Supporting Information...EV G 0 G 1 G 2 0 50 100 Apoptosis (Caspase 3/7 Cleavage) RFU 150 DMSO Tet...
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Supporting Information Olabisi et al. 10.1073/pnas.1522913113 SI Materials and Methods Generation of Stable, Tetracycline-Inducible Transgenic APOL1 T-REx- 293 Cells. pCMV6-entry vector encoding the full-length APOL1 cDNA (RefSeqORF: 1197) with C-terminal MYC and FLAG tags was purchased from Origene. APOL1 variants G1 and G2 were generated using the QuikChange II site-mutagenesis kit (Agilent Technologies). pcDNA4/APOL1-G0, pCDNA4/APOL1-G1, and pcDNA4/ APOL1-G2 were made by digesting all variants of APOL1 genes out of the pcMV6-entry/APOL1 vectors and cloning them into the KpnI and SacII restriction sites of the pcDNA/TO/ myc-HisB vector (Invitrogen) containing the zeocin-resistant gene. After confirmation of the constructs by DNA sequencing (Gen- wiz), T-REx-293 cells (Invitrogen) were transfected with these APOL1 expression constructs using extreme gene 9-transfection reagent (Roche Diagnostics). Single colony cells possessing APOL1 or its variants were picked after 2 wk of zeocin (Invitrogen) selection. The stable cells, referred to as APOL1-G0-REx-293, APOL1-G1-T-REx-293, and APOL1-G2-T-REx-293 cells were obtained. T-REx-293 cells are modified HEK293 cells that stably express high levels of the tetracycline repressor protein (Tet) under the control of the human CMV promoter. Binding of Tet repressor protein to the TetO2 sequences represses transcription of APOL1. In the presence of tet, Tet repressor is unable to bind the Tet operator, thereby inducing transcription of APOL1 gene. Cell Culture Media. T-REx-293 cells were grown under 5% CO 2 at 37 °C in DMEM supplemented with 10% (vol/vol) tetracycline-FBS (Atlanta Biologicals), 0.2 mg/mL zeocin, 2 μg/mL blasticidin, and 1% antibiotic-antimycotic solution. Modified culture media were formulated as variations of DMEM. DMEM contained 5.4 mM KCl, 109.5 mM NaCl, 17.9 mM NaHCO 3 , and 0.9 mM NaH 2 PO 4 . For Complete K + Culture Media (CKCM), all sodium salts were replaced with equimolar potassium salts. In media supplemented with the organic monovalent cation, N-methyl-D-glucamine (NMDG), NaCl was replaced with NMDG·HCT. All media con- tained 1.8 mM CaCl 2 , 0.81 mM MgSO 4 , 1 mM sodium pyruvate, 4 mM L-glutamine, 25 mM glucose, 0.04 mM phenol red, and 0.000045 mM Fe(NO 3 ) 3 and were supplemented with 20 mL/L MEM Amino Acids Solution (Life Technologies) and 10 mL/L MEM Vitamin Solution (Sigma). Media tonicities ranged between 300 and 340 mOsm/kg H 2 O (Osmette A; Precision Systems) with pH values ∼7.5. MEK inhibitor (PD0325901) and p38 inhibitors (Losmapimod, Vx-702, and LY2228820) were from Selleckchem. P38 inhibitor (SB202190) and JNK inhibitor (SP600125) were from Santa Cruz. Cytotoxicity Assays. T-REx-293 cells (1–2 × 10 5 ) were seeded in each well of a 96-well plate. After 12 h of growth, cells were treated with or without 50 ng/mL tetracycline in complete DMEM. Cy- totoxicity and viability were measured 24 h later (Multi Tox-Fluor Multiplex Cytotoxicity assay; Promega) as previously described (58). The cytotoxicity component measures cleavage of cell- impermeant, fluorogenic peptide substrate by dead-cell pro- tease released from cells that have lost membrane integrity. Viability was determined based on cell-permeable fluorogenic peptide substrate that requires cleavage by protease active only in living cells. Apoptosis was measured using the ApoTox-Glo Triplex Assay (Promega), based on cleavage of luminogenic DEVD-peptide substrate by active caspase-3/7. Each treatment condition was run in triplicate, with values averaged to obtain a single data point. Measurement of Intracellular K + with XRpro X-Ray Fluorescence