EMBO/SourceData huggingface dataset:
Figure 7(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).
Figure 1D. Expression distribution of lncORF in both groups by Ribo-seq.H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.
Figure 2A. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance.B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients.E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.
Figure 3B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.
Figure 4D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not.F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.
Figure 5A. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.
Figure 6E. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.
Figure 7C. Flow cytometry of the cell apoptosis ratio under L-OHP with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.E. CCK-8 assay of the inhibition ratio by L-OHP with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector
Figure 8B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a).G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).
f1(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph.(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin.(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
f2(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.(c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence.(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.
f3(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio.(b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.
f4(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.(c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.
f5(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed.(c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.(d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation.(e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.(f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells.(g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCherry-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation.
f6(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained.(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.
Figure 1A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.
Figure 2A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μmB) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.
Figure 3B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.
Figure 7A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.D,E,F) Average (± SEM) Log2(H/M) values for three groups of synaptic proteins (synaptic vesicle, presynaptic, postsynaptic), based on all five experiments. None of the changes observed for these proteins were statistically significant. The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H/M normalization process (Compare with Fig. 6E; see Results for further details).G) log2(H/M) values at t=24 hours for a group of well-studied synaptic proteins, sorted from highest to lowest. P values (two-tailed Welch's t-test) of comparisons with log2(H/M) values for the same proteins at t=0 are shown in parentheses. Proteins that exhibited statistically significant changes (P≤0.05) at t=24h are shown in red. Proteins for which (0.05 < P ≤ 0.10) at t=24h are shown in blue/red. The gray region depicts three standard deviations of log2(H/M) values at t=0 (Fig. 6F), to provide a measure of the method's sensitivity.
Figure 1(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps.(B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).
Figure 2(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.
Figure 3(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.