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  • 94
    TaKaRa hek293 tet on 3g cells
    (a) Western blot showing APOL1 and tubulin levels in kidneys of NPHS1-rtTA/TRE-APOL1 transgenic mice on doxycycline for 3 (G1 and G2) to 17 (G0) weeks. (b) Left panel: Mice were matched for APOL1 transcript levels in NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice Right panel: urine albumin/creatinine ratio (ACR) of NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice after doxycycline diet for 3–6 weeks. (n = 5 per group). Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0029 (G0 vs. G1), P = 0.00612 (G0 vs. G2). (c) Cell toxicity (measured by the ratio of dead to viable cells) assay in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected <t>HEK293</t> cells and normalized to GFP (APOL1) levels. Grey bars represent no doxycycline treated and black (in CTL or G0) or white bars (in G1 or G2) represent doxycycline treated cells. Student’s t-test, P = 0.0031 (G0 vs. G1), P = 0.016 (G0 vs. G2). (d) Violin plots of eGFR (right graph) in a group of low risk (LR) and high-risk (HR) APOL1 genotype patients matched by APOL1 expression level (left graph) (FPKM: fragments per kilobase million) and age, gender and other sequencing parameters (1:3=HR (n = 11): LR (n = 33)). Significance was calculated by conditional logistic regression P = 0.032 (right graph). (e) Correlation between APOL1 transcript levels (by qRT-PCR) and albumin/creatinine ratio for NPHS1-rtTA/TRE-APOL1-G0, G1 or G2 mice. n = 15 per group. The red, white and black diamonds in the graph for the G2 allele represent 3 mice of varying expression of this risk allele and used for analysis in f. (f) Representative PAS-stained kidney images of NPHS1-rtTA/TRE-APOL1-G2 with increasing levels of APOL1 transcript. The diamonds represent the same 3 mice shown in e for the G2 group. (n > 5 per line) (g) APOL1 cell toxicity in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells treated with different concentrations of doxycycline (increasing APOL1 expression), cell toxicity was normalized to GFP (APOL1) expression. (h) Violin plots of glomerular APOL1 transcript level measured by RNA sequencing on microdissected glomeruli from 286 human kidney samples in control (GFR> 60mL/min/1.73m2) and CKD (GFR<60ml/min/1.73m2) patients. Student’s t-test, P = 0.000283. (i) Coomassie gels of urine samples from mice that were placed on doxycycline food for 14 days and then either kept on doxycycline food (on group) or taken doxycycline diet off (off group). Urine samples were taken from day 0, 14, 21 and 31 following initiation of doxycycline food. Quantifications of albumin gel images are shown at the bottom. Intensity of the albumin of Day 21 and 31 were both normalized to that of day 14 (the time of splitting up the groups). All data are presented as means ± s.d. (unless otherwise indicated).
    Hek293 Tet On 3g Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa hek 293 t cells
    (a) Western blot showing APOL1 and tubulin levels in kidneys of NPHS1-rtTA/TRE-APOL1 transgenic mice on doxycycline for 3 (G1 and G2) to 17 (G0) weeks. (b) Left panel: Mice were matched for APOL1 transcript levels in NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice Right panel: urine albumin/creatinine ratio (ACR) of NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice after doxycycline diet for 3–6 weeks. (n = 5 per group). Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0029 (G0 vs. G1), P = 0.00612 (G0 vs. G2). (c) Cell toxicity (measured by the ratio of dead to viable cells) assay in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected <t>HEK293</t> cells and normalized to GFP (APOL1) levels. Grey bars represent no doxycycline treated and black (in CTL or G0) or white bars (in G1 or G2) represent doxycycline treated cells. Student’s t-test, P = 0.0031 (G0 vs. G1), P = 0.016 (G0 vs. G2). (d) Violin plots of eGFR (right graph) in a group of low risk (LR) and high-risk (HR) APOL1 genotype patients matched by APOL1 expression level (left graph) (FPKM: fragments per kilobase million) and age, gender and other sequencing parameters (1:3=HR (n = 11): LR (n = 33)). Significance was calculated by conditional logistic regression P = 0.032 (right graph). (e) Correlation between APOL1 transcript levels (by qRT-PCR) and albumin/creatinine ratio for NPHS1-rtTA/TRE-APOL1-G0, G1 or G2 mice. n = 15 per group. The red, white and black diamonds in the graph for the G2 allele represent 3 mice of varying expression of this risk allele and used for analysis in f. (f) Representative PAS-stained kidney images of NPHS1-rtTA/TRE-APOL1-G2 with increasing levels of APOL1 transcript. The diamonds represent the same 3 mice shown in e for the G2 group. (n > 5 per line) (g) APOL1 cell toxicity in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells treated with different concentrations of doxycycline (increasing APOL1 expression), cell toxicity was normalized to GFP (APOL1) expression. (h) Violin plots of glomerular APOL1 transcript level measured by RNA sequencing on microdissected glomeruli from 286 human kidney samples in control (GFR> 60mL/min/1.73m2) and CKD (GFR<60ml/min/1.73m2) patients. Student’s t-test, P = 0.000283. (i) Coomassie gels of urine samples from mice that were placed on doxycycline food for 14 days and then either kept on doxycycline food (on group) or taken doxycycline diet off (off group). Urine samples were taken from day 0, 14, 21 and 31 following initiation of doxycycline food. Quantifications of albumin gel images are shown at the bottom. Intensity of the albumin of Day 21 and 31 were both normalized to that of day 14 (the time of splitting up the groups). All data are presented as means ± s.d. (unless otherwise indicated).