Technology. Intracellular K + was measured using Icagen XRpro X-ray fluorescence analysis, which provides label-free, direct mea- surements of element content for cell populations grown in multiwell plates, and under standard growth conditions. Elements with atomic number 13 (aluminum) and greater are directly quantified based on characteristic X-ray fluorescence emission (41–43). For ion flux measurements,T-REx-293 cells were seeded in 96-well plates at 10 5 cells per well and grown overnight. Expression of APOL1/APOL1 risk variants was induced by treating cells with tetracycline for 3, 6, or 9 h either in DMEM or CKCM. There were no changes in cell number during this 9-h period. Media were removed, and cells were rinsed quickly with wash buffer containing 150 mM NaCl, 5 mM glucose, 1 mM MgCl 2 , 2 mM CaCl 2 , 0.8 mM NaHPO 4 , 0.8 mM NaH 2 PO 4 , and 25 mM Hepes, pH 7.4. Wash buffer was removed from the plates. Cells were flash frozen on dry ice and shipped to Icagen. Cells were analyzed for K content using XRpro X-ray fluorescence in- struments (www. icagen.com), with element signal references to an internal quantitative standard. Signals were averaged from six biological measurements per treatment condition. Initial Screening of Signaling Pathways with PathScan Signaling Nodes Multi-Target Sandwich ELISA Kit (Cell Signaling). After in- duction of 90% confluent T-REx-293 cells with tetracycline for 9 h, cell lysate was prepared with 1× Triton lysis buffer (Cell Signaling). Then, 1:1 diluted lysates (100 μL) were incubated overnight at 4 °C with capture antibodies against Akt, p-AKT (S473), MEK1, p-p38 MAPK (T180/Y182), STAT3, or p-NF-κB p65 (S536). After removal of unbound lysates, detection anti- bodies against Akt, p-AKT (S473), p-MEK1/2 (S217/221), p38 MAPK, p-STAT3 (Y705), and NF-κB p65 were added to detect the captured target protein. An HRP-conjugated secondary an- tibody was then used to recognize the bound detection antibody. The amount of bound target protein was quantitated from the magnitude of absorbance read at 450 nm by a spectrophotometer (xMark Microplate Spectrophotometer; Bio-Rad). IL-6 Withdrawal Assay. T-REx-293 cells were induced with 50 ng/mL tetracycline for 8 h in serum-free DMEM. An IL-6 withdrawal assay was performed. Briefly, phosphorylation of STAT3 was induced with 10 ng/mL IL-6. After 30 min, IL-6 supplemented media was withdrawn, and cells were rinsed twice with IL-6–free, serum- free DMEM. Cells were then lysed either immediately or after 30 or 60 min. Control cells were not treated with IL-6. Clarified lysates were separated by SDS/PAGE, and levels of p-STAT3 (Y705) and total STAT3 were determined by immunoblotting. Western Blotting. Cells were washed with PBS and lysed in 1× Triton lysis buffer (Cell Signaling). Cell lysates were sonicated and cleared by centrifugation for 10 min at 14,000 × g at 4 °C. Proteins (20– 50 μg) were heated at 97 °C for 5 min in SDS sample buffer plus β-mercaptoethanol, separated by SDS/PAGE (Bio-Rad). PVDF membranes were blocked in 5% (wt/vol) nonfat milk in phosphate buffered saline with Tween for 1 h and then incubated in primary antibodies (1:1,000) overnight at 4 °C, followed by horseradish peroxidase-conjugated antibodies (1:5,000; Santa Cruz Bio- technology). All primary antibodies were from Cell Signaling, except for antibodies against Flag, beta-actin, LC3B (Sigma), and GAPDH-HRP (GeneTex). Signal was developed (Super- Signal West DURA kit; Fisher Scientific) and imaged (Fluo- rChem E; Bio-Techne). Densitometry was performed using FluorChem Q, Version 3.2.2 (Cell Bioscences). Olabisi et al. www.pnas.org/cgi/content/short/1522913113 1 of 6
Transcript of Supporting Information...EV G 0 G 1 G 2 0 50 100 Apoptosis (Caspase 3/7 Cleavage) RFU 150 DMSO Tet...
