cell culture medium complete growth medium  (ATCC)

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Immunoblot detection of APOL1 in podocytes treated in the absence (-INFγ, rightmost lane) or presence of INFγ (Ctrl, leftmost lane). APOL1 is not induced by IFNγ in three clonal populations derived from Celprogen human primary podocytes (C1KO, C2KO, C3KO) in which the APOL1 gene was subjected to CRISPR-mediated inactivation. β-actin served as loading control. B. Preservation of differentiated phenotype in Celprogen podocytes. Upper panels: immunoblot of nephrin in lysates of HEK-293 cells transiently overexpressing nephrin (lane 1), in SV40LgT-immortalized human podocytes (lane 2), in Celprogen primary human podocytes (lane 3) and in podocyte outgrowths from human glomeruli. Lower panels: immunoblots of WT1 and podocin in HEK-293 cells transiently overexpressing WT1 or podocin (lane 1), in SV40LgT-immortalized human podocytes (lane 2) and in Celprogen primary human podocytes (lane 3). β-actin served as loading control for both upper and lower panels. C. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right)) recorded from a representative human primary podocyte incubated 18 h in the absence (lower trace) or presence of IFNγ (10 ng/mL, upper trace). D. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right), recorded first in the absence (red trace) and then after 2 min in the presence of anti-APOL1 aa 1–238 antibody (black trace +Ab), and finally after 3 min of antibody washout (black trace “washout”). Seal resistances at the end of each period were 9.7 GΩ (-Ab), 8.3 GΩ (+Ab) and 4.0 GΩ (washout). E. Capacitance-normalized whole cell currents measured at −60 mV in normal human primary podocytes incubated 24 h in the absence (n=11, left three datasets) or presence of 10 ng/ml IFNγ (n=19, right three datasets); *, p<0.016 vs. minus IFNγ. Each cell was recorded first in the absence and then in the subsequent presence of anti-APOL1 N-terminal domain antibody for 2 min (2 μg/ml, +Ab; n=6 −IFNγ and n=11 +IFNγ), followed by 3 min antibody washout (w/o, n=3 −IFNγ and n 5 +IFNγ). Among the 19 IFNγ-pretreated cells, open circles represent cells for which patch seals did not survive the antibody exposure, black circles connected by black lines depict cells for which patch seals did not survive antibody washout, and black circles connected by red lines show cells for which patch seals survived all three recording conditions. #, p= 0.135, vs. −IFNγ before antibody; ##, p=0.008 vs. +IFNγ before antibody; one-way ANOVA on ranks with Dunnett’s post-test.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Western Blot, Derivative Assay, CRISPR, Preserving, Incubation

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Representative on-cell current traces measured at −Vp = +50 mV in podocytes previously treated 24 h with vehicle (upper trace) or with IFNγ (10 ng/mL, lower trace). B. I-V curve of the predominant unitary conductance class (28 pS) of on-cell patch currents in a representative IFNγ-treated Celprogen human podocyte. C. NPo of predominant unitary current class in native Celprogen podocytes (WT, middle pair of bars) and in two Celprogen knockout cell lines (KO #1 and KO #2), each treated 24 h in the absence (left unfilled bars of each pair) and presence of IFNγ (right bars of each pair; data points overlap in all bars). Values are means ± s.e.m. for indicated (n). *, p<0.05 for WT+IFNγ vs −IFNγ, and for WT+IFNγ vs each KO+IFNγ; unpaired two-way t-tests.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Knock-Out

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes treated without (n=14, white bar, data points overlap) or with 2 μg/ml anti-APOL1 N-terminal domain antibody (n=7, black bar, data points overlap) or nonspecific rabbit IgG (n=6, gray bar) in the pipette solution. Steady-state values at 2–3 min after achievement of seal. *, p=0.003 vs “none”; unpaired two-tailed t-test. B. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes (n=6) exposed to 3 μM recombinant N-terminal fragment of Serum Response-Associated (SRA) of Trypanosoma brucei rhodesiense in the pipette solution, and recorded immediately after establishment of a gigohm seal (gray bar) and ~110 s after seal establishment (black bar; data points overlap). *, p<0.041 vs ~0 sec; unpaired two-tailed t-test. Gigohm resistances were maintained throughout the recording periods.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Transferring, Two Tailed Test, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

doi: 10.4049/jimmunol.2100483

Figure Lengend Snippet: FIGURE 1. AIM2 expression in nor- mal human kidney. (A) Immunoblot probing for AIM2 in two independent human kidney samples using Cell Sig- naling Technology and Sigma-Aldrich Abs. THP-1 cells plus or minus g-IFN are used as controls. Arrows denote AIM2 forms. (B) AIM2 mRNA expres- sion in glomerular fraction normalized to total human kidney by qPCR. Glomeruli were isolated from human kidney tissue by a sieving technique. Mean ± SD, n 5 4. *p < 0.05, Student t test. A represen- tative image of a PAS-stained normal human glomerulus is shown in the lower panel (original magnification 40). (C) Indirect immunofluorescence confocal microscopy probing for AIM2 (red) and WT1, synaptopodin, PDGFRb, and CD31 (green) in paraffin-embedded nor- mal kidney tissue. Nuclear staining using DAPI is shown in merged images (blue). Dashed circle (G) indicates glomeruli in the kidney. Right panel represents mag- nified view of hatched inset. Dual- labeling confocal microscopy shows localization of AIM2 to podocytes. Scale bars, 50 mm. Images are representative of staining performed at least three inde- pendent times. (D) RNA in situ hybridi- zation (RNAScope) probing for AIM2, NHPS1 (nephrin), and CD44 in normal human glomerulus. AIM2 mRNA coloc- alizes with NHPS1 but not CD44 in the normal kidney. (iiii) Magnified views of indicated hatched boxes. Scale bar, 25 mm. Images are representative of experiments performed at least three independent times.

Article Snippet: Podocytes were cultured as per manufacturer’s protocol in Human Podocyte Complete Growth Medium (Celprogen) and on Human Podocyte Cell Culture Extra-cellular Matrix (Celprogen).

Techniques: Expressing, Western Blot, Isolation, Staining, Immunofluorescence, Confocal Microscopy, Labeling, In Situ, RNAscope

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

doi: 10.4049/jimmunol.2100483

Figure Lengend Snippet: FIGURE 2. AIM2 and inflammasome activation in primary human podo- cytes. (A) Immunoblotting for AIM2 and synaptopodin in primary human podocytes. Human kidney and THP-1 cells plus or minus g-IFN stimulation are used as controls. Arrows denote AIM2 forms. (B) ELISA for IL-1b in supernatant of LPS-primed or -unprimed podocytes treated with nigericin (50 mM, 1 h) or transfected with poly(dA:dT) (2 mg/ml, 3 h). PMA-primed THP-1 cells treated with poly(dA:dT) (2 mg/ml, 3 h) were used as a posi- tive control. (C) Immunoblotting probing for caspase-1 (p45 proform and p11, cleaved CARD fragment), proIL-1b and gasdermin D (GSDMD) in unprimed or LPS-primed human podocytes. PMA-primed THP-1 cells treated with nigericin were used as a positive control for inflammasome activation. (D) ELISA for IL-6 secretion in unprimed or LPS-primed human podocytes. PMA-primed THP-1 cells are used as a positive control. Repre- sentative of experiments were performed at least three independent times. nd, not detected.

Article Snippet: Podocytes were cultured as per manufacturer’s protocol in Human Podocyte Complete Growth Medium (Celprogen) and on Human Podocyte Cell Culture Extra-cellular Matrix (Celprogen).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

doi: 10.4049/jimmunol.2100483

Figure Lengend Snippet: FIGURE 5. Gene expression and podocyte proliferation in Aim2/ (B6) mice. (A) Table of top 19 dysregulated genes from nCounter gene expression analy- sis of Aim2/ (B6) mice and Aim21/1 littermates using PanCancer Mouse IO 360 Panel. n 5 3; p < 0.01, BH p 5 0.26. (B) qPCR for Nphs1 and Wt1 gene expression in Aim2/ (B6) mice and Aim21/1 littermates. n 5 35; *p < 0.05, **p < 0.01, Student t test. (C) Phase-contrast podocyte outgrowth from wild- type and Aim2/ (B6) glomeruli (original magnification 40) and (D) quantification of outgrown areas 5 d after seeding. Mean ± SD, n 5 45. ****p < 0.0001 by Student t test. (E) Immunoblotting for AIM2, WT1, and the CDK inhibitor p21 (CDKN1A) in podocytes nucleofected with 5, 25, and 100 ng of a plasmid expressing FLAG-Myc-tagged AIM2. Uncropped immunoblots are shown in Supplemental Fig. 9A. (F) Quantification (densitometry) of WT1 and p21 immuno- blotting. Mock-nucleofection versus FLAG-Myc-AIM2 nucleofected cells (100 ng), n 5 3. * p < 0.05, ** p < 0.01, Student t test. (G) Dual reporter luciferase assay. Primary human podocytes were nucleofected with pGL-Neph-Luc (150 ng), TK-Renilla (15 ng) in the presence of WT1 (isoform A) (100 ng), AIM2 (50 ng), or both for 24 h, and protein lysates were subject to luciferase assay (firefly/Renilla) Mean ± SD, n 5 4 independent experiments. *p < 0.05 by ANOVA with Tukey post hoc test.

Article Snippet: Podocytes were cultured as per manufacturer’s protocol in Human Podocyte Complete Growth Medium (Celprogen) and on Human Podocyte Cell Culture Extra-cellular Matrix (Celprogen).

Techniques: Gene Expression, Western Blot, Plasmid Preparation, Expressing, Luciferase