    Hek 293 T Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher hek 293 tet on cells
    Figure 1. MitoTimer localizes to mitochondria. ( A ) <t>HEK</t> <t>293</t> cells were transfected with pTRE-MitoTimer (MT), exposed to Dox (+) or vehicle (−) for 24 h, then subjected to subcellular fractionation to obtain mitochondria (lanes 1–3) and cytosol (lanes 5–7). As a positive control for the DsRed antibody, which recognizes DsRed as well as MitoTimer, a dish of cells were transfected with mitochondrial-targeted DsRed (DsR, lanes 3 and 7). Blots were probed with antibody to COX IV (mito marker) and RHO (cytosol marker). ( B ) Deconvolved fluorescence image (merged red and green channels) of a transfected and Dox-induced cell showing subcellular localization of MitoTimer. Scale bar: 10 µm. ( C ) Cells were transfected with MitoTimer and induced with Dox for 24 h, then disrupted to obtain whole cell lysate (WCL), cytosol (Cyto), and mitochondria, which were further disrupted to obtain intermembrane space (IMS), matrix (Mtx), inner membrane (IM), and outer membrane (OM). MitoTimer distribution was detected with antibody to DsRed. RHO was used as a cytosol marker, ACO2 as a matrix marker, and COX IV as an inner membrane marker.
    Hek 293 Tet On Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson hek 293 tet on cells
    Stable cell line expresses elevated SUMO and sumoylated proteins. <t>HEK</t> <t>293</t> <t>Tet-On</t> cells were transfected with either pTRE2hyg2-Myc-SUMO or pTRE2hyg2-Myc-Luc vector. Cells stably expressing Myc-SUMO and Myc-Luc were selected with 300 μg/ml hygromycin. After a 48-h incubation with or without 2 μg/ml Dox, whole-cell extracts were resolved by using NuPage gels and probed with anti-Myc (A), anti-SUMO (B), anti-p53 (C), or anti-β-actin (D) antibody. The p53 is marked with a single asterisk, and the sumoylated p53 is marked with double asterisks.
    Hek 293 Tet On Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore hek 293 tet on cells
    Stable cell line expresses elevated SUMO and sumoylated proteins. <t>HEK</t> <t>293</t> <t>Tet-On</t> cells were transfected with either pTRE2hyg2-Myc-SUMO or pTRE2hyg2-Myc-Luc vector. Cells stably expressing Myc-SUMO and Myc-Luc were selected with 300 μg/ml hygromycin. After a 48-h incubation with or without 2 μg/ml Dox, whole-cell extracts were resolved by using NuPage gels and probed with anti-Myc (A), anti-SUMO (B), anti-p53 (C), or anti-β-actin (D) antibody. The p53 is marked with a single asterisk, and the sumoylated p53 is marked with double asterisks.
    Hek 293 Tet On Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (a) Western blot showing APOL1 and tubulin levels in kidneys of NPHS1-rtTA/TRE-APOL1 transgenic mice on doxycycline for 3 (G1 and G2) to 17 (G0) weeks. (b) Left panel: Mice were matched for APOL1 transcript levels in NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice Right panel: urine albumin/creatinine ratio (ACR) of NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice after doxycycline diet for 3–6 weeks. (n = 5 per group). Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0029 (G0 vs. G1), P = 0.00612 (G0 vs. G2). (c) Cell toxicity (measured by the ratio of dead to viable cells) assay in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells and normalized to GFP (APOL1) levels. Grey bars represent no doxycycline treated and black (in CTL or G0) or white bars (in G1 or G2) represent doxycycline treated cells. Student’s t-test, P = 0.0031 (G0 vs. G1), P = 0.016 (G0 vs. G2). (d) Violin plots of eGFR (right graph) in a group of low risk (LR) and high-risk (HR) APOL1 genotype patients matched by APOL1 expression level (left graph) (FPKM: fragments per kilobase million) and age, gender and other sequencing parameters (1:3=HR (n = 11): LR (n = 33)). Significance was calculated by conditional logistic regression P = 0.032 (right graph). (e) Correlation between APOL1 transcript levels (by qRT-PCR) and albumin/creatinine ratio for NPHS1-rtTA/TRE-APOL1-G0, G1 or G2 mice. n = 15 per group. The red, white and black diamonds in the graph for the G2 allele represent 3 mice of varying expression of this risk allele and used for analysis in f. (f) Representative PAS-stained kidney images of NPHS1-rtTA/TRE-APOL1-G2 with increasing levels of APOL1 transcript. The diamonds represent the same 3 mice shown in e for the G2 group. (n > 5 per line) (g) APOL1 cell toxicity in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells treated with different concentrations of doxycycline (increasing APOL1 expression), cell toxicity was normalized to GFP (APOL1) expression. (h) Violin plots of glomerular APOL1 transcript level measured by RNA sequencing on microdissected glomeruli from 286 human kidney samples in control (GFR> 60mL/min/1.73m2) and CKD (GFR<60ml/min/1.73m2) patients. Student’s t-test, P = 0.000283. (i) Coomassie gels of urine samples from mice that were placed on doxycycline food for 14 days and then either kept on doxycycline food (on group) or taken doxycycline diet off (off group). Urine samples were taken from day 0, 14, 21 and 31 following initiation of doxycycline food. Quantifications of albumin gel images are shown at the bottom. Intensity of the albumin of Day 21 and 31 were both normalized to that of day 14 (the time of splitting up the groups). All data are presented as means ± s.d. (unless otherwise indicated).

