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1 Supplementary Information Intrinsic Photosensitivity Enhances Motility of T Lymphocytes by Phan X. Thieu, Barbara Jaruga, Sandeep C. Pingle, Bidhan C. Bandyopadhyay, & Gerard P. Ahern Supplementary Figure 1. Comparison of Ca 2+ responses evoked by T cell receptor mediated signaling and blue light irradiation. Mouse CD4+ T cells were incubated with 5 μg/ml biotinylated anti-cd3 antibody (BD Biosciences) for 10 min at RT followed by cross-linking with 5 μg/ml streptavidin (Pierce Chemical Co.) as indicated. A 30 s pulse of blue light (20 mw cm -2 ) was used to trigger photosignaling. Ca 2+ imaging was performed in Fluo4 loaded cells. The dye was excited with low intensity blue light (0.8 mw cm -2 ) 1
2 Supplementary Figure 2. Light treatment does not affect T cell proliferation. Mouse splenocytes were stimulated with plate bound anti-cd3 antibody (10 µg/ml) for 3 days with or without daily irradiation with full spectrum light. Cell were counted using MTT assay. 2
3 Supplementary Figure 3. Lck activation by light in Jurkat and Lck-null Jurkat cells. Blue light induces phosphorylation of Src kinase as detected with a generic psrc antibody (mouse Y416). The prominent immunoreactive band is absent in J.Cam1.6 (Lck-null) cells confirming predominant activation of Lck. 3
4 Supplementary Figure 4. White light activates Zap-70 in a Lck-dependent manner. Induction of phospho-zap-70 by white light in Jurkat and J.Cam1.6 (Lck-deficient) cells. 4
5 Supplementary Figure 5. Opsin 3 (OPN3) does not contribute to light signaling in Jurkat cells. A, inhibition of Opsin 3 mrna expression in Jurkat cells by sirna. B, inhibition of Opsin 3 mrna expression does not disrupt blue light-evoked Ca 2+ signaling. 5
6 Supplementary Figure 6. Light activates Ca 2+ independently of Cryptochrome proteins. Inhibiting protein expression of cryptochrome 1&2 (CRY1&2) (A) in Jurkat cells does not affect light-evoked Ca 2+ signaling (B). 6
7 Supplementary Figure 7. Blue light irradiation of riboflavin and flavin adenine dinucleotide generates H 2. Solutions containing 5 μm of riboflavin or flavin adenine dinucleotide were irradiated with 600 mj cm -2 blue light with or without docosahexoenoic acid or β-carotene (10 μm). [H 2 ] was measured using a colorimetric Oxired assay (Abcam). 7
8 Supplementary Figure 8. H 2 increases intracellular [Ca 2+ ] in Jurkat cells. Fluo4 fluorescence in individual cells before and after a two-minute exposure to 0, 30, 100 μm and 1 mm H 2 (n=12-15 cells). Non-responding and responding cells are denoted as black and red respectively. Note that H 2 in a concentration dependent manner increases the number of responding cells. Mean F/F o (3.3 ± 0.6 and 4.1 ±0.2) was not significantly different (P=0.15) for 100 μm and 1 mm treatments. F 0 was measured using <10 mj cm -2 blue light that did not affect subsequent fluorescence measured 2 minutes later. 8
9 3b 56 kda 260 mj/cm mj/cm mj/cm mj/cm mj/cm mj/cm 2 PP2 plck (Y394) β-actin 3f pplc-γ1 (Y783) White light PLC-γ1 White light 191 kda 97 kda 3d 59 kda 57 kda 260 mj/cm mj/cm 2 psrc (Y416) 260 mj/cm mj/cm 2 β-actin 3,4e 80 kda 60 kda 480 mj/cm 2 (5 min) 1440 mj/cm 2 (15 min) 6960 mj/cm 2 (2 min) H 2 2mM (2 min) pzap-70 (Y319) 480 mj/cm 2 (5 min) 1440 mj/cm 2 (15 min) 6960 mj/cm 2 (2 min) H 2 2mM (2 min) β-actin 4d 4e H 2 2mM 500 mj/cm 2 0 mj/cm 2 H 2 2mM 500 mj/cm 2 0 mj/cm 2 untransfected 56 kda 56 kda psrc β-actin 0 mj/cm 2 50 mj/cm mj/cm mj/cm mj/cm 2 H 2 2mM psrc β-actin HEK-Lck CHO- Lck Supplementary Figure 9. Full-sized immunoblots for Figures 3b-f, 4d&4e. 9
10 Supplementary Movie 1. Blue light irradiation (5 mw cm -2 ) stimulates an increase in [Ca 2+ ] in Jurkat T cells; 7.5x speed. Supplementary Movie 2. Single cell Ca 2+ and motility response to blue light (488 nm laser 2.7 mw cm -2 ; 20x speed). Supplementary Movie 3. Blue light triggers extension of lamellipodia in Jurkat cells (488 nm laser 2.7 mw cm -2, 37ºC, 20x speed). 10
Intracellular MHC class II molecules promote TLR-triggered innate. immune responses by maintaining Btk activation
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Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow
SUPPLEMENTARY DATA Supplementary Figure Legends Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow cytometry analysis of PMVs labelled with annexin-v-pe (Guava technologies)
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
Plasma exposure levels from individual mice 4 hours post IP administration at the
Supplemental Figure Legends Figure S1. Plasma exposure levels of MKC-3946 in mice. Plasma exposure levels from individual mice 4 hours post IP administration at the indicated dose mg/kg. Data represent
Nature Immunology: doi: /ni.3631
Supplementary Figure 1 SPT analyses of Zap70 at the T cell plasma membrane. (a) Total internal reflection fluorescent (TIRF) excitation at 64-68 degrees limits single molecule detection to 100-150 nm above
Supplementary Figure 1 CD4 + T cells from PKC-θ null mice are defective in NF-κB activation during T cell receptor signaling. CD4 + T cells were
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Supplementary Materials
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot
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SUPPLEMENTARY INFORMATION
doi: 1.138/nature89 IFN- (ng ml ) 5 4 3 1 Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) 1 8 6 4 *** ** Lymph node cells
condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1%
FIGURE LEGENDS Supplementary Fig 1 (A) sumoylation pattern detected under denaturing condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1% SDS in the presence and absence
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Supplementary Materials for
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Supplementary Figure 1: Characterisation of phospho-fgfr-y463 antibody. (A)
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Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after
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Supplementary table and figures
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Supplementary Figure Legends. group) and analyzed for Siglec-G expression utilizing a monoclonal antibody to Siglec-G (clone SH2.1).
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Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Neither the activation nor suppression of the MAPK pathway affects the ASK1/Vif interaction. (a, b) HEK293 cells were cotransfected with plasmids encoding
Supplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. PARP localization patterns using GFP-PARP and PARP-specific antibody libraries GFP-PARP localization in non-fixed (A) and formaldehyde fixed (B) GFP-PARPx
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Figure S1. Loss of Ena/VASP proteins inhibits filopodia and neuritogenesis. (a) Bar graph of filopodia number per stage 1 control and mmvvee (Mena/ VASP/EVL-null) neurons at 40hrs in culture. Loss of all
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Supplementary Figure 1 (Mu) SBP (mmhg) 2 18 16 p Supplementary Figures
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Supporting Information Table of Contents
Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting