Supplementary Information for mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3 Guoli Shi, Stosh Ozog, Bruce E. Torbett, and Alex A. Compton Alex A. Compton, PhD Email:
[email protected] This PDF file includes: Supplementary Materials & Methods Figs. S1 to S4 References for SI
1 www.pnas.org/cgi/doi/10.1073/pnas.1811892115
Supplementary Materials & Methods Cell culture and mTOR inhibition. HeLa (ATCC: CCL-2), HEK293T (ATCC: CRL326), primary human foreskin fibroblasts (ATCC: SCRC-1041), TZM-bl (NIH AIDS Reagent Resource: 8129) and murine embryonic fibroblasts (a gift from M. Diamond) and any derivatives produced in this study were cultivated at 37°C and 5% CO2 in DMEM (Gibco) complemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco) and regularly passaged with the aid of Trypsin-EDTA (0.05%). Rapamycin, Torin 1, bafilomycin A1, MG-132, cycloheximide (Sigma) and EGA (Calbiochem) were reconstituted in DMSO and stored in aliquots at -20°C. Earle’s Balanced Salt Solution (EBSS) without calcium, magnesium, or phenol red was purchased from Gibco. Immunofluorescence confocal microscopy. Cells were seeded (15,000/well) in Lab-Tek II chamber slides (Thermo Fisher) overnight and treated with DMSO, rapamycin (20 uM), or rapamycin and bafilomycin A1 (1uM) for four hours followed by fixation/permeabilization with Cytofix/Cytoperm (BD) for 20 minutes. Primary antibodies were diluted in Odyssey blocking buffer in PBS (Li-Cor) and incubated with cells for 30 minutes at room temperature. Cells were washed and incubated with Alexa Fluor-labeled secondary antibodies for 30 minutes at room temperature. Cells were mounted with ProLong Gold antifade reagent (Life Technologies). Images were acquired with a TCS SP8 confocal laser scanning microscope (Leica). For each experiment, the images were acquired using identical laser power and digital gain settings on the same microscope. 12-bit images with a 1024 x 1024 field size were acquired at 63X or 100X oil-immersion magnification. 2D and 3D images were created and analyzed using ImageJ (Fiji). Colocalization analysis was performed using the “Coloc 2” tool in Fiji. The following antibodies were used: anti-IFITM3 (Abcam, EPR5242), anti-IFITM2/3 (Proteintech, 66081-1-Ig), anti-LAMP1 (Santa Cruz Biotechnology, H5G11, sc-18821), anti-EEA1 (BD, clone 14, 610456), anti-CD63 (Santa Cruz Biotechnology, MX-49, sc5275), and anti-LC3 (MBL, PM036). LysoTracker Deep Red (Life Technologies) was diluted to 50 nM in DMEM + 10% fetal bovine serum and used to stain living cells for 15 minutes prior to immediate imaging. CD34+ HSPC isolation, culture, drug treatment, and analysis. CD34+ HSPC were isolated from cord blood and cultured as outlined as previously described1. Adult CD34+ HSPC isolated from G-CSF-mobilized peripheral blood were obtained from the CoOperative Center for Excellence in Hematology at the Fred Hutchinson Cancer Research Center. Cells were thawed and pre-stimulated in GMP-Stem Cell Growth Medium (CellGenix) for 48 hours with 100 ng/mL each of human TPO, hSCF and Flt-3 ligand (Peprotech). Cells were treated for 4 hours with the indicated doses of rapamycin before addition of VSV-G pseudotyped lentiviral vector carrying the F12-UbC-EGFP transfer vector (Addgene: 14884) at a MOI of 15. Lentivector was incubated with 100,000 CD34+ cells for 30 minutes in 48-well plates and washed three times in PBS + 2% FBS to remove surface-bound virus. Cells were centrifuged onto positively charged confocal grade microscopy slides at 450 x g for 5 min. Cells were fixed in 3.7% confocal grade paraformaldehyde (Thermo Fisher) for 8 minutes and washed three times with confocal 2
