EMBO/SourceData huggingface dataset:
Figure 2(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Figure 3(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).
Figure 4Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).
Figure 5Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Figure 1A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.
Figure 2A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.
Figure 3A-D Comparison of chymotrypsin-like activity from female (n=9) and male (n=10) mice in young (3-5 months old) or older (10-15 months old, female (n=6) and male (n=7)) groups. Each point indicates a single mouse. A) Tissues that did not show significant decline, B) Tissues that show significant decline in females only, C) Tissues that show significant decline in males only or D) in both males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=9 young females, n=6 older females, n=10 young males, n=7 older males). P values obtained by unpaired two-tailed students t-test comparing young and older samples are indicated.
Figure 1(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8.(B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively.(C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).
Figure 2(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.(C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles.(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Figure 3(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.(B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site.(C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies.(D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles.(E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Figure 4(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.(B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles.(C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Figure 5(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.(B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Figure 6(A) GFP-Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy.(B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band).(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.
Figure 1Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).
Figure 2Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue.
Figure 3SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815.SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F).
Figure 4Cells expressing the indicated forms of GFP-TDP-43 were subjected to immunoprecipitation, and the O-GlcNAcylated levels of TDP-43 were examined.   Quantification of (G).
Figure 5Walking speed of 10-day old adult male flies of the indicated genotypes was calculated Mean ± SD. Unpaired two-tailed t-test, biological replicates: Control, n = 63, WT, n = 45, T199A, n = 70, T233A, n = 87; 2TA, n = 43.   .Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.
Figure 6CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).
Figure 1A BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS), demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+ breast cancer. A log rank test was used to calculate P=2.8 e-07 (n= 934, number of events= 216).B BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS) of ER- breast cancer. A log rank test was used to calculate P=0.4481 (n= 322, number of events= 130).C BreastMark microarray platform; correlation of ESRP1 expression with overall survival in tamoxifen-treated patients. A log rank test was used to calculate P=1.218, e-06 (n= 210, number of events= 49).D BreastMark microarray platform; correlation of ESRP1 expression with overall survival in chemotherapy-treated patients. A log rank test was used to calculate the P=0.28 (n= 129, number of events= 21)E The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER+ breast cancer, demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+. A log rank test was used to calculate P=0.00011 (n= 656, number of events= 62).F The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER- breast cancer. Red-high ESRP1 expression; Black-low ESRP1 expression. A log rank test was used to calculate P=0.19 (n=100, number of events=17).
Figure 2A RT-qPCR confirmed the stable knockdown of ESRP1 expression in endocrine-resistant cells using a shRNA lentiviral system (Mission TRC human shRNA constructs-Sigma); 2-control (LCC2 cells transduced with pLKO.1 control vector) and 2C1 and 2C3 (LCC2 cells transduced with pLKO.1 shRNA ESRP1). Clones were selected for expression verification (clone 1 and clone 3); 9-control (LCC9 cells transduced with pLKO.1 control vector), 9C2 and 9C3 (LCC9 cells transduced with pLKO.1 shRNA ESRP1. Clones were selected for expression verification (clone 2 and clone 3). * represents P <0.05; statistically significant. Data (mean ± SD) were calculated using two-way ANOVA based on three independent biological replicates.B Western blot analysis using ESRP1 antibody GTX 131373 (GeneTex). GAPDH is used as the reference control.C Relative cell density was determined by crystal violet assay. Cells were treated with TAM (10-6 M) or ICI (10-9 M) for 6 days in the presence and absence of E2 (β-estradiol; 10-10 M). Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates. D Cell viability was determined using Cell Biolabs CytoSelect™ Cell Transformation Assay using 2-control, 2C3, 9-control and 9C2 cells in CSM-charcoal stripped media, E2 (β-estradiol; 10-10 M) and 10% fetal bovine serum. Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.E Impact of ESRP1 knockdown on in vivo tumor growth. Five million cells (2-control, 2C3, 9-control and 9C2) were implanted into mammary fat pads of athymic mice (five mice per group) in the presence of supplemental estrogen. Tumors were measured weekly using calipers for external measurements. Tumor volume was calculated as L x W2/2, where L is length and W is width (note the different scale for tumor volumes). Data (mean ± SD) were calculated using two-way ANOVA (n= 5 mice per group).
Figure 3A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm.B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis.E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).
Figure 6B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors. A splice graph of the gene's exons is shaded based on expression level and shows the selected splice event outlined in red.
Figure 7A Volcano plots showing the distribution of significant gene expression changes using the HTA 2.0 platform.C Validation of the protein levels of FASN, SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis. GAPDH was used as the loading control. The data are representative of three individual biological replicates.