    Journal: Nature medicine

    Article Title: Transgenic Expression of Human APOL1 Risk Variants in Podocytes Induces Kidney Disease in Mice

    doi: 10.1038/nm.4287

    Figure Lengend Snippet: (a) Western blot showing APOL1 and tubulin levels in kidneys of NPHS1-rtTA/TRE-APOL1 transgenic mice on doxycycline for 3 (G1 and G2) to 17 (G0) weeks. (b) Left panel: Mice were matched for APOL1 transcript levels in NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice Right panel: urine albumin/creatinine ratio (ACR) of NPHS1-rtTA/TRE-APOL1-G0/G1/G2 transgenic mice after doxycycline diet for 3–6 weeks. (n = 5 per group). Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0029 (G0 vs. G1), P = 0.00612 (G0 vs. G2). (c) Cell toxicity (measured by the ratio of dead to viable cells) assay in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells and normalized to GFP (APOL1) levels. Grey bars represent no doxycycline treated and black (in CTL or G0) or white bars (in G1 or G2) represent doxycycline treated cells. Student’s t-test, P = 0.0031 (G0 vs. G1), P = 0.016 (G0 vs. G2). (d) Violin plots of eGFR (right graph) in a group of low risk (LR) and high-risk (HR) APOL1 genotype patients matched by APOL1 expression level (left graph) (FPKM: fragments per kilobase million) and age, gender and other sequencing parameters (1:3=HR (n = 11): LR (n = 33)). Significance was calculated by conditional logistic regression P = 0.032 (right graph). (e) Correlation between APOL1 transcript levels (by qRT-PCR) and albumin/creatinine ratio for NPHS1-rtTA/TRE-APOL1-G0, G1 or G2 mice. n = 15 per group. The red, white and black diamonds in the graph for the G2 allele represent 3 mice of varying expression of this risk allele and used for analysis in f. (f) Representative PAS-stained kidney images of NPHS1-rtTA/TRE-APOL1-G2 with increasing levels of APOL1 transcript. The diamonds represent the same 3 mice shown in e for the G2 group. (n > 5 per line) (g) APOL1 cell toxicity in TRE-GFP/APOL1-G0, G1 or G2 transiently transfected HEK293 cells treated with different concentrations of doxycycline (increasing APOL1 expression), cell toxicity was normalized to GFP (APOL1) expression. (h) Violin plots of glomerular APOL1 transcript level measured by RNA sequencing on microdissected glomeruli from 286 human kidney samples in control (GFR> 60mL/min/1.73m2) and CKD (GFR<60ml/min/1.73m2) patients. Student’s t-test, P = 0.000283. (i) Coomassie gels of urine samples from mice that were placed on doxycycline food for 14 days and then either kept on doxycycline food (on group) or taken doxycycline diet off (off group). Urine samples were taken from day 0, 14, 21 and 31 following initiation of doxycycline food. Quantifications of albumin gel images are shown at the bottom. Intensity of the albumin of Day 21 and 31 were both normalized to that of day 14 (the time of splitting up the groups). All data are presented as means ± s.d. (unless otherwise indicated).

