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

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

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

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

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1, Vero E6, and SRD-12B cells were infected either with the recombinant virus VSVΔG/LASVGP or wild-type VSV (VSVwt) at an MOI of 0.02. Supernatants were collected at different times and titrated by plaque assay. The growth curves shown are the mean result±standard deviation of three independent experiments. B. CHO-K1 Tet-On cells were infected with VSVΔG/LASVGP in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml) at an MOI of 0.02. Supernatants were sampled 24 h and 48 h post-infection and analyzed by plaque assay.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Infection, Recombinant, Plaque Assay, Standard Deviation

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. Schematic representation of constructed α 1 -antitrypsin variants used for generation of stable cell lines. The amino acid sequences of the different motifs introduced into the RCL by recombinant PCR technology are shown in one-letter code. To facilitate their detection, a flag epitope was introduced at the C-terminus as indicated. B. Cellular expression of α 1 -antitrypsin variants. CHO-K1 Tet-On α 1 -ATs cells were induced with doxycycline (Dox, 1 µg/ml) or left untreated. At 24 h post-induction, cell lysates were analyzed by immunoblot analysis using a mouse anti-flag antibody. β-tubulin served as a loading control using a rabbit anti-ß-tubulin antibody. C. Immunofluorescence analysis of α 1 -antitrypsin expression. CHO-K1 Tet-On and α 1 -antitrypsin expressing cells were fixed with 4% PFA and permeabilized using 0.1% Triton X-100. α 1 -antitrypsin variants were visualized with a primary anti-flag mouse antibody and a secondary anti-mouse antibody coupled to rhodamine. Cell nuclei were stained with DAPI.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Construct, Stable Transfection, Recombinant, FLAG-tag, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1 Tet-On cells (lanes 1 and 2), S1P-deficient SRD-12B cells (lanes 3 and 4) and CHO-K1 Tet-On cell lines stably transfected with plasmids encoding α 1 -antitrypsin variants (lanes 5–14) were infected with VSVΔG/LASVGP at an MOI of 0.2 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml) as indicated. At 10 h post-infection cell lysates were analyzed by Western blot analysis using specific antibodies as described in the . B. Efficiency of GP-C cleavage was quantified by infrared fluorescent imaging using the Odyssey infrared imaging system. The amount of GP-2 was quantified compared to total amount of GP-C. Detected GP-2 in induced cell lines was calculated in relation to the corresponding non-induced cells, which were adjusted to 100%. The negative control (SRD-12B cells) was calculated with reference to the positive control (CHO-K1 Tet-On cells). Quantified GP-2 values are means±standard deviations of three independent experiments. Asterisks indicate differences that are statistically significant (**, p<0.002; ***, p<0.0001).

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Stable Transfection, Transfection, Infection, Western Blot, Imaging, Negative Control, Positive Control

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: CHO-K1 Tet-On cells and α 1 -AT variant RRIL CHO cell line were infected with (A) VSVΔG/LASVGP or (B) VSVwt at an MOI of 0.2 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). At 24 h post-infection, virions in cell culture supernatants were pelleted through a 20% sucrose cushion by ultracentrifugation. Cell lysates and purified virions were subjected to SDS-PAGE followed by immunoblotting using antisera specific for LASV GP and VSV proteins, respectively. Intracellular α 1 -AT expression was analyzed using an anti-flag antibody. β-tubulin was used as a loading control.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Variant Assay, Infection, Cell Culture, Purification, SDS Page, Western Blot, Expressing

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1 Tet-On cells, SRD-12B cells and S1P-adapted α 1 -antitrypsin expressing CHO cell lines were infected with VSVΔG/LASVGP at an MOI of 0.02 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). Infected cells were immunostained 24 h post-infection using an antiserum against VSV and HRP-linked secondary antibody. Individual cells and virus spread were visualized by subsequent application of True Blue peroxidase substrate. B. CHO-K1 Tet-On cells, S1P-adapted α 1 -antitrypsin variant RRIL cell line, as well as the furin-specific α 1 -antitrypsin variant RVKR cell line, were infected with Fowl Plague virus (FPV) at an MOI of 0.001 in the presence (+) or absence (−) of doxycycline. At 24 h post-infection, immunostaining was performed as described above using a polyclonal FPV antiserum.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Expressing, Infection, Variant Assay, Immunostaining

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: CHO-K1 Tet-On, SRD-12B and α 1 -AT variant RRIL CHO cell lines were infected with LASV (strain Josiah) at an MOI of 0.1 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). At various times post-infection as indicated, cell culture supernatants were collected and virus yields were determined by TCID 50 . Values shown are mean results±standard deviation of three independent experiments performed in duplicate. Asterisks indicate differences that are statistically significant between induced α 1 -AT variant RRIL cells in comparison to non-induced cells (*, p<0.05).

