EMBO/SourceData huggingface dataset:
Figure 6 (a) Cluster formation assays were conducted in presence or absence of 20µM mCherry-LC3B or mCherry, either pre-mixed with p62 or added 1" after the addition of GST-4xUb. Quantification of particle number (left) and size (right) are shown (b) Cluster formation assays were conduced with mCherry-p62 wt or LIR mutant in presence of GST-4xUb. Left: representative images of the samples two minutes after the addition of GST-4xUb. Middle: quantification of particle count. The dashed line indicates the time point of images displayed on the left. Right: input gel of the samples after reaction. The relative position of marker bands is indicated. For all graphs, averages and SDs from at least three independent replicates are shown(c) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition
Figure 1(A) Solubilized lysosomes cross‐linked with DSP were subjected to affinity chromatography with the antibody against the cytosolic tail of lamp2a (lanes 1 and 2). Eluted proteins were subjected to SDS-PAGE and silver stained (lane 1), or immunoblotted for lamp2a (lane 2). The sequence of the N‐terminus of the 32 kDa band is shown. Part of the solubilized lysosomes were directly subjected to non‐reducing SDS-PAGE (5%) and immunoblotted for lamp2a (lane 3) or PPCA (lane 4). Arrowheads indicate lamp2a multimeric complexes. The 600 kDa (*) and 200 kDa (**) molecular weight complexes of lamp2a were excised and subjected to reducing SDS-PAGE (12%) and silver staining (lanes 5 and 6). Molecular weight (kDa) is indicated on the left.(B) Immunoblot analysis of PPCA in rat liver lysosomes, their membranes (MB) and matrices (MTX) (100 μg of protein) incubated alone (None) or with 5 mM MgCl2. Arrowheads indicate the precursor (54 kDa) and heavy chain (32 kDa) of the mature PPCA.(C) Immunoblot analysis of hsc73 (upper) and PPCA (lower) in membranes from rat liver lysosomes incubated alone (None) or with 5 mM MgCl2, CaCl2, MnCl2 or 2 mM EDTA.(D) Immunoblot analysis of lamp2a co‐immunoprecipitated with PPCA in the absence of chemical cross‐linkers from membranes of liver lysosomes incubated as in (B). Light and heavy chains of IgG are shown. Lane 1 contains 1/10 of the total amount of lysosomal membranes used for immunoprecipitation.(E) Membranes from lysosomes treated as in (D) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre‐immune serum. Carboxypeptidase activity in total lysosomes and in the immunoprecipitates was measured as described under Materials and methods. Values are expressed as percentage of total carboxypeptidase activity in lysosomes present in the immunoprecipitates and are the mean ± SE of three different experiments.
Figure 2Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA. Membranes of lysosomes incubated with increasing concentrations of GAPDH (A) or MgCl2 (B, top panel) as labeled, were analyzed by immunoblot for all forms of lamp2 (upper), lamp2a (middle) or PPCA (lower). The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). (B, bottom panel) Chart showing the effect of increasing concentrations of MgCl2, CaCl2 or EDTA on the degradation of [14C]GAPDH by intact ratliver lysosomes. Values are means ± SE of six different experiments. Differences from the control value were significant at P 0.001 (**) or P 0.01 (*).(C) Immunoblot analysis for PPCA (upper) and cathepsin L (lower) of rat liver lysosomes (100 μg of protein) with high (H+) and low (H−) activity for CMA and of the lysosomal membranes (L.Memb) and matrices (L.Mtx) from both groups of lysosomes (200 μg of protein).(D) Immunoblot analysis for lamp2a, PPCA and cathepsin D, as labeled, of lysosomes (100 μg of protein) from fed or 48 h starved rats and the membranes (L. Memb) and matrices (L. Mtx) of both goups of lysosomes (200 μg of protein). Only mature forms of the cathepsins are shown(E) Correlation between CMA activity, measured as in (B, bottom panel) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats (Starv 20h), highly active (H+) or less active (H−) lysosomes, or lysosomes incubated with 1 mM CaCl2, MgCl2, EDTA, or increasing amounts (2, 5, 10 μg) of GAPDH. Values are expressed as times the value in fed rats (control) and are means ± SE of 3-6 different experiments.
