EMBO/soda-nel huggingface dataset:
plncRNA-encoded pep-AP attenuates the pentose phosphate pathway and sensitizes colorectal cancer cells to Oxaliplatin (2021)
Figure 3

Figure 3. Effect of lnc-AP on chemoresistance depends on its encoded short peptide

A. Knockdown of lnc-AP, as well as the endogenous pep-AP in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, t test).

B. 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).

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).

D. Prediction of potential transcription factors for the source gene of lnc-AP by Match.

E. PCR confirmation of siRNA of the predicted transcription factors in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, t test).

F. PCR analysis of effect of the predicted transcription factors on lnc-AP in HCT116 and SW480 cells (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).

H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test).

I. WB verification of pep-AP attenuation and restoration in HCT116 cells.

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: * 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.
plncRNA-encoded pep-AP attenuates the pentose phosphate pathway and sensitizes colorectal cancer cells to Oxaliplatin (2021)
Figure 4

Figure 4. Screening of potential interacted proteins with pep-AP

A. Schematic diagram of Co-IP combined with LC-MS/MS to screen potential proteins interacted with pep-AP.

B. Silver staining of gels for separating proteins by Co-IP in HEK293T cells.

C. GO annotation of molecular function for pep-AP-interacted proteins. Shown in the upper table and the lower chart, most of the proteins were involved in protein binding, of which TALDO1 with transaldolase activity was quite statistically significant (marked with a red box or a red circle).

D. 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.

E. WB confirmation of the interaction between pep-AP-Flag and TALDO1 in HEK293T, HCT116/L-OHP and SW480/L-OHP cells.

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.

H. IF detection of the colocalization of HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.
plncRNA-encoded pep-AP attenuates the pentose phosphate pathway and sensitizes colorectal cancer cells to Oxaliplatin (2021)
Figure 5

Figure 5. TALDO1 decreases L-OHP sensitivity via promoting PPP

A. 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).

B. Relative NADPH/NADP+ ratio (n = 3, biological replicates, mean±SEM, t test)

C. GSH contents (n = 3, biological replicates, mean±SEM, t test)

D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test)

E. MMP detection. Scale bar, 10 µm.

F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.

G. CCK-8 assay of the inhibition ratio by L-OHP (n = 3, biological replicates, mean±SEM, t test) with down- or up-regulation of TALDO1 in two groups of CRC cells.

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.
plncRNA-encoded pep-AP attenuates the pentose phosphate pathway and sensitizes colorectal cancer cells to Oxaliplatin (2021)
Figure 8

Figure 8. pep-AP sensitizes L-OHP response via suppressing TALDO1 in vivo

A. A flow chart depicting the in vivo experimental design.

B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM).

C. (a) Pictures of the tumors in all groups in B. (b) Further validation of the effect of pep-AP in vivo.

D. Weight of the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test).

E. RT-qPCR quantification of lnc-AP within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test).

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).

I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).

J. A proposed mechanistic model in this study.

Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 1

Figure 1: AICAR but not metformin prevents IFNγ/TNFα-induced myotube atrophy.

(A) Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control.

(B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm.

(C) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width.

(D-E) RT-qPCR analysis of the mRNA levels of MyoD (D) and myogenin (E) 24h after treatment relative to the NT control.

Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 3

Figure 3: AICAR restores anabolic signaling and de novo protein synthesis in cytokine-treated myotubes.

(A) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control.

(B) Radiographic analysis of de novo protein synthesis using 35S-labelling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3).

Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 6

Figure 6: A-769662 prevents iNOS expression and is anti-cachectic.

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width.

(B-C) Rates of glucose consumption (B) and lactate production (C) measured in the media 24h after treatment relative to the DMSO treated control.

(D) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control.

(E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control.

(F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment.

(G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment.

Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 7

Figure 7: AICAR and A-769662 synergistically prevent cytokine-induced myotube atrophy.

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width.

(B) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control.

(C) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the NT control.

Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 8

Figure 8: AICAR partially recovers muscle mass in the C26-mouse model of cancer cachexia.

(A) Representative images of overall hind limb musculature. Scale bar represents 1cm.

(B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC.

(C) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm.

(D-E) RT-qPCR analysis of Atrogin-1/MAFbx (D) and MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3).

Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls.
The AMPK agonist AICAR, but not metformin, prevents inflammation-associated cachectic muscle wasting (2018)
Figure 9

Figure 9: AICAR completely prevents muscle wasting in response to LPS injection.

(A) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm.

(B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B) and MuRF1 (C) mRNA expression from the tibialis anterior of four mice per cohort (n = 4).

Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls.
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway (2022)
Figure 1

Figure 1.

SCAI-deficient cells display major hallmarks of FA gene dysfunction

A. Schematic outline of genome-scale CRISPR-Cas9 screen for genes whose KO sensitizes human RPE-1 cells to MMC. LD20, 20% lethal dose; NGS, next generation sequencing.

B. DrugZ analysis of sgRNA depletion in CRISPR screen in (A) following low-dose MMC treatment (n=2 technical replicates). FA genes are highlighted in blue; SCAI is highlighted in red.

C. Immunoblot analysis of U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO cells stably reconstituted with Strep-HA-SCAI (U2OS/SCAI KO/Strep-HA-SCAI).

D. Clonogenic survival of U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).

E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments).

F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown.

G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test).

H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test).

I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm.

J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test).

K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway (2022)
Figure 2

Figure 2.

