EMBO/soda-nel huggingface dataset:
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 8

Figure 8. Endouc associates with monosomes and plays a key role in regulating CHOP post-translation.

A. HEK 293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 hr and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 hr. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysate from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analysed by SDS-PAGE, and subjected to Western blot using antibodies against Flag and S6.

B, C. The lysates from control (pCS2), Endouc-expressing (Endouc), and EndoucK242A-expressing (EndoucK242A) cells were applied to PPA. The resulting fractions from 1-10 collected from sucrose gradient were subsequently subjected to RT-qPCR assay to quantify (B) exogenous huORFchop-tagged luc (luc mRNA) and (C) endogenous CHOP transcripts (CHOP mRNA). The distribution of relative abundance of luc and CHOP transcripts contained in each fraction was determined.

D, E. HEK 293T cells were co-transfected with pCS2-Endouc and phuORFchop-luc for 24 hr and then subjected to DMSO or TH treatment for 2 hr. The lysates of transfected cells were then applied to PPA. Total RNAs were isolated from heavy polysome fractions followed by performing 5`RACE to characterize the nucleotide sequences of truncated (D) exogenous huORFchop-luc and (E) endogenous huORFchop-CHOP transcripts. The first sequence at N-terminus of RNA samples from the truncated forms derived from exogenous huORFchop-luc (n=14) and endogenous huORFchop-CHOP transcripts (n=11) was determined; the percentages of positive colonies containing various 5'UTR lengths (truncated forms) of luc and CHOP transcripts among total colonies examined were calculated.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 10

Figure 10. Comparison between Cap-dependent and IRES-dependent translation of the huORFchop-transcript containing reporter.

A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%.

G. The luc activity driven by the indicated A-capped monocistronic mRNA in the presence of Endouc or EndoucK242A in HEK 293T cells. The m7G-capped Rluc mRNA was used as an internal control to normalize the activities of A-cap Fluc transcripts. The egfp mRNA was used as a negative control, and the luc activity of cells transfected with egfp mRNA and A-cap Fluc mRNA containing full-length huORFchop-1-105-nt was set to 1. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005, ***: P <0.001).
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 1

Figure 1 - Replication fork progression is unsteady after Chk1 loss.

A Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1.

B Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control.

C Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.

D Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.

E U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.

Data information: The bars/numbers on top of the distribution clouds indicate the median; different letters indicate significant differences, calculated by unpaired t-test.
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 2

Figure 2 - Chk1 loss generates replication-associated obstacles that impair nascent DNA elongation.

A-C U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm. (B) IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. (C) Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean IdU track lengths (B) or mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.

D DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.

Data information: Different letters indicate significant differences, calculated by two-way ANOVA with a Tukey post-test (B-C) or unpaired t-test (D).
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 3

Figure 3 - High CDK activity prevents Polη-dependent bypass of replication barriers created by Chk1 loss.

A Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers.

B IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control.

C Percentage of origin firing (mean+SD) from DNA fibers in (A) (n=2). The dotted line represents the mean percentage of origin firing in control samples.

Data information: The bars on top of the distribution clouds indicate the median; different letters indicate significant differences, calculated by one-way ANOVA with a Bonferroni post test (B) or Repeated Measures ANOVA with Newman-Keuls post test (C).
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 4

Figure 4 - Serine 687 phosphorylation status determines GFP-Polη function at replication barriers created by Chk1 loss.

A Schematic representation of human Polη showing conserved, putative CDK phosphorylation S/TP sites and cyclin-binding domains (RxL). POL: polymerase domain, UBZ: ubiquitin-binding zinc finger, NLS: nuclear localization signal, PIP: PCNA-binding domain.

B Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm.

C Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control.

D IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition.

E Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. >150 cells/sample were analyzed in 4 independent experiments. p<0.05 was considered significant.

F Representative images of cells with GFP-Polη foci used for quantification in (E). The number of foci per cell for each representative nucleus is indicated. Scale bar: 10 μm.

G Model that depicts the effect of CDK-dependent phosphorylation of Polη on fork elongation. In Chk1-deficient cells, replication barriers accumulate and recruit Polη. CDKs, whose activity is high in Chk1-deficient cells, phosphorylate Polη, impeding its engagement in fork elongation. Upon CDK inhibition, Polη bypasses replication barriers created by Chk1 loss, promoting fork elongation.

Data information: The bars on top of the distribution clouds indicate the median; different letters indicate significant differences, calculated by one-way ANOVA with a Bonferroni post test.
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 5

Figure 5 - Replication fork progression and origin firing are independent variables in Chk1-depleted cells.

A IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells treated or not with a CDC7 inhibitor (CDC7i). >380 (track length) and >600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition.

B IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from >290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency >700 DNA fibers from 3 independent experiments were scored.

C IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.

D IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.

Data information: The bars on top of the distribution clouds indicate the median; different letters indicate significant differences, calculated by one-way ANOVA with a Bonferroni post test (track lengths) or Repeated Measures ANOVA with Newman-Keuls post test (% of origins).
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 6

Figure 6 - Replication barrier bypass in Chk1-deficient cells does not alleviate DNA damage.

A Scheme of the tools used to revert DNA replication phenotypes caused by Chk1 deficiency. CDK inhibition rescues impaired fork elongation and excess origin firing; NS supplementation or GFP-Polη overexpression specifically rescue reduced fork elongation; CDT1 depletion or CDC7 depletion/inhibition specifically rescue excess origin firing.

B Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics.

