Supplementary Material s41

1 2 1Supplementary material

2Tumour hypoxia promotes melanoma growth and metastasis via High Mobility Group Box-1

3and M2-like macrophages

5Roman Huber1,†, Barbara Meier1, Atsushi Otsuka1,†, Gabriele Fenini1, Takashi Satoh1, Samuel

6Gehrke1, Daniel Widmer1, Mitchell P Levesque1, Joanna Mangana1, Katrin Kerl1, Christoffer

7Gebhardt3,4, Hiroko Fujii2, Chisa Nakashima2, Kenji Kabashima2, Yumi Nonomura2, Reinhard

8Dummer1, Emmanuel Contassot1,‡,*, and Lars E. French1,‡,*

101. Department of Dermatology, University Hospital Zürich, Zürich 8091, Switzerland

112. Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan

123. Skin cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany

134. Department of Dermatology, Venereology and Allergology, University Medical Center

14 Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim, Germany

16Content:

17 - Supplementary methods

18 o Control of knock-down efficiency and stability

19 o In vitro cell proliferation and apoptosis

20 - Supplementary figures

21 o Fig S1. Assessment and validation of HIF1 and HMGB1 detection and

22 localization by immunofluorescence.

23 o Fig. S2. Selection of HMGB1 knock-downs and stability of silencing efficiency

24 over time in vitro and in vivo

25 o Fig. S3. Validation of B16 cells transduced with lamin-specific shRNA as control.

26 o Fig. S4. The in vitro growth properties of B16 cells transduced with shRNA

27 specific to lamin or HMGB1 are identical.

3 1 4 5 6 28Supplementary methods 29

30Control of knock-down efficiency and stability

31To determine the knock-down stability of the B16-F10 mouse melanoma cell-line transduced

32with shRNA specific to HMGB1, shHMGB1-B16 as well as shLamin-B16 were cultured at

3337°C in 5 % CO2 in cDMEM medium (DMEM supplemented with 1 % L-glutamine and 10 %

34fetal bovine serum) and 1x105 cells were lysed at day 0, 7, 14, 21 and 28 in 10mM Tris pH

357.5, 1% NP-40, 150mM NaCl, 5mM EDTA with protease inhibitors (Roche). The cells lysates

36were subjected to Western-blotting using a rabbit polyclonal anti--actin (Cell Signaling) or a

37rabbit polyclonal anti-HMGB1 antibody (Abcam). Secondary antibodies were coupled to

38alkaline phosphatise (AP). AP detection was performed using the NBT/ BCIP substrate kit

39(Promega, Madison, WC). Blots were scanned using the CanonScan 9950F scanner

40(Canon, Tokyo, Japan).

42In vitro cell proliferation and apoptosis

43To compare in vitro proliferation of B16-F10 stably expressing shRNA specific to HMGB1 or

5 44lamin, 1.5 ×10 cells were cultured at 37°C in 5 % CO2 in DMEM supplemented with 1 % L-

45glutamine and 10 % fetal bovine serum (cDMEM). At days 0, 1, 2 and 3 mitochondrial

46dehydrogenase activity of living cells was measured by incubation with 10 % MTT (Sigma-

47Aldrich) for 4 h at 37°C. Optical densities were measured by the SpectraMax190 plate

48reader (Molecular Devices).

49Proliferation was measured using CFSE. Cells were incubated with PBS + 1 µM CFSE

50(Thermo Fisher Scientific, Waltham, MA) for 10 min at 37°C and seeded in 6-well plates. At

51days 0, 3, 6 and 8 cells were detached and CFSE-related fluorescence intensity was

52determined by flow cytometry. Acquisition was performed with a FACS Canto II (Becton-

53Dickinson) and sample analysis was done using the FACS DIVA software (Becton-

54Dickinson).

55To assess apoptosis in transfected cells by flow cytometry, cells were detached at day 0, 3, 6 7 2 8 9 10 56and 9 and stained with 1.0 µg/ml propidium iodide (Sigma-Aldrich) and Annexin V (Becton-

57Dickinson) for 15 min on ice. Acquisition was performed with a FACS Canto II (Becton-

58Dickinson) and analysed using the FACS DIVA software (Becton-Dickinson).

11 3 12 13 14 59Supplementary figures

60 a Metastatic melanoma cell line, hypoxic condition, control IgGs 61 DAPI Hif-1α HMGB1 DAPI HMGB1 Hif-1α HMGB1 Hif-1α

Metastatic melanoma cell line, normoxic conditions

Metastatic melanoma cell line, hypoxic conditions

b Metastatic melanoma cell line, normoxic conditions HMGB1 HIF1α HMGB1 HIF1α DAPI

100µm Metastatic melanoma cell line, hypoxic conditions

100µm

15 4 16 17 18 62Fig. S1. Hypoxia induces detectable HIF1 and HMGB1 relocalisation in human metastatic

63melanoma cell lines. (a) Immunofluorescence co-labelling with anti-HIF1 and anti-HMGB1

64antibodies of a metastatic melanoma cell line after 72 hrs in hypoxic conditions or left in normoxia. (b)

65Higher magnification of melanoma cells cultured in the same conditions as in (a). HIF1a is detectable