Supporting InformationOlabisi et al. 10.1073/pnas.1522913113SI Materials and MethodsGeneration of Stable, Tetracycline-Inducible Transgenic APOL1 T-REx-293 Cells. pCMV6-entry vector encoding the full-length APOL1cDNA (RefSeqORF: 1197) with C-terminal MYC and FLAG tagswas purchased from Origene. APOL1 variants G1 and G2 weregenerated using the QuikChange II site-mutagenesis kit (AgilentTechnologies). pcDNA4/APOL1-G0, pCDNA4/APOL1-G1, andpcDNA4/ APOL1-G2 were made by digesting all variants ofAPOL1 genes out of the pcMV6-entry/APOL1 vectors and cloningthem into the KpnI and SacII restriction sites of the pcDNA/TO/myc-HisB vector (Invitrogen) containing the zeocin-resistant gene.After confirmation of the constructs by DNA sequencing (Gen-wiz), T-REx-293 cells (Invitrogen) were transfected with theseAPOL1 expression constructs using extreme gene 9-transfectionreagent (Roche Diagnostics). Single colony cells possessing APOL1or its variants were picked after 2 wk of zeocin (Invitrogen)selection. The stable cells, referred to as APOL1-G0-REx-293,APOL1-G1-T-REx-293, and APOL1-G2-T-REx-293 cells wereobtained. T-REx-293 cells are modified HEK293 cells that stablyexpress high levels of the tetracycline repressor protein (Tet)under the control of the human CMV promoter. Binding of Tetrepressor protein to the TetO2 sequences represses transcriptionof APOL1. In the presence of tet, Tet repressor is unable to bindthe Tet operator, thereby inducing transcription of APOL1 gene.
Cell Culture Media. T-REx-293 cells were grown under 5% CO2 at37 °C in DMEM supplemented with 10% (vol/vol) tetracycline-FBS(Atlanta Biologicals), 0.2 mg/mL zeocin, 2 μg/mL blasticidin, and1% antibiotic-antimycotic solution. Modified culture media wereformulated as variations of DMEM. DMEM contained 5.4 mMKCl, 109.5 mM NaCl, 17.9 mM NaHCO3, and 0.9 mM NaH2PO4.For Complete K+ Culture Media (CKCM), all sodium salts werereplaced with equimolar potassium salts. In media supplementedwith the organic monovalent cation, N-methyl-D-glucamine(NMDG), NaCl was replaced with NMDG·HCT. All media con-tained 1.8 mM CaCl2, 0.81 mM MgSO4, 1 mM sodium pyruvate,4 mM L-glutamine, 25 mM glucose, 0.04 mM phenol red, and0.000045 mM Fe(NO3)3 and were supplemented with 20 mL/LMEM Amino Acids Solution (Life Technologies) and 10 mL/LMEM Vitamin Solution (Sigma). Media tonicities ranged between300 and 340 mOsm/kg H2O (Osmette A; Precision Systems) withpH values ∼7.5. MEK inhibitor (PD0325901) and p38 inhibitors(Losmapimod, Vx-702, and LY2228820) were from Selleckchem.P38 inhibitor (SB202190) and JNK inhibitor (SP600125) were fromSanta Cruz.
Cytotoxicity Assays. T-REx-293 cells (1–2 × 105) were seeded ineach well of a 96-well plate. After 12 h of growth, cells were treatedwith or without 50 ng/mL tetracycline in complete DMEM. Cy-totoxicity and viability were measured 24 h later (Multi Tox-FluorMultiplex Cytotoxicity assay; Promega) as previously described(58). The cytotoxicity component measures cleavage of cell-impermeant, fluorogenic peptide substrate by dead-cell pro-tease released from cells that have lost membrane integrity.Viability was determined based on cell-permeable fluorogenicpeptide substrate that requires cleavage by protease active onlyin living cells. Apoptosis was measured using the ApoTox-GloTriplex Assay (Promega), based on cleavage of luminogenicDEVD-peptide substrate by active caspase-3/7. Each treatmentcondition was run in triplicate, with values averaged to obtain asingle data point.
Measurement of Intracellular K+ with XRpro X-Ray FluorescenceTechnology. Intracellular K+ was measured using Icagen XRproX-ray fluorescence analysis, which provides label-free, direct mea-surements of element content for cell populations grown in multiwellplates, and under standard growth conditions. Elements with atomicnumber 13 (aluminum) and greater are directly quantified based oncharacteristic X-ray fluorescence emission (41–43). For ion fluxmeasurements,T-REx-293 cells were seeded in 96-well plates at 105
cells per well and grown overnight. Expression of APOL1/APOL1 riskvariants was induced by treating cells with tetracycline for 3, 6, or 9 heither in DMEM or CKCM. There were no changes in cell numberduring this 9-h period. Media were removed, and cells were rinsedquickly with wash buffer containing 150 mM NaCl, 5 mM glucose,1 mM MgCl2, 2 mM CaCl2, 0.8 mM NaHPO4, 0.8 mM NaH2PO4,and 25 mM Hepes, pH 7.4. Wash buffer was removed from theplates. Cells were flash frozen on dry ice and shipped to Icagen. Cellswere analyzed for K content using XRpro X-ray fluorescence in-struments (www. icagen.com), with element signal references toan internal quantitative standard. Signals were averaged from sixbiological measurements per treatment condition.
