EMBO/soda-nel huggingface dataset:
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 3

PI5P4K2s Localize on Autophagosomes and Regulate Autophagy

(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).

(C) HeLa cells stably expressing GFP-mRFP-LC3 treated as in (A) were analyzed on a Cellomics ArrayScan system as previously described (mean ± SEM).

(D) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA and transfected with HA-ATG12 and ATG5 for the last 16 hr (mean ± SEM).

(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.

(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 4

PI5P4K2A and PI5P4K2C Regulate Accumulation of Mutant Huntingtin Exon 1

(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).

(C) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV-PI5P4K2C overexpression construct for 48 hr were transfected with EGFP-httQ74 for the last 48 hr. Percentage of cells with EGFP-positive aggregates is shown in the graph; n = 500 cells (mean ± SEM). Cells expressing control vector typically have 25% of aggregates and we set the control at 100% to enable comparison and statistics from independent experiments.

(D and E) Percentage of cells with EGFP-positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP-httQ74 for the last 48 hr (D), or in COS7 and SKNSH cells (E), transfected with PI5P4K2C plasmid together with EGFP-httQ74 for the last 48 hr.

(F and G) WT (Atg5+/+) or autophagy-deficient cells lacking the key autophagy gene Atg5 (Atg5−/−) MEFs were treated with PI5P4K2C siRNA (5 days) or PI5P4K2C plasmid (48 hr) together with EGFP-httQ74 for 48 hr. The percentages of transfected cells with EGFP-httQ74 aggregates were assessed and shown in the graph; n = 500 cells (mean ± SEM). Representative confocal images are shown in (G). See also Figures S3 and S4.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 5

PI(5)P Triggers Autophagy in the Absence of VPS34 Activation

(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells pretreated with Wm (200 nM, 2 hr), then loaded with 10 μM PI(5)P for 1 hr (in the presence of Wm and BAF) (mean ± SEM).

(C) Quantification of numbers of GFP-LC3 vesicles in HeLa cells stably expressing GFP-LC3 treated as in (A) and then shifted to starvation media (HBSS) or complete media (Basal) for 2 hr (in the presence of Wm) (mean ± SEM).

(D) HeLa cells transfected with HA-ATG12 and ATG5 treated with 200 nM Wm as in (A) and subjected to western blot analysis with anti-HA antibody to detect free ATG12 and the ATG5-ATG12 complex (mean ± SEM). ATG5-ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti-ATG12 antibody (bottom).

(E and F) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected with control, PI5P4K2A, 2B, and 2C siRNA and treated with Wm (200 nM, 2 hr in the presence of BAF) (mean ± SEM).

(G and H) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA, transfected with HA-ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr (mean ± SEM).

(I) HeLa cells stably expressing GFP-LC3 treated with control, PI5P4K2A, 2B, and 2C siRNA, were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr (in the presence of Wm) (mean ± SEM).

(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3 vesicles per cell is shown in (K) (mean ± SEM).

(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3)P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 6

PI(5)P Recruits Proteins Required for the Initiation of Autophagosome Formation

(A) HeLa cells transfected with GFP-WIPI2B or GFP-DFCP1 preloaded with indicated concentrations of PI(5)P for 1 hr, starved in HBSS for 1 hr, and then incubated with Wm in HBSS were tracked by time-lapse microscopy for 10 min after the addition of Wm. Quantification of WIPI2B or DFCP1 vesicles (percentage of those at the starting time [T0]) during the treatments are shown in the graphs.

(B-D) HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA were transfected with GFP-WIPI2B (B), GFP-WIPI2D (C), or GFP-DFCP1 (D); starved in HBSS (1 hr); and then incubated with Wm in HBSS. WIPI2 or DFCP1 structures were tracked and quantified as in (A).

(E) Lysates from HeLa cells stably expressing GFP-WIPI2B were incubated with agarose beads coated with PI, PI(5)P, and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls.