Figure 3(A) Rates of protein degradation in skin fibroblasts from wild‐type (control) or PPCA(−/−) mice maintained in the presence (serum +) or absence of serum (serum −). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of four different experiments.(B) Degradation of [14C]GAPDH after 30 min incubation with lysosomes isolated from wild‐type and PPCA(−/−) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods. Where indicated, 25 μg of RNase A were added. Values are means ± SE of four different experiments. Differences from the control value were significant to P 0.001 (**).(C) Immunofluorescent staining for lamp2a and lamp1 of skin fibroblasts from wild‐type and PPCA(−/−) mice. Gain for lamp2a images (top) was 1/8 of the one for lamp1 (bottom) to visualize the punctate pattern of lamp2a in PPCA(−/−) mice. Inset: detail of the vesicular distribution of lamp2a. The gain in wild type was eight times the gain in PPCA(−/−) mice). Bars: 100 μm (top), 50 μm (bottom), 10 μm (inset).(D) Immunoblot analysis for lamp2a, all lamp2s, lamp1 or cathepsin D (cath D) of homogenates (75 μg of protein), lysosomes (10 μg of protein) and lysosomal membranes (L.MB) and matrices (L.MTX) (5 μg of protein) isolated from wild‐type (WT) and PPCA(−/−) skin fibroblasts. The bottom chart shows the densitometric quantification of the membrane proteins (means + SE) from four immunoblots similar to those shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).
Figure 4(A) Immunoblot analysis for lamp2a, all lamp2s, lamp1, transferrin receptor (TFR) or cathepsin D (cath D) of liver homogenates (100 μg of protein) from wild‐type (WT), PPCA(−/−) and β‐galactosidase(−/−) (GM) mice. Right: densitometric quantification of three immunoblots (means + SE) similar to the ones shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).(B) Immunocytochemistry for lamp2a (left), lamp1 (center) or lamp2s (right) in hematoxylin and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells.(C) Immunofluorescence for lamp2a (top) and P‐glycoprotein (bottom) in mouse liver sections. The right panel shows merged images. Bar: 100 μm. Inset shows detailed area at higher magnification. Bar: 50 μm.
Figure 5(A) Autofluorography of the sequential immunoprecipitation of lamp2a first (left) and all the remaining isoforms of lamp2 second (right) from metabolically labeled wild‐type and PPCA(−/−) mouse skin fibroblasts. Values are means ± SE of three different experiments similar to the ones shown in the upper panels.(B) Immunoblot analysis for lamp2a (top) and all lamp2 isoforms (bottom) in lysosomalmembranes from wild‐type (WT) or PPCA(−/−) mouseskinfibroblasts at different times after incubation at 37°C. The open arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). Right shows the densitometric quantification (mean ± SE) of four different experiments similar to the one shown here. Values are expressed as percentage of remaining lamp2a, and are means ± 1 SE of three different experiments.
Figure 6(A) Rates of total protein degradation in the presence and absence of serum of normal skin fibroblasts (CTR), GS cells (None) and GS cells supplemented with the precursor forms of native PPCA (+PPCA), a catalytic mutant of PPCA (+PPCA S/A) or cathepsin L (Cath L). Values are means ± SE of six different experiments. Differences from the control value were significant for P 0.001 (**) and P 0.05 (*).(B) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.(C) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.(D) Immunocytochemistry for lamp2a of liver (top) and kidney (bottom) sections of age‐matched wild‐type (WT), PPCA(−/−) and bone marrow transplanted (BMT) mice. Bar, 50 μm (liver) or 100 μm (kidney).
Figure 7(A) Left: amino acid sequences of the C‐terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse. The proposed cleavage site for PPCA in lamp2a is highlighted. Right: immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 (mouse; upper panel) and NIH 3T3 cells (human; lower panel) stably transfected with wild‐type (FL) and mutated (GL) HA‐lamp2a. The arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region.(B) Total rates of protein degradation in the presence and absence of serum in non‐transfected HEK293 cells (WT) and in the transfected HEK293 clones described in (A). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different experiments.(C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.
Figure 1(D) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. (
Figure 2(C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.(E) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.
Figure 3(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.
Figure 4(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).
Figure 6(D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.(E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.
Figure 7(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S
Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.
Figure 2C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).
Figure 3Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.
Figure 4Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).
Figure 5Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.
Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.