EMBO/soda-nel huggingface dataset:
Shp2 signaling suppresses senescence in PyMT‐induced mammary gland cancer in mice (2015)
Figure 4

Shp2 activates Src, Fak, and Mek1/Erk to regulate senescence in PyMT tumor cells

SA‐β‐gal staining on PyMT tumor cells treated with control, Shp2 inhibitor GS493 (15 μM), Src inhibitor PP2 (2.5 μM), Fak inhibitor TAE226 (0.5 μM), or Mek1 inhibitor U0126 (20 μM). Scale bar, 100 μm.

Quantification of the numbers of senescent cells in the presence or absence of the inhibitors, as shown in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01.

Quantification of the numbers of spheres in the presence or absence of the inhibitors, as in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01.

Western blot analysis of pY527, pY416 and total Src, pY925 and total Fak, pT202/pT204 and total Erk1/2, and α‐tubulin in PyMT tumor cells with or without treatment with 5 μM GS493.

qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control cells and those treated with the indicated inhibitors. Error bars represent SEM (n = 3).

Summary of the deregulated genes by the chemical inhibition of Shp2, Src, Fak, and Mek1 signaling. Arrows represent downregulation of genes. Statistical significance was assessed by Student's unpaired t‐test.

Source data are available online for this figure.
Shp2 signaling suppresses senescence in PyMT‐induced mammary gland cancer in mice (2015)
Figure 5

The Shp2‐regulated gene expression programs predict the survival of patients with breast cancers

A Kaplan-Meier survival analysis of human patients (n = 3,935) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 (see ).

B-D Kaplan-Meier survival analyses of the human patients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes.

E, F Kaplan-Meier survival analyses of human patients (n = 3,935) divided into two groups based on the gene expression level of Skp2 or Aurka. HR, hazard ratio.

Data information: Statistical significance was assessed by the log‐rank test.
Shp2 signaling suppresses senescence in PyMT‐induced mammary gland cancer in mice (2015)
Figure 6

Shp2 ablation inhibits mammary gland tumor formation in PyMT mice

Kaplan-Meier analyses of tumor formation for control (PyMT;Shp2fl/fl, blue, n = 33), heterozygous Shp2 mutant (PyMT;MMTV‐Cre;Shp2+/fl, green, n = 15), and homozygous Shp2 mutant (PyMT;MMTV‐Cre;Shp2fl/fl, also called PyMT;coShp2, orange, n = 34) mice. ***P 0.001 (log‐rank test).

Mammary gland whole‐mounts from control PyMT and PyMT;MMTV‐Cre;Shp2fl/fl mice at indicated ages. The characteristic lymph node of the mammary gland fat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm.

Quantification of premalignant lesions in mammary glands of control PyMT and PyMT;MMTV‐Cre;Shp2fl/fl mice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test).
Shp2 signaling suppresses senescence in PyMT‐induced mammary gland cancer in mice (2015)
Figure 7

Late‐stage primary tumors and metastases in PyMT;coShp2 mice develop from tumor cells that escaped recombination

Immunostaining of EYFP and CK8 on mammary gland sections from PyMT;MMTV‐Cre;Shp2+/fl;EYFP−/fl and PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl mice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8.

Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from PyMT;Shp2fl/fl, PyMT;MMTV‐Cre;Shp2+/fl, and PyMT;MMTV‐Cre;Shp2fl/fl mice. Scale bar, 100 μm. n = 8.

qRT-PCR analyses of Shp2 mRNA levels in tumor tissues from PyMT;Shp2fl/fl (n = 8), PyMT;MMTV‐Cre;Shp2+/fl (n = 6), and PyMT;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles.

Western blot analysis of Shp2 protein levels in tumor tissues from PyMT;Shp2fl/fl, PyMT;MMTV‐Cre;Shp2+/fl, and PyMT;MMTV‐Cre;Shp2fl/fl mice.
Shp2 signaling suppresses senescence in PyMT‐induced mammary gland cancer in mice (2015)
Figure 8

Shp2 ablation induces senescence in mammary gland tumors in PyMT mice

Shp2 inhibition prevents tumor formation at early states and inhibits tumor growth at late stages in PyMT mice.

Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary gland tumors from control PyMT;Shp2fl/fl;EYFP−/fl, PyMT;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl mice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary gland tumors of PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl mice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5.

Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary gland tumors from control PyMT;Shp2fl/fl;EYFP−/fl, PyMT;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl mice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5.

qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP− cells isolated by FACS from PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl tumors. Error bars represent SEM (n = 5).

Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) PyMT;MMTV‐Cre;Shp2fl/fl mice. The bar indicates the duration of treatment.