    Article Snippet: HEK293 Tet-On 3G cells (Clontech) were cultured in Eagle MEM with 10% Tet system approved fetal bovine serum (FBS), 10% L-glutamine and 1% penicillin-streptomycin.

    Techniques: Western Blot, Transgenic Assay, Transfection, Expressing, Sequencing, Quantitative RT-PCR, Staining, RNA Sequencing Assay

    (a) Immunogold electron microscopy (EM) of APOL1 in human kidney podocytes. APOL1 localizes to plasma membrane (red circles) and intracellular vesicles (blue arrows). Scale bar, 200nm. (b) Double immunofluorescence micrographs of cultured human podocytes with APOL1 (red) and intracellular organelle markers (green) (EEA1-early endosome, Rab7-late endosome, Rab11-recycle endosome, LC3-autophagic vacuoles, LAMP2-lysosomes). Scar bar, 11μm. (c) Quantification of colocalization correlation using Pearson r correlation through ImageJ coloc2 function. n = 5 separate experiments, 3–10 cells for each genotype were analyzed. Data are presented as means ± s.e.m. (d) Representative frame from the supplementary video 1 of spinning disk confocal microscopy analysis of GFP-APOL1 (green) and RFP-Rab7 (late endosome) (red) in transfected HEK293 cells. Arrowheads, overlapping puncta of GFP-APOL1 and RFP-Rab7. Scale bar, 5μm. (e–f) Representative fluorescence images of confocal microscopy analysis of endogenous. (e) Rab7 and (f) LC3 immunofluorescence stain in cultured low risk genotype (G0/G0) and high risk genotype (G1/G2) human podocytes and quantification showing increased stain in G1/G2 cells. n = 5 separate experiments, 3–10 cells for each genotype were analyzed. Scale bars, 11μm. (g) Representative transmission electron micrographs from control, TRE-APOL1-G0, TRE-APOL1-G1 and TRE-APOL1-G2 transfected HEK293 cells. Examples of autophagy-related compartments include: **(two black asterisks): Late endosomes/MVB (MVB), *(one black asterisk): autophagosomes (APG), ** (two white asterisks): autolysosomes (AUT), * (one white asterisk): amphisomes (AMP). Scale bars, 0.5μm. (h) Quantification showing the number of each type of vesicle per section. n = 7 analyzed sections. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.032, 0.0057, 0.011 for G0, G1, G2 comparing to control (AUT); while P = 0.024,0.00027, 0.00903 for G0, G1, G2 comparing control (AMP). (i) Quantification showing the relative percentage of each compartment. n = 7 analyzed sections. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0064 (G2 vs. CTL, APG), 0.0068 and 0.0.01005 (G0 and G2, respectively, vs. CTL, AUT), 0.034 and 0.00044 (G0 and G2, respectively, vs. CTL, AMP). (j) Representative frames from the supplementary videos 1 and 2 of spinning disk confocal microscopy analysis of GFP-APOL1-G0 or GFP-APOL1-G2 (green) and RFP-Rab7 or RFP-Rab11 (red) in transfected HEK293 cells. Scale bar, 5 μm. All data are presented as means ± s.d. (unless otherwise indicated).

    Journal: Nature medicine

    Article Title: Transgenic Expression of Human APOL1 Risk Variants in Podocytes Induces Kidney Disease in Mice

