Figure S4.
Sorting of time series according to the presence/absence of a fluorescent signal, analysis of quiescence entry in cells CUP1p-expressing transcriptional repressors, and in xbp1Δ cells.(A) Sorting of mixed time series from cells expressing mTFP1-tagged transcription factors and control cells without the mTFP1 construct that were loaded in the same microfluidics device (see Fig. 7). (B–E) Analysis of quiescence entry in strains bearing Gln3-mKOκ, Rtg1-mNeptune2.5, Sfp1–mScarlet-I, Xbp1-mNeonGreen, and Cdc10-mCyOFP1 after CUP1p-expression of mTFP1 C-terminally tagged WT Xbp1 (n = 6; OAM458), the DNA-binding domain mutant xbp1-EE (n = 7; OAM466), the Xbp1-related transcriptional repressor Stb3 (n = 4; OAM497), or the cell cycle repressor Whi5 (n = 7; OAM455) at starvation onset. (B) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-XBP1-mTFP1 during quiescence entry. Cells whose Xbp1-mTFP1 levels exceeded two SDs from the mean Xbp1-mTFP1 levels were excluded. (C) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-xbp1-EE-mTFP1 during quiescence entry. (D) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-STB3-mTFP1 during quiescence entry. (E) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-WHI5-mTFP1 during quiescence entry. (F) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1–mScarlet-I, Gln3-mKOκ, Rtg1-mNeptune2.5, Msn2-mNeonGreen, and Stb3-mTFP1) in six-color xbp1Δ cells (n = 4; 500 cells, OAM500). Unlike XBP1 strains, quiescence entry in xbp1Δ cells could not be described as three main clusters; instead, TPML sorted xbp1Δ cells into four clusters (four-color bar) and cells undergoing ectopic divisions. (G) Percentage of cells entering ectopic proliferation during starvation in control (Ctrl; OAM502) and xbp1Δ (OAM500) cells. (H) Schematic quantification of quiescence entry in control (OAM502) and xbp1Δ (OAM500) cells, including the percentage of cells with ectopic divisions in each cluster. Red arrows indicate paths with <75% viability upon return to rich medium (n > 3). (I) Viability of direct G1 and post-mitotic Q-cells in WT (left) and xbp1Δ cells (right) upon return to rich medium after 20-h starvation. Gray area on plot = period in rich medium. Red arrowhead = onset of starvation. Red star = P < 0.05, K-S test. Solid lines with shaded area = average ± 95% confidence intervals. Bar plots = mean + SD.

Sorting of time series according to the presence/absence of a fluorescent signal, analysis of quiescence entry in cells CUP1p-expressing transcriptional repressors, and in xbp1Δ cells. (A) Sorting of mixed time series from cells expressing mTFP1-tagged transcription factors and control cells without the mTFP1 construct that were loaded in the same microfluidics device (see Fig. 7). (B–E) Analysis of quiescence entry in strains bearing Gln3-mKOκ, Rtg1-mNeptune2.5, Sfp1–mScarlet-I, Xbp1-mNeonGreen, and Cdc10-mCyOFP1 after CUP1p-expression of mTFP1 C-terminally tagged WT Xbp1 (n = 6; OAM458), the DNA-binding domain mutant xbp1-EE (n = 7; OAM466), the Xbp1-related transcriptional repressor Stb3 (n = 4; OAM497), or the cell cycle repressor Whi5 (n = 7; OAM455) at starvation onset. (B) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-XBP1-mTFP1 during quiescence entry. Cells whose Xbp1-mTFP1 levels exceeded two SDs from the mean Xbp1-mTFP1 levels were excluded. (C) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-xbp1-EE-mTFP1 during quiescence entry. (D) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-STB3-mTFP1 during quiescence entry. (E) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1, Rtg1, Gln3, and Xbp1) in cells expressing CUP1p-WHI5-mTFP1 during quiescence entry. (F) Average time series per cluster for cell cycle (Cdc10 signal) and stress markers (Sfp1–mScarlet-I, Gln3-mKOκ, Rtg1-mNeptune2.5, Msn2-mNeonGreen, and Stb3-mTFP1) in six-color xbp1Δ cells (n = 4; 500 cells, OAM500). Unlike XBP1 strains, quiescence entry in xbp1Δ cells could not be described as three main clusters; instead, TPML sorted xbp1Δ cells into four clusters (four-color bar) and cells undergoing ectopic divisions. (G) Percentage of cells entering ectopic proliferation during starvation in control (Ctrl; OAM502) and xbp1Δ (OAM500) cells. (H) Schematic quantification of quiescence entry in control (OAM502) and xbp1Δ (OAM500) cells, including the percentage of cells with ectopic divisions in each cluster. Red arrows indicate paths with <75% viability upon return to rich medium (n > 3). (I) Viability of direct G1 and post-mitotic Q-cells in WT (left) and xbp1Δ cells (right) upon return to rich medium after 20-h starvation. Gray area on plot = period in rich medium. Red arrowhead = onset of starvation. Red star = P < 0.05, K-S test. Solid lines with shaded area = average ± 95% confidence intervals. Bar plots = mean + SD.

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