(F-H) Cell extracts from HeLa cells stably expressing GFP-WIPI2B were incubated for 3 hr with PI(5)P-containing liposomes (F and H) or PI(3)P-containing liposomes (G and H) before a pull-down experiment using the indicated beads. PS-containing liposomes were used as internal controls for both competition assays in (F and G).

(I) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5)P and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 7

PI(5)P Is a Master Regulator of Autophagy in Response to Glucose Starvation

(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).

(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM).

(C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr.

(D) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope.

(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM).

(G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm.

(H) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3)P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.
Noncanonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis (2019)
Figure 1

Figure 1. Epithelial-to-mesenchymal transition proteins are associated with sensitivity to Plk1 inhibitors in non-small cell lung cancer cell lines in vitro in an independent dataset. Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536, GSK461364, BRD-K70511574, and GW-843682X), based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database (Li et al, 2017). (A) Proteins with expression levels that correlate with area under the curve (AUC) values for at least two drugs are included in the graph. The color and size of the data point represent the direction and significance (p value) of the correlation. Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity (i.e., proteins in red are more highly expressed in resistant cell lines). (B) AUC values for two Plk1 inhibitors are compared with expression of selected proteins (cMet, E-cadherin, β-catenin, and Snail); each data point represents an individual non-small cell lung cancer cell line. E-cadherin, β catenin, and cMet protein expression correlated with drug resistance.
Noncanonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis (2019)
Figure 2

Figure 2. TGF-β-induced mesenchymal phenotype increases sensitivity to Plk1 inhibition. Three epithelial/resistant non-small cell lung cancer cell lines were treated with 5 ng/ml TGF-β for 14 days to induce a mesenchymal phenotype, which was confirmed with Western blot (left) and qPCR (right) analysis of epithelial-to-mesenchymal transition (EMT) markers (A). Parental and TGF-β isogenic cell lines were treated with 50nM volasertib for 72 hours. Cells were then harvested, and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β-actin (B). Apoptosis was measured by the Apo-BrdU assay (C) and DNA damage was measured by the Comet assay (D). Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparison are indicated (*p < 0.01).
Noncanonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis (2019)
Figure 3

Figure 3. Activation of cMet is differentially regulated in epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines following Plk1 inhibition and knockdown. (A) Three pairs of TGF-β-treated isogenic NSCLC cell lines (H1975, HCC4006, and HCC366) and two mesenchymal NSCLC cell lines (Calu6 and H1792) were incubated with 50nM volasertib for 24 hours, lysed, and subjected to reverse phase protein array analysis. Experiments were done in triplicate. (B) Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text). (C) The same cell lines treated identically were subjected to immunoblotting for the indicated proteins (upper) with densitometric quantification normalized with β-actin (lower). (D) Epithelial (red text) and mesenchymal (blue text) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control and subjected to Western blotting with the indicated antibodies. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-sided Student t test are indicated by *p < 0.05. (E) H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.
Noncanonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis (2019)
Figure 6

Figure 6. Constitutively active cMet expression reduces sensitivity to Plk1 inhibition. (A) Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay. (B) Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control. (C) Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated. (D) Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.
Noncanonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis (2019)
Figure 8

Figure 8. Plk1 inhibition or silencing decreases vimentin phosphorylation and regulates cMet phosphorylation via β1-integrin. (A) Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. (B) Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. (C) Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. (D) Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. (E) Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after treatment with fibronectin for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. (F) Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins. (G) Schematic summary of the proposed mechanism by which cMet drives primary and acquired resistance to volasertib in NSCLC and possible treatment combinations to overcome this resistance.
Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis (2012)
f1

a, b, Representative images of GFP–LC3 puncta (autophagosomes) in skeletal (vastus lateralis) (a) and cardiac (b) muscle from GFP–LC3 transgenic mice at serial time points after exercise. Scale bar, 20 &mgr;m. c, Quantification of data (mean ± s.e.m. of 10 tissue sections) in a and b. **P  0.01, ***P  0.001 (one-way ANOVA). d, Western blot analysis of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in indicated tissue from mice at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively. e, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise. IP, immunoprecipitate; WB, western blot; WCL, whole cell lysates.
Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis (2012)
f2