66only when cells are kept under low oxygen and is stabilized in both nucleus and cytosol whereas

67HMGB1 exhibit different localization from nuclear (normoxia) to cytosolic (hypoxia). (a) and (b) show 2

68independent metastatic melanoma cell lines and are representative of experiments repeated 3 times

69with each. 70

19 5 20 21 22 71 a 1.5 lamin shRNA 72 HMGB1 shRNA

1.0 WT-B16 73 T C   -

0.0 Sequence: 1 2 1 2 4 5

2.0 lamin shRNA HMGB1 shRNA 1.5 T C

 1.0 WT-B16  - 2 0.5

0.0 Clone: 1 2 3 4 5 6 1 3 5 7 8 9 11 14 15 16 17 19

b shLamin shHMGB1 WT-B16 cl1s2 cl17s5

-actin

0 7 4 1 8 0 7 4 1 8 D D 1 2 2 D D 1 2 2 D D D D D D

shLamin-B16 c DAPI HMGB1 DAPI HMGB1

shHMGB1-B16

23 6 24 25 26 74Fig. S2. Validation and selection of clones based on HMGB1 knock-down efficiency and

75stability. (a) B16 cells were transduced with 2 sequences of shRNA specific to lamin and 4 HMGB1-

76specific shRNA sequences. Quantitative PCR was performed on in vitro expanded

77transduced/selected cells (puromycin). B16 cells transduced with shRNA specific to lamin (sequence

782) or HMGB1 (sequence 5) were subsequently cloned by the limiting dilution method. Quantitative

79PCR was performed on in vitro expanded transduced/selected clones (puromycin). Results are

80expressed as 2-CT and standardized to wild-type B16 for which a 2-CT value of 1 was attributed

81(dashed line). (b) Cultures of B16 clones transduced with lamin-specific shRNA (clone 1 of sequence

822) or HMGB1 (clone 17 of sequence 5) were harvested, lysed and subjected to western-blot analysis

83using anti-HMGB1 and anti--actin antibodies at the indicated time points. (c) B16 cells transduced

84with lamin-specific shRNA (clone 1 of sequence 2) or HMGB1 (clone 17 of sequence 5) were injected

85s.c. to C57BL/6 mice and the resulting tumours were dissected at day 13 and stained with an anti-

86HMGB1 antibody. Nuclei were visualized using DAPI. Pictures are representative of 7 tumours per

87group. 88

27 7 28 29 30 89

400 shLamin cl1s2 )

3 wild-type m 300 m (

u 200 l o v

m 100 u T

0 0 6 8 9 10 11 12 13 Days after tumor inoculation 90

95Fig. S3. Exclusion of off-target effects upon transduction of B16 cells with shRNA to lamin.

96Wild type and lamin shRNA-expressing B16 cells (cl1s2, control) were injected s.c. into C57BL/6 mice

97and displayed comparable tumour growth in vivo (n=5 mice/group). Representative results of 3

98independent experiments are presented.

31 8 32 33 34 99

100 101 3 a b B16-F10 101 shLamin-B16 101 2 shHMGB1-B16 102 101 1 103 101 0 d e

104 t n u

o 109 c

105 s l l e C 106 108 0.5 shLamin c shHMGB1 0

107 y 0.4 10 a s D

u t 0.3 l

108 a 6 d 10 v e

s i D l 0.2 a O

109 m r 5 o 0.1 10 n 0 2 4 6 8 10 12 14 110 0.0 Days

112 Day 0 Day 3 Day 6 Day 9 d 90 90 90 90 s

113 l l 80 e 80 80 80 c 70 e 70 70 70 v i t 114 i 60 s 60 60 60 o

50 50 50 I 15 15 15 P 15 /

115 V 10 n 10 10 10 i x

e 5 n 5 5 5 116 n A 0 0 0 0 117

119 e 0.4 shLamin shHMGB1 0

a 0.3 e D s

a o t e

121 l d e

r 0.2 e

s i H l a D

122 L m

r 0.1 o n 123 0.0

125Fig. S4. The in vitro growth properties of B16 cells transduced with shRNA to lamin and

126HMGB1 are identical. (a) B16 cells transduced with shRNA specific to lamin or HMGB1 were labelled

127with CFSE and collected after 72 and 144 h and analysed by flow cytometry. (b) B16 cells transduced

35 9 36 37 38 128with lamin- or HMGB1-specific shRNA were counted over a 15-day culture period. (c) Proliferation of

129B16 cells transduced with lamin shRNA (white histograms) or HMGB1 shRNA (black histograms) was

130assessed over a 3-day culture period using the MTT assay. (d) At day 0, 3, 6 and 9, B16 cells

131transduced with shRNA specific to lamin (shLamin) or HMGB1 (shHMGB1) were harvested, stained

132with Annexin V and propidium iodide (PI) and analyzed by flow cytometry. Annexin V +/PI+ cells were

133considered as late apoptotic. As a positive control, wild-type B16 cells (WT) were treated with the

134apoptosis inducer staurosporin. (e) Viability of B16 cells transduced with shRNA specific to lamin

135(white histograms) or HMGB1 (black histograms) was assessed over a 3-day culture period using an

136LDH release assay. The mean +/- SEM of 3 independent cultures is shown.

39 10 40