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: 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: On-cell patch recording revealed G2 knock-in mouse podocyte cation currents with unitary conductance of 25.2±2.5 pS and NPo of 1.41±0.24 (n=3, −Vp = +50 mV, data not shown), similar to values recorded in on-cell patches of IFNγ-treated Celprogen human G0 podocytes ( ).

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: On-cell patch recording revealed G2 knock-in mouse podocyte cation currents with unitary conductance of 25.2±2.5 pS and NPo of 1.41±0.24 (n=3, −Vp = +50 mV, data not shown), similar to values recorded in on-cell patches of IFNγ-treated Celprogen human G0 podocytes ( ).

Techniques: Transferring, Two Tailed Test, Recombinant