Tumor growth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) PyMT mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5).
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 1

Figure 1. Inhibition of stromal TGFβR2 reduces IL-6 production and tumor cell STAT3 activation in PDA. (A) Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control). (B) ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown. (C-D) KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group). (E-K) KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. P values vs. control by t test are indicated.
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 2

Figure 2. CAFs are the major source of IL-6 in PDA. (A) Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown. (B) Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control. (C) Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 3

Figure 3. CAF-secreted IL-6 is regulated by TGFβ and activates STAT3 in pancreatic cancer cells. (A-C) Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A). Conditioned media (CM) was collected mouse IL-6 ELISA (B-C). P value by ANOVA is shown. (D-F) NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA. P values by t test are shown. (G-H) CAF-PC2 cells were treated with TGFβ (30 ng/ml) (G) or IL-1α (1 ng/ml) (H) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA. P values by t test were shown. (I-J) KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) (I), normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J). (K-N) 3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml). Scale bars = 50 μm. n = 5/group, P values by t test are shown.
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 4

Figure 4. TGFβ-induced IL-6 from CAFs inhibits NK cell activity and promotes pancreatic cancer. (A-F) Saline and 2G8-treated MiaPaca-2 and Colo375 xenografts were subjected to RNA-seq analysis for mouse gene expression changes. A heat map was generated with gene clustering (A). IPA showing pathways most effected by 2G8 (B-C). Molecules most significantly by 2G8 treatment. Y-axis values shown are log10(FPKM+1) of the transcript levels (D-F). (G) An in vitro NK cell cytotoxicity assay was performed. Human NKL cells were used as effector cells, and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM (control), conditioned medium collected from TGFβ (30 ng/ml)-treated human pancreatic CAFs (CAF-PC2; CAF TGFβ-CM), CAF TGFβ-CM + IL-6 antibody (100 ng/ml) or normal DMEM + TGFβ (TGFβ). Living cells were labeled with CFSE, and dead cells were labeled with 7-AAD. Samples were analyzed by flow cytometry. Cytotoxicity percentage was calculated using the formula (7-AAD positive cells %) / (7-AAD positive cells % + CFSE positive cells %) x 100%. n = 4/group, P values by t test are shown. (H-I) Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H&E-stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions. P values by t test are shown.
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 5

Figure 5. Inhibition of stromal TGFβR2 reverses immunosuppression in PDA. (A-U) KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Immune landscape changes were detected by immunohistochemistry for macrophage makers F4/80 (total, A-C), iNOS (M1, D-F), ARG1 (M2, G-I), CD11b (myeloid-derived suppressor cells, J-L) and T cell markers CD3 (M-O), CD8 (P-R) and FOXP3 (regulatory T cells, S-U) in KIC and KPC mice. Scale bars = 50 μm. n = 4/group, P values by t test are shown. (V-X) Spatial distribution analysis of CD8-positive T cells after 2G8 treatment. Details are described in Appendix Fig S7. Sample images of KPC tumors are shown (V). Substantial cytotoxic T cell infiltration was observed after 2G8 treatment. Histological score was calculated using the following formula (W-X): low IHC cells % + 2x (medium IHC cells %) + 3x (high IHC cells %). Scale bars = 500 μm. n = 4/group, P values vs. control by t test are shown.
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 6

Figure 6. Loss-of-function mutations in TGFBR2 are common in PDA. (A-B) KIC mice were treated in a survival study with rat IgG Mac48 (control) or 2G8. Median overall survival of rat IgG treatment was 72 days, while median overall survival of 2G8 treatment was 63 days (B). (C) Tissues from (A) were stained for Ki67. Scale bars = 50 μm. (D) Mutation profile of TGFBR2 in PDA patient samples from two independent sources (UTSW and TCGA). (E) Overall survival of PDA patients from TCGA with and without TGFBR2 mutation. (F) Loss-of-function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs (mut1 and mut2) in the mouse PDA cell line BMFA3 derived from KPfC model. Control cell line (Tgfbr2wt) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) were treated with TGFβ (30 ng/ml) for 5 hours. Cell lysates were harvested and western blotting for P-SMAD2, SMAD2, SMAD4, P-ERK1/2, ERK1/2, P-P38, P38, and actin was performed. (G) Immunofluorescence was performed to study the expression and localization of E-cadherin and SMAD4 after TGFβ treatment (30 ng/ml for 24 hours) in control cell line and two Tgfbr2-mutant cell lines. Scale bars = 50 μm (fluorescent), scale bars = 100 μm (brightfield).
Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer (2019)
Figure 7