Initial Screening of Signaling Pathways with PathScan SignalingNodes Multi-Target Sandwich ELISA Kit (Cell Signaling). After in-duction of 90% confluent T-REx-293 cells with tetracycline for9 h, cell lysate was prepared with 1× Triton lysis buffer (CellSignaling). Then, 1:1 diluted lysates (100 μL) were incubatedovernight at 4 °C with capture antibodies against Akt, p-AKT(S473), MEK1, p-p38 MAPK (T180/Y182), STAT3, or p-NF-κBp65 (S536). After removal of unbound lysates, detection anti-bodies against Akt, p-AKT (S473), p-MEK1/2 (S217/221), p38MAPK, p-STAT3 (Y705), and NF-κB p65 were added to detectthe captured target protein. An HRP-conjugated secondary an-tibody was then used to recognize the bound detection antibody.The amount of bound target protein was quantitated from themagnitude of absorbance read at 450 nm by a spectrophotometer(xMark Microplate Spectrophotometer; Bio-Rad).
IL-6 Withdrawal Assay.T-REx-293 cells were induced with 50 ng/mLtetracycline for 8 h in serum-free DMEM.An IL-6 withdrawal assaywas performed. Briefly, phosphorylation of STAT3 was inducedwith 10 ng/mL IL-6. After 30 min, IL-6 supplemented media waswithdrawn, and cells were rinsed twice with IL-6–free, serum-free DMEM. Cells were then lysed either immediately or after30 or 60 min. Control cells were not treated with IL-6. Clarifiedlysates were separated by SDS/PAGE, and levels of p-STAT3(Y705) and total STAT3 were determined by immunoblotting.
Western Blotting.Cells were washed with PBS and lysed in 1×Tritonlysis buffer (Cell Signaling). Cell lysates were sonicated and clearedby centrifugation for 10 min at 14,000 × g at 4 °C. Proteins (20–50 μg) were heated at 97 °C for 5 min in SDS sample buffer plusβ-mercaptoethanol, separated by SDS/PAGE (Bio-Rad). PVDFmembranes were blocked in 5% (wt/vol) nonfat milk in phosphatebuffered saline with Tween for 1 h and then incubated in primaryantibodies (1:1,000) overnight at 4 °C, followed by horseradishperoxidase-conjugated antibodies (1:5,000; Santa Cruz Bio-technology). All primary antibodies were from Cell Signaling,except for antibodies against Flag, beta-actin, LC3B (Sigma),and GAPDH-HRP (GeneTex). Signal was developed (Super-Signal West DURA kit; Fisher Scientific) and imaged (Fluo-rChem E; Bio-Techne). Densitometry was performed usingFluorChem Q, Version 3.2.2 (Cell Bioscences).
Olabisi et al. www.pnas.org/cgi/content/short/1522913113 1 of 6
Immunofluorescence. Cells grown in chamber slides (Nunc Lab-Tek IICC2 Chamber Slide System; Thermo Scientific) were treated as in-dicatedand then rinsedoncewithPBSand then fixedwith coldmethanolfor 4min at−20 °C. Cells were blocked with BlockAid blocking solution(Life Technologies) for 1 h at 37 °C and then incubated overnight withspecified primary antibodies at 4 °C, followed by three washes with PBS,and incubation with appropriate secondary antibodies conjugated withCy3 (1:300; Invitrogen) for 1 h at 37 °C. Cell nuclei were counterstainedwith Hoechst 33342 (NucBlueLive ReadyProbes Reagent; Life Tech-nologies). Coverslips were mounted, and fluorescent signals were ac-quired with a confocal laser scanning microscope (LSM 880; Zeiss).
Quantitative Real-Time PCR.Total RNAwas isolated fromT-REx-293cells using an RNeasy plus Mini kit according to the manufacturer’sinstructions (Qiagen). One microgram of total RNA was reversetranscribed using the first-strand synthesis system (Roche). Quanti-tative PCR was set up in SYBR Green PCR master mix (AppliedBiosystems) and carried out in an ABI Prism 7300 Real Time PCRSystem (Applied Biosystems). The primer sequences were 5′-CG-GACAGCTTGAACAGAATGT-3′ (GP130-Fwd), 5′-ACCATCC-CACTCACACCTCA-3′ (GP130-Rev), 5′-TCCCTGGAGAAGA-GCTACGA-3′ (beta-actin-Fwd), and 5′-AGCACTGTGTTGGCG-TACAG (beta-actin-Rev).
