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. 2020 Aug 10;219(10):e201911120. doi: 10.1083/jcb.201911120

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© 2020 Khalil et al.

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Figure S5. — Hemichannel-ADORA1 loop in other collective but not single-cell invasion models and downstream AKT activation. (a) Distribution of Cx43, β-catenin, and F-actin in NMuMG spheroids during collective invasion in 3D collagen after 68 h. Images represent maximum intensity projections from a 3D confocal stacks. Arrowheads, focal Cx43 hemichannel localization in leader cells. (b) Bright-field images of NMuMG spheroids embedded in 3D collagen for 68 h in the presence or absence of CBX. Arrowheads, protrusive leader cells. (c) Cumulative median length of invasion per spheroid (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) from 42 to 75 stands from six (control) and eight (CBX) spheroids (one experiment). P values, two-tailed unpaired Mann–Whitney test. (d) Bright-field images of NMuMG spheroids in 3D collagen after 48 h culture in the presence of PSB36 (5 or 20 µM). (e) Mean number of leader cells per spheroid and SEM from seven to nine spheroids per condition (one experiment). P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (f) HPLC analysis of purines released from individualized 4T1 cells into the supernatant after 24 h of invasion in 3D collagen. Values represent concentrations of each purine from one experiment. (g) Speed of 4T1 leader vs individual cells in 3D collagen. Values represent the medians (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) of six leader cells (one experiment) and 19 individual cells tracked over 24 h (two independent experiments). P value, two-tailed unpaired Mann–Whitney test. (h and i) Percentage of individualized 4T1 cells with elongated morphology after 12, 24, and 40 h in collagen and treatment with CBX (h) and PSB36 (i). Data show the means and SEM from 17–33 cells per condition from two (h) or one (i) experiments. DPCPX, 8-cyclopentyl-1,3-dipropylxanthine. (j) Distribution of AKT, phospho-AKT473, and F-actin in MMT spheroid invasion culture. 3D confocal maximum intensity projections after 24 h of culture. Dashed line indicates the region of analysis in k. (k) Distribution of AKT and phospho-AKT473 and F-actin along extending protrusions of leader cells. Data represent the mean intensities and SEM of F-actin, AKT, and phospho-AKT473 from at least 12 leader cells from one experiment. (l) Phospho-AKT473 levels in response to 24 h of PSB36 treatment during 4T1 spheroid culture in 3D collagen. (m) Phospho-AKT473 levels in of 4T1 and MMT 2D cultures after ADO treatment for 0.5, 1, 3, 6, and 12 h. Numbers in (l, m) represent the ratio of phospho-AKT473 over total AKT mean intensities from one experiment. Scale bars: 50 µm (a, b, d, and j), 25 µm (a and j, insert).