wash buffer (PBS + 0.1% saponin + 1% BSA). Cells were stained with anti-IFITM2/3 (Proteintech, 66081-1-Ig), and anti-LAMP1 Antibody (Thermo Fisher, PA1-654A) overnight at 4°C. Slides were washed three times and incubated for one hour with Alexa Fluor-conjugated secondary antibodies for one hour at room temperature. Slides were mounted for three days with ProLong Gold Antifade Reagent (Thermo Fisher, P36930). Confocal microscopy analysis of endosomal markers was performed using a Zeiss 710 laser scanning confocal microscope at 64X oil-immersion magnification at a 2-fold digital zoom with a 1024 x 1024 field size. For establishing voltage and aperture parameters, snap images were captured for each channel using control slides stained only with secondary antibody. Images were collected in 16-slice intervals and 0.2 µm apart. At least 50 cells were imaged for each condition. Z-stacks were analyzed with Imaris (Bitplane) using the ImarisCell analysis module to quantify number and intensity of IFITM2/3 and LAMP-1+ vesicles. Statistical analysis of vesicle quantity and staining intensity was conducted using Prism 7 (GraphPad) and evaluated with the Kruskal-Wallis test using Dunn’s multiple comparison correction. Reverse transcription quantitative polymerase chain reaction (RT-qPCR). HeLa cells were treated with DMSO or rapamycin (20 uM) for four hours and total RNA was extracted with Tri-Reagent (Sigma). RNA was incubated with Turbo DNAse I (Ambio) for one hour at 37°C followed by first-strand cDNA synthesis using the SuperScript III kit (Invitrogen). Quantitative PCR of cDNA templates was run on a DNA Engine Opticon 2 detection system using Power SyberGreen PCR Master Mix (Applied Biosciences) paired with the following primer sets: 5’GAATCACACTGTCCAAACCTTC-3’ and 5’-CATAGGCCTGGAAGATCAGC-3’ (IFITM3); 5’-GAAATCCCATCACCATCTTCCAGG-3’ and 5’GAGCCCCAGCCTTCTCCATG-3’ (GAPDH).
3
5 5 1010
5 5 1010
5
10105
3 3 1010
3 3 1010
103 3
4.3 4.3
APC-A APC-A
APC-A APC-A
APC-A APC-A
4
10104
22.5 22.5
4 4 1010
28.9 28.9
4 4 1010
10
2
10102
2
10102
2 2 1010
00
0 0
0 0 0 0 0 0 50K 50K 100K 100K 150K 150K 200K 200K 250K 250K FSC-A FSC-A Living Living DMSO_IFN250-ON_009.fcs DMSO_IFN250-ON_009.fcs Event Count: 6657 Event Count: 6657
50K 50K
00 50K 50K 100K 100K 150K 150K 200K 200K 250K 250K FSC-A FSC-A Living Living Rapa30_IFN250-ON_027.fcs Rapa30_IFN250-ON_027.fcs Event Count: 5460 Event Count: 5460
100K 100K 150K 150K 200K 200K 250K 250K FSC-A FSC-A
Living Living Rapa20_IFN250-ON_018.fcs Rapa20_IFN250-ON_018.fcs Event Count: 7384 Event Count: 7384
20170911 Rapa Torin WT ATG9.jo
A
NoNoIFN-! IFN No IFN No drug
Layout: Rapa v T
IFN-! + DMSO
IFN IFN+ +Rapamycin Rapamycin(20 (20uM) uM)
37.6
9.5
IFITM3
0.4
IFN-!(30uM) IFN IFN++Rapamycin Rapamycin(30 uM) + Rapa 30 uM
IFN-! + Rapa 20 uM
IFN IFN+ +DMSO DMSO
Sample shSCR Exp shSCR Exp shSCR Exp shSCR Exp
Drug duration:
5.2
% 2_DMSO_014.fcs 83.1 2_Rapa 1 uM_015.fcs 90.4 2_Rapa 5 uM_016.fcs 87.7 2_Rapa 20 uM_017.fcs 65.2
Gate Living Living Living mid
4hours hourspost-drug post-drug 4 4hr
Mean:APC-A 110 102 80.5 26.8
Sample shSCR Exp shSCR Exp shSCR Exp shSCR Exp shSCR Exp
293T IFNb Rapa
FSC 42.9
28.2
2_DMSO Torin_023.fcs 2_Torin 1 uM_018.fcs 2_Torin 5 uM_019.fcs 2_Torin 20 uM_020.fcs 2_Torin 50 uM_021.fcs
% 58.2 87.6 88 88.2 61.9
Mean:APC-A 59.8 101 130 88.2 35.8
Gate Living Living Living Living a
Mean:APC-A 59.8 101 130 88.2 35.8
11.0 88hr hours post-drug 8 hours post-drug
10
0
10
1
mid
2
10 APC-A
10
3
10
4
100
101
a
28.9
22.5
0
10
2
3
% IFITM3+ cells following IFN-!