C Representative images of data shown in (B). Scale bar: 50 μm.

D DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments.

E Representative images of data shown in (D). Scale bar: 25 μm.

F Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null.

G Representative images of data shown in (F). Scale bar: 25 μm.

Data information: The bars on top of the distribution clouds indicate the median; different letters indicate significant differences, calculated by one-way ANOVA with a Bonferroni post test (D) or Repeated Measures ANOVA with Newman-Keuls post test (B, F).
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 7

Figure 7 - Replication barrier bypass in Chk1-deficient cells does not ameliorate cellular fitness.

A Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments.

B Sensitivity of U2OS cells transduced with shRNA targeting Chk1 (shChk1) or non-targeting shRNA (shScr) and Lenti-GFP-Polη or Lenti-GFP (-). Cell number was determined 7 days after transduction (5 days after maximal downregulation). Data represent the mean (+SD) of 3 independent experiments.

Data information: Different letters indicate significant differences, calculated by Repeated Measures ANOVA with Newman-Keuls post test.
Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing (2019)
Figure 8

Figure 8 - CDC45-dependent generation of replication barriers compromise the fitness of Chk1-deficient cells.

A Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control.

B IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition.

C IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control.

D Percentage of origin firing (mean+SD) from DNA fibers in C (n=2). >490 fibers per condition were scored.

E Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments.

F Representative image of data shown in (E). Scale bar: 100 μm.

G Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments.

H Representative image of data shown in (G). Scale bar: 100 μm.

Data information: The bars on top of the distribution clouds indicate the median and different letters indicate significant differences, calculated by one-way ANOVA with a Bonferroni post test (B-C) or Repeated Measures ANOVA with Newman-Keuls post test (D-E, G).
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf1

(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining. (b) Schematic representation of vBcl-2, Beclin1, PI(3)KC3 and UVRAG protein structure. BD, Bcl-2 binding domain; Acc, accessory domain; BH1, Bcl-2 homologue 1. (c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag–UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5. (d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3)KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3)KC3. (e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1–V5 (lanes 2 and 4) together with Flag–UVRAGΔCCD (lanes 1 and 2) or Flag–UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. (f) Beclin1 binds UVRAG through its CCD. After transfection with Flag–UVRAG together with full-length or fragments of Beclin1–V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. (g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag–UVRAG and empty vector or Flag–UVRAG and Beclin1–V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag. (h) UVRAG directly interacts with Beclin1. MBP–UVRAGCCD (lane 1), His6–Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP–UVRAGCCD, His6–Beclin1CCD and complex between these two. (i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD–UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf2

(a) The Bcl-2 binding domain of Beclin1 is sufficient to bind vBcl-2. At 48 h posttransfection with wild-type and mutant Beclin1–V5, together with HA–vBcl-2, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-HA. (b) The loss of Beclin1 interaction of vBcl-2AAA mutant. At 48 h posttransfection with wild-type HA–vBcl-2 or vBcl-2AAA mutant, together with Beclin1–V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5, followed by immunoblotting with anti-HA. (c) vBcl-2 interaction with wild-type UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag–UVRAG together with Beclin1–V5 and HA–vBcl-2 or vBcl–2AAA mutant, WCLs of 293T cells were used for immunoprecipitation with anti-HA followed by immunoblotting with anti-Flag. (d) vBcl-2 interaction with PI(3)KC3 in the presence of Beclin1. At 48 h posttransfection with HA–vBcl-2 together with Flag–PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunprecipitation with anti-HA followed by immunoblotting with anti-Flag. (e) PI(3)KC3 N-terminal C2 domain binds Beclin1. At 48 h posttransfection with Beclin1–V5 together with full-length or fragments of Flag–PI(3)KC3, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. CAT, catalytic domain. (f) Beclin1CCD and Beclin1ECD are sufficient for binding PI(3)KC3. At 48 h posttransfection with Flag–PI(3)KC3 together with full-length or fragments of Beclin1–V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. CT, C terminus (aa 337–448). (g) PI(3)KC3 interaction with UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag–UVRAGΔCCD or UVRAGCCD together with HA– PI(3)KC3 and Beclin1–V5, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA. (h) Schematic representation of the Beclin1–Bcl-2–UVRAG–PI(3)KC3 multiprotein complex. BH, Bcl-2 homologue; BH3B, BH3 binding motif. The raw data for a−d, f and g are presented in the Supplementary Information, Fig. S5.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf3

(a) UVRAG expression and LC3 process in HCT116 cells. The same protein amounts of 293T, NIH3T3, HCT116 and MCF7 cells were used for immunoblotting with anti-UVRAG and anti-tubulin. WCLs of 293T cells transfected with Flag-tagged UVRAG were included as controls. WCLs of HCT116.vector and HCT16.UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti-LC3 and anti-tubulin. The raw data for this experiment is presented in the Supplementary information, Fig. S5. (b) Electron microscopy of autophagy in HCT116 cells. HCT116.vector and HCT116.UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy. Scale bars (from left to right panels) represent 2 nm, 0.5 nm, 0.2 nm and 0.2 nm. The arrows indicate autophagosome or autolysosome. (c) UVRAG induces autophagosome formation in HCT116 cells. After transfection with GFP–LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions or starved for 3 h. GFP–LC3 was detected using an inverted fluorescence microscope. Arrows indicate autophagosomes. The scale bars represent 10 μm. (d) After transfection with GFP–LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf4

(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments. (b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP–LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.