a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle’s balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2. b, Quantification of GFP–LC3 puncta (autophagosomes) in MEFs of indicated genotype in normal growth conditions or starvation conditions. Data represent mean ± s.e.m. for 100 cells per well of triplicate samples per condition. Similar results were observed in three independent experiments. c, Representative images of GFP–LC3 puncta (autophagosomes) in skeletal and cardiac muscle of GFP–LC3 wild-type and GFP–LC3 BCL2 AAA mice before exercise, after 80 min exercise, or after 75% of maximal exercise capacity. Scale bar, 20 &mgr;m. d, Quantification of data (mean ± s.d. of 4 mice per experimental group) in c. e, Western blot analysis of LC3-I/II and p62 levels in indicated tissue from mice of indicated genotype at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively. f, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest (−) or after 30 min of exercise. WCL, whole cell lysates. NS, not significant. *P  0.05, **P  0.01, ***P  0.001, one-way ANOVA for comparison between groups; †P  0.001, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.
Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis (2012)
f3

a, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 5 mice per group. b, Representative haematoxylin and eosin (H E) and periodic acid-Schiff (PAS) staining in tibialis anterior muscle sections from mice of indicated genotype. Scale bar, 20 &mgr;m. c, d, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (∼900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9–11 mice per group from three independent cohorts; similar results were observed in each cohort. e, Representative images of GLUT4 immunofluorescence staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise. Scale bar, 20 &mgr;m. For b and e, similar results were observed in 3 mice per group. f, Western blot analysis of AMPK phosphorylation (p-AMPK (Thr 172)) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise. g, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 4–6 male mice per group. h, Soleus muscle 14C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P  0.05, **P  0.01, ***P  0.001, one-way ANOVA for comparison between groups; †P  0.05, ††P  0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.
Stearate-derived very long-chain fatty acids are indispensable to tumor growth (2022)
Figure 1

Figure 1. Tumor growth is more vulnerable to ACC1-KO, rather than FASN-KO

A scheme to show de novo synthesis of palmitate and fatty acid elongation achieved by enzymes, ELOLVs, KAR, HACDs, and TECR.

Western blot verification of knockout of FASN, ACC1, and/or ACC2 in MDA-MB-231 cells.

Cell proliferation rate of MDA-MB-231 and HeLa cells with knockout of FASN, ACC1, and/or ACC2.

Colony formation ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2.

Invasion ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2. The right panel shows the pictures of invasive cells, and the cell numbers are quantified in the left panel. Scale bar: 20 μm.

Tumor formation ability in nude mice of MDA-MB-231 and HeLa cells with gene knockout as indicated.

Data information: In (C-E), data are from triplicate experiments, and all experimental data are verified in at least two independent experiments. In (F), n = 4 for MDA-MB-231-derived tumors, n = 6 for HeLa-derived tumors. Error bars represent mean ± SD. *P < 0.01, **P < 0.01 (t-test); black asterisk shows comparison with wild type group.
Stearate-derived very long-chain fatty acids are indispensable to tumor growth (2022)
Figure 4

Figure 4. Knockout of elongation enzymes suppreses tumor growth

Western blot verification of knockout of KAR or TECR in MDA-MB-231 cells.

B,C. Cell proliferation rate and colony formation ability of MDA-MB-231 cells with knockout of KAR or TECR. Error bars represent mean ± SD. Data are from triplicate experiments, and all experimental data were verified in at least two independent experiments. **P < 0.01 (t-test); asterisk shows comparison with wild type group.

D,E. Tumor formation ability in nude mice of MDA-MB-231 cells with gene knockout, as indicated. Error bars represent mean ± SD (n = 6 for D, n = 8 for E).

F. Relative cellular levels of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. Each colored datum shows the mean from three independent cultures.

G. Relative cellular levels of lipid-containing fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. The top curve plot indicates the fraction of the corresponding fatty acid. Each colored datum shows the mean from three independent cultures.

H. Mass isotopomer analysis of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6-glucose for 48 h. Error bars represent mean ± SD. Data are from three independent cultures.