and tSMAD1/5/8 proteins in HuH7 control cells treated with DMSO (vehicle) and increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). Lower panel . Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation (* p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, ° p < 0.05 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle. ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). ( D ) Quantification of HAMP gene expression in sh-CTR cells treated with DMSO and tunicamycin (1.5 µg/mL). Data are means ± standard deviation of three independent experiments. (** p < 0.01; Student’s t -test).

Article Snippet: Total protein extracts were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes (BioRad, Milan, Italy), and then incubated with the required combinations of the following antibodies: rabbit anti-SEC23B (1:1000; SAB2102104; Sigma Aldrich); SEC23A; Rabbit anti-Ferritin (1:1000; Abcam, ab75973); polyclonal rabbit Anti-Transferrin Receptor 2 (1:1000, Abcam, ab80194); monoclonal rabbit anti-HFE (1:1000.

Techniques: Western Blot, Standard Deviation, Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: SEC23B Loss-of-Function Suppresses Hepcidin Expression by Impairing Glycosylation Pathway in Human Hepatic Cells

doi: 10.3390/ijms23031304

Figure Lengend Snippet: Schematic models of the mechanism of iron overload in SEC23B loss-of-function hepatic cells . Left panel . At physiological conditions, SEC23B mediates the anterograde trafficking of correctly folded proteins and post-translational modification. Correctly processed proteins (TFR2, HJV, HFE) associated in the membrane complex that mediates activation of BMP/SMADs pathway. The activation of the complex results in the phosphorylation of SMAD1/5/8 that in turn mediates the activation of the transcription of the BMP target genes, HAMP , SMAD6 , ID1 , and ID3 . Right panel . SEC23B loss-of-function determines the decreased glycosylation of membrane proteins TFR2, HFE, and HJV (light colored), which accounts for the reduced expression and stabilization of the membrane complex. Therefore, BMP6 failed to activate the pathway, and this finally causes the suppression of HAMP gene transcription.

Article Snippet: Total protein extracts were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes (BioRad, Milan, Italy), and then incubated with the required combinations of the following antibodies: rabbit anti-SEC23B (1:1000; SAB2102104; Sigma Aldrich); SEC23A; Rabbit anti-Ferritin (1:1000; Abcam, ab75973); polyclonal rabbit Anti-Transferrin Receptor 2 (1:1000, Abcam, ab80194); monoclonal rabbit anti-HFE (1:1000.

Techniques: Modification, Activation Assay, Expressing

Journal: eLife

Article Title: Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate retriever-mediated recycling on endosomes

doi: 10.7554/eLife.69709

Figure Lengend Snippet:

Article Snippet: antibody , Transferrin receptor (Rabbit polyclonal) , Proteintech , 10084–2-AP RRID: AB_2240403 , WB (1:2000).

Techniques: Imaging, Plasmid Preparation, Recombinant, Knock-In, Software