Sample shSCR Exp shSCR Exp shSCR Exp shSCR Exp
10 APC-A
10
8/31/18 6:44 6:44 PM PM 8/31/18 Living
3_DMSO_001.fcs 3_Rapa 1 uM_002.fcs 3_Rapa 5 uM_003.fcs 3_Rapa 20 uM_004.fcs
% 89.3 90.5 90.4 90.3
Mean:APC-A 2928 3079 2995 1369
104
24 24hrhours post-drug 24 hours post-drug
Sample shSCR Exp 3_DMSO 2_009.fcs shSCR Exp 3_Torin 1 uM_005.fcs shSCR Exp 3_Torin 5 uM_006.fcs shSCR Exp 3_Torin 20 uM_007.fcs shSCR Exp 3_Torin 50 uM_008.fcs
% 86.6 90.5 81.2 84.3 81.2
Mean:APC-A 3264 2914 3136 3022 1617
IFN DMSO IFN Rapa 20 Rapamycin
80
30 uMIFN Rapa 30 20 uM DMSO 0 10 10
60 40
103
4.3
100
4
102 APC-A
10
5
Page 11 of of 11 Page 2
20
Living
3
APC-A
104
(FlowJo v9
105
0
4 hr 4 hr
B 293T-IFITM Rapa Torin 1 Frequency
30 uM 20 uM 10 uM DMSO
0 102
103
104
88 hr hr Drug duration Sample HeLa Rapa HeLa Rapa HeLa Rapa HeLa Rapa
Torin1_DMSO 30_002.fcs Torin1_Torin 10_004.fcs Torin1_Torin 20_005.fcs Torin1_Torin 50_007.fcs
24 hr 24 hr
% 90.2 90.5 82.5 82.9
Mean:APC-A 3397 3102 2035 1064
105
Living
IFITM3
Rel.Relative IFITM3Infection protein
C 100 80 60
3/30/18 3:10 40 PM
Page 1 of 2
(FlowJo v9.
20 0
D293TR IFITM3
293T D R ΔUb
293T D R Δ17-18
293T D R Δ17-20
293TR D IFITM2
293TR D IFITM1
293T: IFITM3 ΔLys Δ17-18 Δ17-20 IFITM2 IFITM1 WT
Fig. S1. (A) HEK293T cells were seeded in 24-well plates (50,000/well) overnight and treated with 250 IU/mL interferon-b for 24 hours. DMSO or the indicated concentration of rapamycin was added for four, eight, or 24 hours. Cells were fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. Error bars indicate standard error from three experiments. (B) HeLa cells were seeded in 24-well plates (50,000/well) overnight and treated with DMSO or the indicated concentration of Torin 1 for four hours. Cells were fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. (C) HEK293T cell lines stably expressing FLAGtagged IFITM3, IFITM3 mutants, IFITM2, or IFITM1 were seeded in 24-well plates (50,000/well) overnight and treated with DMSO or rapamycin (30 uM) for four hours. Cells were fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. MFI from univariate histograms of IFITM3 or FLAG expression were normalized and averaged, with expression in the DMSO condition of each cell line set to 100. Error bars indicate standard error from three to five experiments.