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Variant Assay, Infection, Cell Culture, Standard Deviation

Journal: Nature Communications

Article Title: ZBP1 not RIPK1 mediates tumor necroptosis in breast cancer

doi: 10.1038/s41467-021-23004-3

Figure Lengend Snippet: a FVB/NJ mice at three weeks post-implantation with MVT-1 cells were treated weekly with vehicle or Necrostatin-1 (Nec-1; i.v .) until week 5. Left panel shows the representative images of H&E stained 5-week tumors of vehicle or Nec-1 treated mice. Scale bar, 2 mm. Right panel shows the percentage of tumor necrosis area (TN) of the total tumor area from mice at 5-week. Data are presented as mean values ± SEM. b Representative H&E and immunohistological images of phospho-MLKL (p-MLKL) antibody staining of tumor sections from mice implanted and treated as in Fig. a . Scale bar, 50 µm. c MVT-1 CRISPR/Cas9 control (CRISPR CT) or MVT-1 RIPK1 knock out (RIPK1 KO) cells were generated by using the CRISPR/Cas9 system. Left panel shows the representative H&E image of 4-week tumors from FVB/NJ mice implanted with the MVT-1 CRISPR CT or MVT-1 RIPK1 KO cells. Scale bar, 2 mm. Right panel shows the percentage of tumor necrosis area (TN) of the total tumor area after 4-weeks. Data are presented as mean values ± SEM. d Representative H&E and immunohistological images of p-MLKL antibody staining of 4-week tumor sections from mice implanted as in Fig. c . Scale bar, 50 µm. e Western blotting analysis of tumor lysates from mice implanted as in Fig. c using the indicated antibodies. Western blotting analysis representative of three independent experiments. Two-sided student’s t test was used to determine the statistical significance of differences between groups. Differences with P values < 0.05 were considered significant. Source data are provided as a Source Data file.

Article Snippet: MVT-1 inducible RIPK1 KO stable cells were generated using the Lenti-X tet-on 3G CRISPR-Cas9 system from Clontech (Takara Bio).

Techniques: Staining, CRISPR, Knock-Out, Generated, Western Blot

Journal: Nature Communications

Article Title: ZBP1 not RIPK1 mediates tumor necroptosis in breast cancer

doi: 10.1038/s41467-021-23004-3

Figure Lengend Snippet: a Western blotting analysis representative of three independent experiments. MVT-1 CRISPR/Cas9 control (CRISPR CT) or ZBP1 knock out (ZBP1 KO) tumor lysates were blotted using the indicated antibodies (upper panel). Tumor growth curve by measuring tumor volume of FVB/NJ mice implanted with MVT-1 CRISPR CT or ZBP1 KO cells (lower panel). b Left panel shows the representative images of H&E stained tumors at 5-weeks post-implantation as in Fig. a . Scale bar, 2 mm. Right panel shows the percentage of tumor necrosis area (TN) of the total tumor area from mice at 5-weeks post-implantation. Data are presented as mean values ± SEM. c Representative images of H&E and immunohistological stained sections with phospho-MLKL (p-MLKL) or cleaved caspase-3 (cl.Casp-3) antibodies of 5-week tumor sections from mice implanted as in Fig. a . Scale bar, 50 µm. d Western blotting analysis is representative of three independent experiments. 5-week tumor lysates from mice implanted as in Fig. a was determined by using the indicated antibodies. e Left panel shows the representative images of H&E stained lung sections from mice implanted as in Fig. a showing lung metastasis. Scale bar, 2 mm. Right panel shows the quantification of metastatic foci in lungs from mice at 5-week post-implantation. Data are presented as mean values ± SEM. Two-sided student’s t test was used to determine the statistical significance of differences between groups. Differences with P values < 0.05 were considered significant. Source data are provided as a Source Data file.

Article Snippet: MVT-1 inducible RIPK1 KO stable cells were generated using the Lenti-X tet-on 3G CRISPR-Cas9 system from Clontech (Takara Bio).

Techniques: Western Blot, CRISPR, Knock-Out, Staining