    doi: 10.1038/nm.4287

    Figure Lengend Snippet: (a) Immunogold electron microscopy (EM) of APOL1 in human kidney podocytes. APOL1 localizes to plasma membrane (red circles) and intracellular vesicles (blue arrows). Scale bar, 200nm. (b) Double immunofluorescence micrographs of cultured human podocytes with APOL1 (red) and intracellular organelle markers (green) (EEA1-early endosome, Rab7-late endosome, Rab11-recycle endosome, LC3-autophagic vacuoles, LAMP2-lysosomes). Scar bar, 11μm. (c) Quantification of colocalization correlation using Pearson r correlation through ImageJ coloc2 function. n = 5 separate experiments, 3–10 cells for each genotype were analyzed. Data are presented as means ± s.e.m. (d) Representative frame from the supplementary video 1 of spinning disk confocal microscopy analysis of GFP-APOL1 (green) and RFP-Rab7 (late endosome) (red) in transfected HEK293 cells. Arrowheads, overlapping puncta of GFP-APOL1 and RFP-Rab7. Scale bar, 5μm. (e–f) Representative fluorescence images of confocal microscopy analysis of endogenous. (e) Rab7 and (f) LC3 immunofluorescence stain in cultured low risk genotype (G0/G0) and high risk genotype (G1/G2) human podocytes and quantification showing increased stain in G1/G2 cells. n = 5 separate experiments, 3–10 cells for each genotype were analyzed. Scale bars, 11μm. (g) Representative transmission electron micrographs from control, TRE-APOL1-G0, TRE-APOL1-G1 and TRE-APOL1-G2 transfected HEK293 cells. Examples of autophagy-related compartments include: **(two black asterisks): Late endosomes/MVB (MVB), *(one black asterisk): autophagosomes (APG), ** (two white asterisks): autolysosomes (AUT), * (one white asterisk): amphisomes (AMP). Scale bars, 0.5μm. (h) Quantification showing the number of each type of vesicle per section. n = 7 analyzed sections. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.032, 0.0057, 0.011 for G0, G1, G2 comparing to control (AUT); while P = 0.024,0.00027, 0.00903 for G0, G1, G2 comparing control (AMP). (i) Quantification showing the relative percentage of each compartment. n = 7 analyzed sections. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0064 (G2 vs. CTL, APG), 0.0068 and 0.0.01005 (G0 and G2, respectively, vs. CTL, AUT), 0.034 and 0.00044 (G0 and G2, respectively, vs. CTL, AMP). (j) Representative frames from the supplementary videos 1 and 2 of spinning disk confocal microscopy analysis of GFP-APOL1-G0 or GFP-APOL1-G2 (green) and RFP-Rab7 or RFP-Rab11 (red) in transfected HEK293 cells. Scale bar, 5 μm. All data are presented as means ± s.d. (unless otherwise indicated).

    Article Snippet: HEK293 Tet-On 3G cells (Clontech) were cultured in Eagle MEM with 10% Tet system approved fetal bovine serum (FBS), 10% L-glutamine and 1% penicillin-streptomycin.

    Techniques: Electron Microscopy, Immunofluorescence, Cell Culture, Confocal Microscopy, Transfection, Fluorescence, Staining, Transmission Assay

    (a) Representative Western blot analysis and (b) quantification of LC3 and GFP in TRE-APOL1 transfected HEK293 cells under fed (F), starved (S) and starved+chloroquine treatment (SCQ) conditions. Top panel shows LC3I and LC3II bands on the same gel. Due to high LC3I content in the cells, different exposure times of the individual rows of LCI and LCII (see solid and dashed arrowed lines, respectively, pointing to the lower panels) were utilized for clear and quantifiable visibility of the bands. Data are presented as means ± s.e.m. and Student’s t-test compared to TRE-APOL1-G0 transfected cells. (c) Quantification of autophagosomes (AP) and autophagolysosomes (AL) by transmission EM and their ratio in transfected HEK293 cells. n = 35, 32, 54 and 42 cells for control, G0, G1 and G2 transfected cells were analyzed, respectively. All data are presented as means ± s.e.m. and Student’s t-test, P = 0.0013 (left panel), 0.025 (right panel) compare to non-transfected (CTL) + TRE-APOL1-G0 transfected cells. (d,e) LC3 staining of low risk (G0/G0) and high-risk (compound heterozygous G1/G2) genotype human podocytes under fed (F), starved (S) and starved plus chloroquine (SCQ) conditions (d) and quantification of this data (e). Scale bars, 11μm. n = 3, 9–20 cells were analyzed per condition. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0462 (left panel), 0.0104 (right panel) compared to low risk genotype (G0/G0) podocytes.