Preventing FA pathway activation alleviates ICL hypersensitivity of SCAI-deficient cells

A. Schematic outline of genome-scale CRISPR-Cas9 screen for genes whose KO suppresses MMC hypersensitivity of SCAI KO cells. LD80, 80% lethal dose; NGS, next generation sequencing.

B. MaGECK analysis of sgRNA enrichment in CRISPR screen in (A) following MMC treatment (n=3 technical replicates; false discovery rate (FDR) of 0.2 indicated by dotted line). FA genes are highlighted in blue.

C. GO term analysis of significantly enriched genes (FDR<0.2) in CRISPR-Cas9 screen in (A), using Reactome pathways from PANTHER16.0.

D. Immunoblot analysis of FANCA siRNA knockdown efficiency in U2OS cells.

E. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or FANCA siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).

F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test).

G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test).

H. Immunoblot analysis of the indicated U2OS cell lines.

I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).

J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).

K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway (2022)
Figure 3

Figure 3.

SCAI promotes replication-coupled ICL repair

A. Current model of ICLpt repair in Xenopus egg extracts (Amunugama et al, 2018).

B. pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting.

C. pICLpt was replicated in egg extracts in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. The p97 inhibitor NMS-873 (p97i; 200 μM), was supplemented to the reaction where indicated. Note that in the absence of p97 activity, CMGs are no longer unloaded from the plasmid, leading to accumulation of replication intermediate products (RI) (Fullbright et al, 2016). OC, open circular; SC, supercoiled.

D. Samples from (C) were recovered via plasmid pulldown at the indicated time points as in (B) and analyzed by immunoblotting.

E. pICLpt was replicated in mock- or SCAI-C-depleted egg extracts, and samples collected at the indicated time points were analyzed by immunoblotting.

F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.

G. Schematic of pICLpt illustrating the SapI site, which is regenerated upon replication-coupled repair (Knipscheer et al, 2009).

H. Quantification of SapI regeneration in Mock or SCAI-depleted extracts. Note that plasmids containing a SapI site but no ICL account for ~5-7% of each pICLpt preparation. Primary data are shown in Figure EV2F,G. A representative of two independent experiments is shown.

I. Schematic of intermediates and extension products generated by AflIII digest of pICLpt.

J. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were digested with AflIII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.

K. Schematic of intermediates and extension products generated by PsiI+XhoI double digest of pICLpt.

L. Samples in (J) were digested with PsiI+XhoI and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.

M. Quantification of extensions with deletions in (L) at 240 min (mean±SEM; n=6 independent experiments; **, p<0.01; two-tailed paired t-test).
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway (2022)
Figure 4

Figure 4.

SCAI interacts with Polζ but protects against erroneous ICL repair independently of Polζ

A. Analysis of SCAI-interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI-C and SCAI-N immunoprecipitates (IPs) compared to an IgG control, plotted against the P-value (n=4 independent experiments; permutation-based FDR<0.01, s0=2).

B. SCAI, REV7, REV1 and REV3 immunoprecipitates (IP) in NPE were immunoblotted with the indicated antibodies. Asterisks denote antibody cross-reactivity.

C. Whole proteome MS analysis of mock- versus SCAI-depleted egg extracts. The volcano plot shows the difference in abundance of proteins between the mock reaction and SCAI-depleted samples (with either N or C antibodies) (x-axis), plotted against the p-value resulting from two-tailed Student's t-test (y-axis). Proteins significantly down-regulated (FDR<5%) in SCAI-depleted extracts are highlighted in red (n=4 biochemical replicates; FDR<5% corresponds to a permutation-based FDR-adjusted q-value of <0.05).

D. GFP IPs from U2OS or U2OS/GFP-SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP-SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1-Polζ complex that are independent of REV3.

E. Schematic of SCAI interaction with the REV1-Polζ complex. The SCAI-interacting region of human REV3 and its alignment with a homologous region in human AHDC is indicated. NTD, N-terminal domain; PCD, positively charged domain; Pol, polymerase domain; CTD, C-terminal domain.

F. GFP IPs from U2OS cells transfected with indicated REV3-GFP expression constructs were immunoblotted with indicated antibodies.

G. As in (F), except that U2OS or U2OS/GFP-SCAI cells were transfected with FLAG-NLS-REV3(995-1194) expression plasmid.

H. Mock-, SCAI-, REV1- or SCAI/REV1-depleted egg extracts were analyzed by immunoblotting with indicated antibodies. Asterisks denote antibody cross-reactivity.

I. pICLpt was replicated in egg extracts immunodepleted with the indicated antibodies and processed as in Figure 3J,L.

J. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting.
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway (2022)
Figure 5

Figure 5.

SCAI prevents Polθ-mediated MMEJ during ICL repair

A. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies. Replication samples were amplified by PCR and analyzed by next-generation sequencing for deletions and insertions. Results from two independent experiments are shown.

B. Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross-reactivity.

C. pICLpt was replicated in extracts immunodepleted as in (B), digested with PsiI+XhoI or AflIII and resolved on a denaturing polyacrylamide gel.

D. Immunoblot analysis of Polθ siRNA knockdown efficiency in U2OS cells.

E. Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I. Scale bars, 10 µm.

F. Quantification of radial chromosomes in (E) (mean±SD; 1197, 241, 155 and 90 metaphase cells analyzed per condition (left to right) pooled from 3 independent experiments; *, p<0.05, two-tailed t-test).

G. Quantification of chromosomal breaks/gaps in (E) (mean±SEM; 150, 150, 114 and 90 metaphase cells analyzed per condition (left to right) pooled from 3 independent experiments; ****, p<0.0001, Mann-Whitney U test).

H. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).