Figure 7. TGFβR2 blockade has therapeutic efficacy in TGFβR2-mutant PDA due to the inhibition of stromal TGFβ signaling. (A-C) Control cell line (Tgfbr2wt) (A) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) (B-C) were treated with TGFβ at different time points, and cell growth (MTT) assays were performed. P value by t test is shown. NS, not significant. (D-F) Subcutaneous tumors established from control cell line (D) and two Tgfbr2-mutant cell lines (E-F) in C57Bl/6 mice received rat IgG Mac48 (control) or 2G8 (each 30 mg/kg 2x/week, n = 4/group). Therapy started at day 12 post tumor cell injection; mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are shown. (G-L) Control cell line (Tgfbr2wt; G&I) and the Tgfbr2-mutant cell line (Tgfbr2mut1; H&J) were orthotopically implanted in C57Bl/6 mice. Mice received rat IgG Mac48 or 2G8, n = 9-10/group for survival study (each 30 mg/kg 2x/week). Mice were sacrificed when they became moribund. Tumors were harvested and weighed (K-L). P values by t test are indicated. (M-X) Immunohistochemistry with tumor samples from (G-H) for Ki67 (M-O), P-STAT3 (P-R), ZAP70, and CD3 (S-X) was performed. ZAP70 is a common marker for NK cells and T cells and was stained with a brown chromogen, and CD3 is a specific T-cell marker and was stained with a blue chromogen. Brown signal was converted to red signal and blue signal was converted to green by ImageJ (V); therefore NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 4/group, P values vs. control by t test are shown.
Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut (2021)
Figure 3

Figure 3. Downregulation of ACE2 upon SARS-CoV-2 infection.

A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.

B. Dot plots displaying the expression changes of known SARS-CoV-2 entry determinants for both infected and bystander cells during the course of infection (mock, 12 and 24 hpi) in colon (left) and ileum organoids (right). The dot size represents the percentage of cells expressing the gene; the color represents the average relative expression across the cell type excluding zeros.

C. Pearson correlation of gene expression values with the amount of SARS-CoV-2 genome (y-axis) vs the maximal log2 fold change (x-axis) across conditions. This plot is generated by comparing both 12 and 24 hpi to mock. Top 5 high correlated and anticorrelated genes and known SARS-CoV-2 entry determinants are highlighted in blue.

D. Correlation of SARS-CoV-2 expression with ACE2 expression across all infected cell types from ileum organoids at 24 hpi. (n=146) Gray area represents the standard error to the fitted line.

E. Global-scaled log normalized expression values of ACE2 in each cell type for mock infected and SARS-CoV-2 infected cells in ileum organoids at 12 hpi and 24 hpi. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Immature Enterocyte 2 Mock n=377 , 12h n=390 & 24h n=270,Stem Cells Mock n=184 , 12h n=219 & 24h n=115,TA Mock n=248 , 12h n=241 & 24h n=128,Immature Enterocyte 1 Mock n=452 , 12h n=559 & 24h n=280,Cycling TA Mock n=187 , 12h n=240 & 24h n=214,Enterocyte 1 Mock n=249 , 12h n=374 & 24h n=140,Goblet Cells Mock n=12 , 12h n=24 & 24h n=5,Secretory TA Mock n=7 , 12h n=59 & 24h n=2,Enteroendocrine cells Mock n=26 , 12h n=30 & 24h n=15).

F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.

G. Correlation of the relative expression SARS-CoV-2 and ACE2 for infected cells from the multiplex in situ RNA hybridization shown in (F). Each dot represents a cell.
Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut (2021)
Figure 6

Figure 6. SARS-CoV-2 infection impairs interferon-mediated signaling.

A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.

B. Quantification of the mean fluorescence intensity of samples in (A). Each dot represents a cell.

C. Schematic depicting the infection and interferon addition to evaluate ISG15 induction following SARS-CoV-2 and astrovirus infection.

D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test.

E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um.

F. Schematic depicting the T84 MX-1 mcherry expressing cells and their response to IFN or SARS-CoV-2 infection.

G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.
Rewiring Phospholipid Biosynthesis Reveals Resilience to Membrane Perturbations (2022)
Figure 1

Fig 1. Rewiring PE and PC synthesis to expose lipid trafficking routes.

A) Scheme depicting the topology of the CDP-DAG -and Kennedy (red) pathways producing PE and PC in wild-type (WT) yeast cells (left panel), and an example of a rewired condition in which PE synthesis is directed to the mitochondrial inner membrane (MIM) and PC synthesis to peroxisomes (pex) (right panel). Black arrows indicate PS (dashed) and PE (dotted) lipid transport that must occur to enable the sequential enzymatic reactions.

B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.
Rewiring Phospholipid Biosynthesis Reveals Resilience to Membrane Perturbations (2022)
Figure 2

Fig 2. Rewired yeast cells are viable and produce all phospholipids.

A) Cells of the indicated genotypes were pre-cultured in medium with 10mM ethanolamine and 10mM choline and subsequently in medium lacking them for 5 hours, before being diluted in the indicated medium for growth curve analysis. Emp. vec. refers to cells bearing an empty vector. Data shown are the mean ±SEM of three independent replicates, except for Psd(MIM) (two experiments).

B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON).

C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.