    Journal: Nature medicine

    Article Title: Transgenic Expression of Human APOL1 Risk Variants in Podocytes Induces Kidney Disease in Mice

    doi: 10.1038/nm.4287

    Figure Lengend Snippet: (a) Representative Western blot analysis and (b) quantification of LC3 and GFP in TRE-APOL1 transfected HEK293 cells under fed (F), starved (S) and starved+chloroquine treatment (SCQ) conditions. Top panel shows LC3I and LC3II bands on the same gel. Due to high LC3I content in the cells, different exposure times of the individual rows of LCI and LCII (see solid and dashed arrowed lines, respectively, pointing to the lower panels) were utilized for clear and quantifiable visibility of the bands. Data are presented as means ± s.e.m. and Student’s t-test compared to TRE-APOL1-G0 transfected cells. (c) Quantification of autophagosomes (AP) and autophagolysosomes (AL) by transmission EM and their ratio in transfected HEK293 cells. n = 35, 32, 54 and 42 cells for control, G0, G1 and G2 transfected cells were analyzed, respectively. All data are presented as means ± s.e.m. and Student’s t-test, P = 0.0013 (left panel), 0.025 (right panel) compare to non-transfected (CTL) + TRE-APOL1-G0 transfected cells. (d,e) LC3 staining of low risk (G0/G0) and high-risk (compound heterozygous G1/G2) genotype human podocytes under fed (F), starved (S) and starved plus chloroquine (SCQ) conditions (d) and quantification of this data (e). Scale bars, 11μm. n = 3, 9–20 cells were analyzed per condition. Data are presented as means ± s.e.m. and Student’s t-test, P = 0.0462 (left panel), 0.0104 (right panel) compared to low risk genotype (G0/G0) podocytes.

    Article Snippet: HEK293 Tet-On 3G cells (Clontech) were cultured in Eagle MEM with 10% Tet system approved fetal bovine serum (FBS), 10% L-glutamine and 1% penicillin-streptomycin.

    Techniques: Western Blot, Transfection, Transmission Assay, Staining

    (a) Representative Western blot analysis of caspase3 (Casp3) and cleaved caspase3 (cCasp3) following transient transfection with TRE-APOL1-G0/G1/G2. UV exposure served as apoptosis positive control. n = 5. (b) Representative Western blot analysis of caspase1 (Casp1) and cleaved caspase1 (cCasp1) following transient transfection of TRE-APOL1-G0/G1/G2. GFP served as an APOL1 expression reference, α-tubulin as loading control and LPS as pyroptosis positive control. Western blot analysis of mature IL1β in medium from the same transfected HeLa cells. (c) Cell toxicity (measured by propidium iodide staining) in stably transfected TRE-GFP/APOL1-G1 HEK293 cells, with (APOL1) or without (CTL) doxycycline and in the presence of the indicated caspase1 inhibitors (concentration see Methods). Experiments were done in triplicates. Data are presented as means ± s.e.m, and Student’s t-test, P = 0.0015 (Ac-YVAD-CHO), 0.00018 (VX765) compared to APOL1. (d) Cell toxicity (measured by LDH release to the medium) in stably transfected TRE-GFP/APOL1-G1 HEK293 cells with (APOL1) or without (CTL) doxycycline and with indicated inhibitors (concentration see Methods). A representative experiment out of three is presented; each experiment was done in triplicates. All data are presented as means ± s.e.m, and Student’s t-test, P < 0.05.

    Journal: Nature medicine

    Article Title: Transgenic Expression of Human APOL1 Risk Variants in Podocytes Induces Kidney Disease in Mice

    doi: 10.1038/nm.4287

    Figure Lengend Snippet: (a) Representative Western blot analysis of caspase3 (Casp3) and cleaved caspase3 (cCasp3) following transient transfection with TRE-APOL1-G0/G1/G2. UV exposure served as apoptosis positive control. n = 5. (b) Representative Western blot analysis of caspase1 (Casp1) and cleaved caspase1 (cCasp1) following transient transfection of TRE-APOL1-G0/G1/G2. GFP served as an APOL1 expression reference, α-tubulin as loading control and LPS as pyroptosis positive control. Western blot analysis of mature IL1β in medium from the same transfected HeLa cells. (c) Cell toxicity (measured by propidium iodide staining) in stably transfected TRE-GFP/APOL1-G1 HEK293 cells, with (APOL1) or without (CTL) doxycycline and in the presence of the indicated caspase1 inhibitors (concentration see Methods). Experiments were done in triplicates. Data are presented as means ± s.e.m, and Student’s t-test, P = 0.0015 (Ac-YVAD-CHO), 0.00018 (VX765) compared to APOL1. (d) Cell toxicity (measured by LDH release to the medium) in stably transfected TRE-GFP/APOL1-G1 HEK293 cells with (APOL1) or without (CTL) doxycycline and with indicated inhibitors (concentration see Methods). A representative experiment out of three is presented; each experiment was done in triplicates. All data are presented as means ± s.e.m, and Student’s t-test, P < 0.05.

    Article Snippet: HEK293 Tet-On 3G cells (Clontech) were cultured in Eagle MEM with 10% Tet system approved fetal bovine serum (FBS), 10% L-glutamine and 1% penicillin-streptomycin.