4
A
B
IFITM3 LAMP1
IFITM3 EEA1
PCC = 0.22 +/- 0.12
PCC = 0.41 +/- 0.12
DMSO
Rapamycin
Rapa + BafA1
IFITM3
EEA1
Merge
C Frequency
Rapamycin (20 uM) 6h 4h 2h 1h 30m 0m 0 102
103
104
105
IFITM3
103
104
105
0 102
LAMP1-RFP
LC3
103
104
105
EEA1 Merge
Rapa + BafA1
DMSO
IFITM3
0 102
LAMP1
D
6h 4h 2h 1h 30m 0m
6h 4h 2h 1h 30m 0m
LC3
Fig. S2. (A) HeLa cells were treated with DMSO for four hours and fixed/permeabilized, stained with anti-IFITM3 and anti-LAMP1 or anti-EEA1, and imaged by immunofluorescence confocal microscopy. The average Pearson’s correlation coefficient or R (PCC) and standard deviation were calculated for ten cells per staining combination, identified by selecting regions of interest (ROI). (B) HeLa cells were treated with DMSO, rapamycin (20 uM), or rapamycin and bafilomycin A1 (1 uM) for four hours and fixed/permeabilized, stained with anti-IFITM3 and anti-EEA1, and imaged by immunofluorescence confocal microscopy. (C) HeLa cells were treated with DMSO or rapamycin (20 uM) for the durations indicated. Cells were fixed/permeabilized, stained with anti-IFITM3, anti-LAMP1, or anti-EEA1, and analyzed by flow cytometry. Univariate histograms from one experiment is shown, representative of three. (D) TZM-bl cells were transfected with LAMP1-RFP for 24 hours. Cells were treated with DMSO or rapamycin (20 uM) for four hours and fixed/permeabilized, stained with anti-IFITM3 and antiLC3, and imaged by immunofluorescence confocal microscopy. Scale bars are 10 microns.
5
1 1 fA 1 1 fA fA Ba Ba in BafA + cin Ba d + y yc d O am a + rve rve O am a + rved ved S S p r p p p a DM Ra Ra Sta St DM Ra Ra Sta Sta
A 15
IFITM3
15
LC3-I LC3-II
LC3 Tubulin
50
HFF
TZM-bl
1 1 20170911 fATorin WT iATG9.jo fA Rapa Torin WT ATG9.jo n 20170911 in Rapa Ba Ba yc yc O am a + SO am a + S p p p p M HeLa WT DM Ra Ra D Ra Ra
B 115
15
Frequency
ATG9a LC3-I LC3-II 100 Living
50
101
10 2 APC-A
103
Tubulin
104
Sample % Mean:APC-A HeLa and ATG90 KO HeLa_WT DMSO_024.fcs 90.2 54.6 HeLa and ATG90 KO HeLa_WT Rapa_025.fcs 86.3 26.6
100
Living
Rapamycin DMSO ATG9a KO
101
101
102
10 2 APC-A
103
103
104
Layout: WT v ATG9 KO HeLa Layout: WT v ATG9 KO HeLa
Sample % Mean:APC-A HeLa and ATG90 KO HeLa_WT DMSO_024.fcs 90.2 54.6 HeLa and ATG90 KO HeLa_WT Rapa_025.fcs 86.3 26.6
10 0
104
10 1
101
105
Living
102
102 APC-A
103
103
104
104
105
Sample % Mean:APC-A HeLa and ATG90 KO HeLa_ATG9 KO DMSO_026.fcs 91 60.2 HeLa and ATG90 KO HeLa_ATG9 KO Rapa_027.fcs 94.7 24.2
10 0 Living
10 1
102 APC-A
103
Sample % Mean:APC-A HeLa and ATG90 KO HeLa_ATG9 KO DMSO_026.fcs 91 60.2 HeLa and ATG90 KO HeLa_ATG9 KO Rapa_027.fcs 94.7 24.2
104
This should accompany a western blot looking at LC3 and p62 in the same cells. This should accompany a western blot looking at LC3 and p62 in the same cells.
IFITM3
HeLa WT
IFITM3
ATG9a KO
C DMSO
Rapa + BafA1
Effect of Raptor Rictor silencing
PCC = 0.04 +/- 0.23
PCC = 0.45 +/- 0.08 IFITM2/3 CD63
IFITM2/3 CD63
(FlowJo v9.9.5)
(FlowJo v9.9.5)
80 60 40 20
to
to r
siRNA:
r
0
Actin
ic
Rictor
Page 1 of 1
R
40
Actin
** ns
Page 1 of 1 100
tro l
140
Raptor
E
ap
40
r tor PM pto10/6/17ic4:41 Ra R
on
140
C
R
siRNA:
Rel.IFITM3 IFITM3 protein Protein Rel.
