1. Probing Mammalian Cell Size Homeostasis by Channel-Assisted Cell Reshaping 
Giulia Varsano, Yuedi Wang, Min Wu 
Cell Reports 
Volume 20, Issue 2, Pages (July 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 20, 397-410DOI: (10.1016/j.celrep.2017.06.057)
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3. Figure 1
Controlling Mammalian Cell Geometry Using Microfabricated Channels
(A) Schematic drawing of microfabricated polydimethylsiloxane (PDMS) channel for manipulating the shape of mammalian cells. The cells are seeded into reservoirs connected to channels designed with three different widths (6, 4, and 2 μm) and a fixed height (9 μm).
(B) Representative cytosolic GFP/DIC overlay image of cells in the 6 μm wide channels adopting the geometry of the channel.
(C) Time-lapse sequence of a cell crawling into a 6 μm wide channel. The images are shown as cytosolic GFP/DIC overlays.
(D) Time-lapse images of cells expressing GFP-H2B (green) and mCherry-labeled-alpha-tubulin (mCherry-α-tubulin, magenta) that are crawling into a 4 μm wide channel (upper panel) and being excluded from a 2 μm wide channel (lower panel). The white dashed line represents the boundary between the reservoir and the channel. Scale bars, 10 μm.
(E) Representative x-y, x-z, and y-z confocal images of cytosolic GFP-expressing cells in 6 and 4 μm wide channels. The white dashed lines indicate the y-z and the x-z planes. Scale bars, 7 μm.
See also Figure S1 and Movies S1 and S2.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Characterization of Cell Cycle and Cell Size Information for Cells in the Channels
(A) Time series of a single cytosolic GFP-expressing cell growing and dividing in a 6 μm wide channel. The stages of pre-division and just after cytokinesis are highlighted.
(B) Quantification of the cell-cycle duration for cells grown in tissue culture dish in respect to 6 and 4 μm channels. The data are represented as mean ± SD. The error bar stands for SD.
(C) Montages of cytosolic GFP-expressing cells from the same lineage undergoing consecutive cell divisions in the channel as well as in the reservoir.
(D) Quantification of the cell-cycle duration for cells in (C).
(E) Histogram of the length, basal area, and volume distributions of asynchronous populations in 4 and 6 μm wide channels.
(F) Volume distributions of asynchronous populations obtained by cell sizing system or calculated from cell length in the 6 μm wide channels. Insets: mean, mode, and CV (SD/mean). Scale bars, 10 μm.
See also Figure S2.
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5. Figure 3 Cell Size Homeostasis in the Channel
(A) Representative images of cytosolic GFP-expressing cells dividing in 6 and 4 μm wide channels. Snapshots of before and after cytokinesis are shown in the image.
(B) Quantification of the relative length ratio of sibling daughter cells in 6 and 4 μm wide channels. The value of 1.5 was chosen as the threshold of asymmetric cell division.
(C) Basal area distribution of newborn cells in 4 and 6 μm wide channels.
(D) Snapshots of pre-division, mitosis, and newborn cells. Scale bars, 10 μm.
(E and F) Histogram of the lengths (E) and basal area distributions (F) of pre-division cells in 4 and 6 μm wide channels.
See also Movie S2.
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6. Figure 4 Two-Tier Control of Cell Size Homeostasis at G1/S Transition
(A) Representative time-lapse images of cells stably expressing the cell-cycle indicator, FUCCI, dividing symmetrically (upper panel) and asymmetrically (lower panel) in 4 μm wide channels. The frame interval is 10 min. Scale bars, 10 μm. Fucci traces of the four cells are shown on the right. TG1, time of G1 duration.
(B) Scatterplot and single linear fitting of G1 duration and basal area at birth in 4 μm (left), combined (middle), and 6 μm (right) wide channels. Fitting on binned data are shown.
(C) Same as (B) except that segmental linear fitting was used.
(B and C) Grey dots, single cell data from 4 μm wide channels. The orange dots show single cell data from 6 μm wide channels. The open circle shows binned data. The lines show linear fitting. The dashed lines show 95% confidence interval (CI). The slopes, goodness of fit (R2), and Pearson correlation coefficient (r) with p values are shown on the graph. See also Figures S3–S5 and Movies S4 and S5.
(D) Comparison of daughter cells basal area differences at birth and at G1/S transition. The siblings from asymmetrical division are shown in red (n = 10 pairs), and those from symmetrical divisions in green (n = 12 pairs).
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7. Figure 5 Identification of Cryptic G1/S Size Checkpoint
(A) Scatterplot and two-segment linear fitting of basal area extension in the G1 and basal area at birth in 4 μm (left), combined (middle), and 6 μm (right) wide channels. Fitting on binned data are shown.
(B) Scatterplot and two-segment linear fitting of basal area at the G1/S transition and basal area at birth in 4 μm (left), combined (middle), and 6 μm (right) wide channels. Fitting on binned data are shown. Black dashed line, reference line y = x.
(A and B) Grey dots, single cell data from 4 μm wide channels. The orange dots show the single cell data from 6 μm wide channels. The open circle shows binned data. The lines show linear fitting. The dashed lines show 95% CI. The slopes and goodness of fit (R2) are shown on graph.
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8. Figure 6 Cell Size Homeostasis after Drug-Induced Cell Size Reduction
(A) Histogram of the basal area distributions of newborn cells in 6 μm wide channels without (empty black) or with 72 hr pre-treatment with 5 μm LY (cyan).
(B) Schematic representation of the experimental strategy applied for drug-induced size reduction.
(C) Representative images of FUCCI PM-GFP expressing cells without (CTRL) or with LY pre-treatment (LY) in 6 μm wide channel. LY cells are kept with fresh medium without drug after seeding in the channel. Newborn cell length is highlighted by white dashed lines. Scale bars, 10 μm.
(D) Scatterplot and two-segment linear fitting of the G1 duration versus basal area at birth of LY pre-treated cells in 6 μm wide channels (left). The same data on the left overlaid with 4 μm wide channels values from Figure 4C are shown in the middle. The same data as the middle, but only binned data and line fitting are shown on the right.
(E) Basal area extension in G1 versus basal area at birth plot.
(F) Basal area extension in G1/S versus basal area at birth plot.
(E and F) Arranged similar to (D).
(D–F) Cyan dots, single cell data of LY pre-treated cells in 6 μm wide channels. The open circle represents binned data. The lines show linear fitting. The dashed lines show 95% CI.
See also Figure S6.
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9. Figure 7 Cell Size Homeostasis in RAW 264.7 Macrophages
(A) Left, representative x-y, x-z, and y-z confocal images of RAW expressing cytosolic GFP in 6 μm wide channels. The white dashed lines indicate the y-z and the x-z planes. A time series of a single cytosolic mCherry-expressing RAW cell growing and dividing in a 6 μm wide channel is shown on the right. Scale bars, 10 μm.
(B) Scatterplot and two-segment linear fitting of basal area at G1 versus basal area at birth of RAW cells in 6 μm wide channels (left). The binned data on the left are overlaid with RBL values in 6 or 4 μm wide channels from Figure 5B (middle and right).
(C) Scatterplot and two-segment linear fitting of basal area extension in G1 versus basal area at birth of RAW cells in 6 μm wide channels (left). The binned data on the left are overlaid with RBL values in 6 or 4 μm wide channels from Figure 5A (middle and right). The green dots show single cell data of RAW cells in 6 μm wide channels. The open circle shows binned data. The lines show linear fitting. The dashed lines show 95% CI.
See also Figure S7.
Cell Reports  , DOI: ( /j.celrep )
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