    Techniques: Western Blot, Transfection, Positive Control, Expressing, Staining, Stable Transfection, Concentration Assay

    Figure 1. MitoTimer localizes to mitochondria. ( A ) HEK 293 cells were transfected with pTRE-MitoTimer (MT), exposed to Dox (+) or vehicle (−) for 24 h, then subjected to subcellular fractionation to obtain mitochondria (lanes 1–3) and cytosol (lanes 5–7). As a positive control for the DsRed antibody, which recognizes DsRed as well as MitoTimer, a dish of cells were transfected with mitochondrial-targeted DsRed (DsR, lanes 3 and 7). Blots were probed with antibody to COX IV (mito marker) and RHO (cytosol marker). ( B ) Deconvolved fluorescence image (merged red and green channels) of a transfected and Dox-induced cell showing subcellular localization of MitoTimer. Scale bar: 10 µm. ( C ) Cells were transfected with MitoTimer and induced with Dox for 24 h, then disrupted to obtain whole cell lysate (WCL), cytosol (Cyto), and mitochondria, which were further disrupted to obtain intermembrane space (IMS), matrix (Mtx), inner membrane (IM), and outer membrane (OM). MitoTimer distribution was detected with antibody to DsRed. RHO was used as a cytosol marker, ACO2 as a matrix marker, and COX IV as an inner membrane marker.

    Journal: Autophagy

    Article Title: MitoTimer

    doi: 10.4161/auto.26501

    Figure Lengend Snippet: Figure 1. MitoTimer localizes to mitochondria. ( A ) HEK 293 cells were transfected with pTRE-MitoTimer (MT), exposed to Dox (+) or vehicle (−) for 24 h, then subjected to subcellular fractionation to obtain mitochondria (lanes 1–3) and cytosol (lanes 5–7). As a positive control for the DsRed antibody, which recognizes DsRed as well as MitoTimer, a dish of cells were transfected with mitochondrial-targeted DsRed (DsR, lanes 3 and 7). Blots were probed with antibody to COX IV (mito marker) and RHO (cytosol marker). ( B ) Deconvolved fluorescence image (merged red and green channels) of a transfected and Dox-induced cell showing subcellular localization of MitoTimer. Scale bar: 10 µm. ( C ) Cells were transfected with MitoTimer and induced with Dox for 24 h, then disrupted to obtain whole cell lysate (WCL), cytosol (Cyto), and mitochondria, which were further disrupted to obtain intermembrane space (IMS), matrix (Mtx), inner membrane (IM), and outer membrane (OM). MitoTimer distribution was detected with antibody to DsRed. RHO was used as a cytosol marker, ACO2 as a matrix marker, and COX IV as an inner membrane marker.

    Article Snippet: HEK 293 Tet-On cells were maintained in DMEM 1X + GlutaMax media (Gibco, 10569-010) containing 10% tetracycline-free fetal bovine serum (Clontech, 631106), 5% antibiotic-antimycotic (Gibco, 15240) and 5% D-glucose.

    Techniques: Transfection, Fractionation, Positive Control, Marker, Fluorescence

    Stable cell line expresses elevated SUMO and sumoylated proteins. HEK 293 Tet-On cells were transfected with either pTRE2hyg2-Myc-SUMO or pTRE2hyg2-Myc-Luc vector. Cells stably expressing Myc-SUMO and Myc-Luc were selected with 300 μg/ml hygromycin. After a 48-h incubation with or without 2 μg/ml Dox, whole-cell extracts were resolved by using NuPage gels and probed with anti-Myc (A), anti-SUMO (B), anti-p53 (C), or anti-β-actin (D) antibody. The p53 is marked with a single asterisk, and the sumoylated p53 is marked with double asterisks.

    Journal:

    Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

    doi: 10.1073/pnas.0402889101

    Figure Lengend Snippet: Stable cell line expresses elevated SUMO and sumoylated proteins. HEK 293 Tet-On cells were transfected with either pTRE2hyg2-Myc-SUMO or pTRE2hyg2-Myc-Luc vector. Cells stably expressing Myc-SUMO and Myc-Luc were selected with 300 μg/ml hygromycin. After a 48-h incubation with or without 2 μg/ml Dox, whole-cell extracts were resolved by using NuPage gels and probed with anti-Myc (A), anti-SUMO (B), anti-p53 (C), or anti-β-actin (D) antibody. The p53 is marked with a single asterisk, and the sumoylated p53 is marked with double asterisks.