l ro
10/6/17 4:41 PM ont
C
D
Fig. S3. (A) SDS-PAGE and western blot analysis of whole cell lysates derived from TZM-bl (left) and primary HFF (right) treated with DMSO, rapamycin (20 uM), rapamycin and bafilomycin A1 (1 uM), EBSS medium, or EBSS medium and bafilomycin A1 (1 uM) for four hours. Immunoblotting was performed with anti-IFITM3, and anti-LC3. Tubulin served as loading control. (B) SDS-PAGE and western blot analysis of whole cell lysates derived from HeLa (WT and ATG9a KO) treated with DMSO, rapamycin (20 uM), or rapamycin and bafilomycin A1 (1 uM) for four hours. Immunoblotting was performed with anti-ATG9a and antiLC3. Tubulin served as loading control. HeLa (WT and ATG9a KO) were treated with rapamycin (20 uM) for four hours and fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. Univariate histograms of IFITM3 expression are provided from one experiment, representative of three. (C) HeLa cells were treated with DMSO or rapamycin (20 uM) and bafilomycin A1 (1 uM) for four hours and fixed/permeabilized, stained with anti-IFITM3 and anti-CD63, and imaged by immunofluorescence confocal microscopy. The average Pearson’s correlation coefficient or R (PCC) and standard deviation were calculated for ten cells per staining combination. (D) SDS-PAGE and western blot analysis of whole cell lysates from HeLa cells transfected with siRNA (non-targeting control, Raptor, or Rictor) for 72 hours. Immunoblotting was performed with anti-Raptor and anti-Rictor. Actin served as loading control. (E) HeLa cells were transfected with siRNA (non-targeting control, Raptor, or Rictor) for 72 hours and fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry.
6
A
pQCXIP-FLAG
I
T FI
M1 I
T FI
M2 I
T FI
B
M3
1 A1 fA in af in Ba yc yc +B 1 O O pam pa + A1 am pa A S S f f p Ra Ba Ra Ba DM Ra DM Ra
15
15
IFITM1 Proteintech 60074-1-Ig
15
IFITM2 Proteintech 66137-1-Ig
15
IFITM3 Abcam EPR5242 ab109429
IFITM2/3 25
*
IFITM2/3 (high intensity)
15
55
Actin
40
IFITM1 Proteintech 60074-1-Ig
shRNA Control
HeLa
shRNA IFITM3
IFITM3 Abcam EPR5242 ab109429
15
IFITM2/3 Proteintech 66081-1-Ig
15 55
Actin
40 293T
C
W
T
15
IF
IT
M3
KO
D
W
T
IF
I
3 TM
KO
IFITM3
IFITM3
IFITM3
IFITM2/3
IFITM1
IFITM2
IFITM3 / IFITM2/3
15
IFITM1 / IFITM3
IFITM2 / IFITM3
Actin
Actin
Actin
40
40 HeLa
15
55
55
55 40
E
fA fA cin Ba cin Ba + my a + my O O a a a S S p p p p DM Ra Ra DM Ra Ra
TZM-bl TZM-bl WT
IFITM3 KO
Fig. S4. (A) SDS-PAGE and western blot analysis of whole cell lysates derived from 293T cells transfected with pQCXIP-FLAG-IFITM1, -IFITM2, or -IFITM3 for 48 hours. Immunoblotting was performed with anti-IFITM1, anti-IFITM2, anti-IFITM3, and anti-IFITM2/3. Actin served as loading control. (B) SDS-PAGE and western blot analysis of whole cell lysates derived from HeLa cells stably expressing shRNA (scrambled control or IFITM3) treated with DMSO, rapamycin (20 uM) alone, rapamycin and bafilomycin A1 (1 uM), or bafilomycin A1 (1 uM) alone for four hours. Immunoblotting was performed with anti-IFITM2/3 and enhanced to reveal bands of higher molecular weight (marked with asterisk). Bands specific to IFITM2 and IFITM3 are indicated with red and green arrows, respectively. Actin served as loading control. (C) SDSPAGE and western blot analysis of whole cell lysates derived from HeLa (WT and IFITM3 KO). Immunoblotting was performed with anti-IFITM3 and anti-IFITM2/3. Actin served as loading control. (D) SDS-PAGE and western blot analysis of whole cell lysates derived from TZM-bl (WT and IFITM3 KO). Immunoblotting was performed with anti-IFITM3 and anti-IFITM1. Actin served as loading control. (E) SDS-PAGE and western blot analysis of whole cell lysates derived from TZM-bl (WT and IFITM3 KO) treated with DMSO, rapamycin (20 uM), or rapamycin plus bafilomycin A1 (1 uM). Immunoblotting was performed with anti-IFITM3 and anti-IFITM2. Actin served as loading control.
7
References 1. Wang, C.X., et al. Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells. Blood 124, 913-923 (2014).
8