Fig. S1. Schematic representation of tetracycline-inducible APOL1 expression vector stably transfected into T-REx-293 cells. The * represents mutations thatconstitute G1 (S342 and I 384M) and G2 (deletion of N388 and Y389).
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1G0G
LC3-I
LC3-II
LC3-II/I
Tet:
Chloroquine:
- -- -
++ +
+ - -- -
++ +
+
0.8 1.1 1.6 1.7 0.9 1.9 1.7 2.6
Fig. S2. Chloroquine increased G1 APOL1-induced LC3-II accumulation. G0 or G1 APOL1–T-REx-293 cells were tet-induced (50 ng/mL) in the presence orabsence of chloroquine (5 μM) for 9 h. Autophagy flux was quantitated at 9 h by measuring LC3-II/LC3-I in cell lysates.
DMSO Te
t
Tet +CQ(1uM)
Tet +CQ(5uM)
Tet +Wortmanin(1uM)
Tet +Wortmanin(10uM)
0.0
0.5
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1.5
2.0
Cytotoxicity/Viability
Fig. S3. Autophagy inhibitors do not reduce G1 APOL1 cytotoxicity in T-REx-293 cells. Cytotoxicity: viability ratio of G1 APOL1–T-REx-293 cells after tet in-duction (50 ng/mL) in the presence or absence of autophagy inhibitors chloroquine (CQ) or wortmannin. All treatments lasted 24 h. Each bar represents mean ±SD of experimental triplicates.
EV G0 G1 G20
50
100
150
Apoptosis(Caspase3/7Cleavage)RFU DMSO
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Fig. S4. Expression of G1 or G2 APOL1 does not increase apoptosis in T-REx-293 cells. Quantitation of apoptosis (caspase-3/7 cleavage) after tet-induction ofG0, G1, or G2 APOL1 expression for 24 h. Each bar represents mean ± SD.
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Flag-APOL1
STAT3
STAT3
DAPIDAPI DAPI++
-OSM
+OSM
EV
-OSM
+OSM
G0
G1
G2
-OSM
+OSM
-OSM
+OSM
Fig. S5. Representative confocal micrographs of maximum intensity projection (15 optical Z-stacked images) of STAT3 nuclear localization G0, G1, or G2 T-REx-293 cells that were tet-induced for 9 h, followed by treatment or not with OSM (10 ng/mL, 10 min). EV are empty vector control cells. All images were collectedunder identical settings and were processed similarly. (Scale bars: 100 μm.)
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Fig. S6. Expression of G1 or G2 APOL1 in T-REx-293 cells activates MAPK signaling pathways. After inducing expression of G0, G1, or G2 APOL1 in T-REx cellsfor 9 h using tetracycline, cell lysates were immunoblotted for the p38, JNK, and ERK and their substrates. Note that ATF2 is a substrate of both p38 MAPKand JNK.
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Fig. S7. MEK and JNK are activated after 6 h of expression of G1 or G2 APOL1 in T-REx-293 cells. After tetracycline-induced expression of G0, G1, or G2 APOL1for the specified time points, cell lysates were immunoblotted for phospho-MEK (S217/221), MEK (total), and phospho-JNK.
LC3-I
LC3-II
LC3-II/I
B-actin
Tet:SB202190, duration(hr):
G2
--- - 9
0.63 0.83 0.59 0.94 0.96 0.93
9 6 3+ + + +
Fig. S8. Inhibition of p38 MAPK does not reduce G2 APOL1-induced autophagy. G2 APOL1–T-REx-293 cells were induced or not with tet (50 ng/mL) for 9 h(total). Cells were exposed to SB202190 (10 μM) for 3, 6, or 9 h. Autophagy was quantitated in all cells after 9 h.
LC3-II/I
LC3-ILC3-II
B-actin
DMEM:CKCM:
Tet:
0.47 0.60 0.75 0.75 1.00 1.05 0.84 1.06
--+
+-+
-+-
++-
+-+
++-
+-+
++-
EV G0 G1 G2
Fig. S9. Elevated extracellular [K+] does not reduce G1 or G2 APOL1-induced autophagy. G0, G1, or G2 APOL1–T-REx-293 cells were induced with tet for 9 h inDMEM or CKCM. Autophagy was quantitated in all cells after 9 h.
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