    Article Snippet: HEK 293 Tet-On cells were cultured in DMEM supplemented with 10% Tet-system approved FBS (BD Biosciences/Clontech) and 100 μg/ml of antibiotic G418. pTRE2hyg2-Myc-SUMO and pTRE2hyg2-Myc-Luc were transfected into HEK 293 Tet-On cells by using FuGENE 6 (Roche Molecular Biochemicals).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Incubation

    Myc-tagged SUMO and sumoylated proteins mainly localized in the nucleus. After Myc-SUMO stably expressed HEK 293 Tet-On cells were induced with 2 μg/ml Dox for 48 h, cells were fixed with 3.7% formaldehyde and stained with anti-Myc primary antibody and FITC-conjugated secondary antibody. DAPI was used to stain nuclei.

    Journal:

    Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

    doi: 10.1073/pnas.0402889101

    Figure Lengend Snippet: Myc-tagged SUMO and sumoylated proteins mainly localized in the nucleus. After Myc-SUMO stably expressed HEK 293 Tet-On cells were induced with 2 μg/ml Dox for 48 h, cells were fixed with 3.7% formaldehyde and stained with anti-Myc primary antibody and FITC-conjugated secondary antibody. DAPI was used to stain nuclei.

    Article Snippet: HEK 293 Tet-On cells were cultured in DMEM supplemented with 10% Tet-system approved FBS (BD Biosciences/Clontech) and 100 μg/ml of antibiotic G418. pTRE2hyg2-Myc-SUMO and pTRE2hyg2-Myc-Luc were transfected into HEK 293 Tet-On cells by using FuGENE 6 (Roche Molecular Biochemicals).

    Techniques: Stable Transfection, Staining

    Cellular sumoylated proteins show a similar pattern to in vitro isolated sumoylated proteins in 2D gel electrophoresis. Whole cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO were resolved by using 7-cm 2D gel and probed with anti-Myc antibody (A). The Myc-SUMO is marked with an arrow. The in vitro isolated sumoylated proteins were resolved by using 18-cm 2D gel and silver stained (B). Biotin–SUMO is also marked with an arrow.

    Journal:

    Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

    doi: 10.1073/pnas.0402889101

    Figure Lengend Snippet: Cellular sumoylated proteins show a similar pattern to in vitro isolated sumoylated proteins in 2D gel electrophoresis. Whole cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO were resolved by using 7-cm 2D gel and probed with anti-Myc antibody (A). The Myc-SUMO is marked with an arrow. The in vitro isolated sumoylated proteins were resolved by using 18-cm 2D gel and silver stained (B). Biotin–SUMO is also marked with an arrow.

    Article Snippet: HEK 293 Tet-On cells were cultured in DMEM supplemented with 10% Tet-system approved FBS (BD Biosciences/Clontech) and 100 μg/ml of antibiotic G418. pTRE2hyg2-Myc-SUMO and pTRE2hyg2-Myc-Luc were transfected into HEK 293 Tet-On cells by using FuGENE 6 (Roche Molecular Biochemicals).

    Techniques: In Vitro, Isolation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Expressing, Staining

    hnRNP A1, hnRNP F, and hnRNP K are sumoylated. The whole-cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO or Myc-Luc were immunoprecipitated with either anti-Myc (A), anti-hnRNP F (B), or hnRNP K (C) antibody. The immunoprecipitates were resolved by NuPage gels and probed with anti-hnRNP A1 (A) or anti-Myc (B and C) antibody. Sumoylated hnRNP proteins are marked with arrows. The lower bands in B are caused by IgG heavy chain cross-reacting with the secondary antibody.

    Journal:

    Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

    doi: 10.1073/pnas.0402889101

    Figure Lengend Snippet: hnRNP A1, hnRNP F, and hnRNP K are sumoylated. The whole-cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO or Myc-Luc were immunoprecipitated with either anti-Myc (A), anti-hnRNP F (B), or hnRNP K (C) antibody. The immunoprecipitates were resolved by NuPage gels and probed with anti-hnRNP A1 (A) or anti-Myc (B and C) antibody. Sumoylated hnRNP proteins are marked with arrows. The lower bands in B are caused by IgG heavy chain cross-reacting with the secondary antibody.

    Article Snippet: HEK 293 Tet-On cells were cultured in DMEM supplemented with 10% Tet-system approved FBS (BD Biosciences/Clontech) and 100 μg/ml of antibiotic G418. pTRE2hyg2-Myc-SUMO and pTRE2hyg2-Myc-Luc were transfected into HEK 293 Tet-On cells by using FuGENE 6 (Roche Molecular Biochemicals).

    Techniques: Expressing, Immunoprecipitation