EMBO/soda-nel huggingface dataset:
Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy (2016)
Figure 3

Figure 3. Interaction between Ulk1 and PPM1D and role of phospho-Ulk1637 in autophagy.

(A) Physical interaction between endogenous Ulk1 and endogenous PPM1D. PPM1D+/+ MEFs were treated with etoposide (10 µM) or left untreated for 6 hr. Cells were then lysed and immunoprecipitated with an anti-Ulk1 antibody or a control IgG. Immune complexes and total lysates (1.8% input) were analyzed by western blotting using anti-PPM1D and anti-Ulk1 antibodies. (B) Interaction between endogenous Ulk1 and recombinant PPM1D. Lysates from healthy PPM1D+/+ MEFs were incubated with GST-PPM1D or GST. Binding molecules were then analyzed by western blotting using anti-Ulk1 and anti-GST antibodies. “Total lysate” indicates 7% of the lysates that were incubated with GST fusion protein. (C) In vitro Ulk1 dephosphorylation assay. Immunoprecipitated Ulk1 from PPM1D+/+ MEF lysates was incubated with GST-PPM1D (1 µg) with or without a phosphatase inhibitor cocktail for 1 hr. Then, the extent of Ulk1 dephosphorylation was analyzed by western blotting using anti-phospho-Ulk1637 and anti-phospho-Ulk1757 antibodies. Semiquantitative analyses are shown in Fig. EV2B. (D) The presence of cytosolic PPM1D puncta in etoposide-treated PPM1D+/+ MEFs. PPM1D+/+ MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with an anti-PPM1D antibody, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (green; upper panels) and Hoechst 33342 (blue; lower panels) are shown. Arrowheads indicate cytoplasmic PPM1D puncta. (E) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide-treated PPM1D+/+ MEFs. PPM1D+/+ MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with anti-PPM1D and anti-Ulk1 antibodies, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (red; left), anti-Ulk1 (green; middle), and a merged image (right) are shown. Magnified images of the areas within the dashed squares are shown at the bottom. Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1. (F–J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (F–H) Ulk1/Ulk2 DKO MEFs that were stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM). Then, the cell lysates were collected time-dependently, and the expression of each protein was examined by western blotting. Asterisks in the Ulk1 blots are nonspecific bands. Semiquantitative analyses are shown in Fig. EV2E. (I, J) The indicated MEFs were treated with or without etoposide (10 µM) for 6 hr, followed by immunostaining with an anti-LC3 antibody. Representative images are shown in (I). LC3 puncta are markedly observed in DKO MEFs transfected with wild-type HA-Ulk1. Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A, respectively. (J) The population of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.
Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy (2016)
Figure 4

Figure 4. Involvement of PPM1D in Ulk1 activity and Ulk1/DFCP1 puncta formation.

(A–D) Ulk1 puncta formation was induced by etoposide in a PPM1D-dependent manner. The indicated MEFs were treated with etoposide (10 µM) with or without GSK2830371 (20 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images are shown in (A). Ulk1 puncta are observed in etoposide-treated PPM1D+/+ MEFs. (B–D) The proportion of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01, NS: not significant. (E–I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (E, F) Cells were immunostained with an anti-Ulk1 antibody (E), and the population of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment) (F). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01. (G–I) Cell lysates were collected in a time-dependent manner, and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting. Semiquantitative analyses are shown in Fig. EV3D. (J) EGFP-DFCP1-expressing PPM1D+/+ MEFs were treated with etoposide (10 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images of EGFP-DFCP1 (green; left), anti-Ulk1 (red; center), and a merged image (right) are shown. The nuclei were stained with Hoechst 33342 in the merged image. A magnified image of the area within the dashed square is also shown. Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. (K, L) The role of Ulk1 dephosphorylation at Ser637 on etoposide-induced DFCP1 puncta formation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were transfected with EGFP-DFCP1 and treated with etoposide (10 µM) for the indicated hours, followed by immunostaining with an anti-HA antibody. Representative merged images of EGFP-DFCP1 (green), anti-HA (red), and Hoechst 33342 (blue) are shown in (K). Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. The number of DFCP1 puncta in each cell was calculated and is shown in (L) (n > 8 cells in each experiment). Red lines indicate the mean values. **p < 0.01, NS: not significant.
Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy (2016)
Figure 5

Figure 5. Impaired autophagy and decreased cell viability in PPM1D-deficient thymocytes after X-ray irradiation.

(A) PPM1D-dependent dephosphorylation of Ulk1 at Ser637, induction of autophagy, and degradation of Noxa. PPM1D+/+ and PPM1D−/− primary thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were lysed and the expression of each protein was examined by western blotting. α-Tubulin was used as a loading control. Semiquantitative analyses are shown in Fig. EV5B. (B–F) Suppression of X-ray-induced autophagy in PPM1D−/− thymocytes. (B, C) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were immunostained with an anti-LC3 antibody. Nuclei were stained with Hoechst 33342. Representative images are shown in (B). LC3 puncta can be seen in irradiated PPM1D+/+ thymocytes (arrowheads). In (C), the proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+ thymocyte. (E) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were stained with Cyto-IDTM. Representative flow cytometric data are shown. Quantitative data are shown in Fig. EV5C. Cyto-ID fluorescence in irradiated PPM1D+/+ thymocytes was higher than that in the other thymocytes. (F) Analysis of autophagic flux. The indicated thymocytes were X-ray irradiated (5 Gy) in the presence or absence of chloroquine (120 µM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. Semiquantitative analyses are shown in Figure EV4D. (G–J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (G) Thymocytes were X-ray irradiated (5 Gy), and 6 hr later, cell death was determined using the PI assay. Data represent the mean  ± SD (n = 3 independent thymi). *p < 0.05, **p < 0.01, NS: not significant. (H, I) Thymocytes were X-ray irradiated (5 Gy), and 3 hr later, the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses are shown (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01. (J) Indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, caspase3/7 activity was examined using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer’s protocol. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01. (K) The effect of Noxa siRNA on thymocytes. The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs. After 12 hr, cells were X-ray irradiated (5 Gy), and 6 hr or 10 hr later, the population of dead cells among the GFP-positive cells was determined using the PI assay. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, NS: not significant.
LINC00839 promotes colorectal cancer progression by recruiting RUVBL1/Tip60 complexes to activate NRF1 (2022)
Figure 1

Figure 1 - LINC00839 is highly expressed in CRC and positively associated with poor outcome in patients.

A The expression of LINC00839 in CRC tissues and normal mucosa tissues in the UALCAN database.

B, C, D The expression of LINC00839 in unpaired CRC and normal samples in the LnCAR database (GSE37364, GSE71187, and GSE33113).

E, F The expression levels of LINC00839 in tumour tissues and NATs from the CRC cohort (n = 45). nmCRC denotes CRC patients without metastases, and mCRC denotes CRC patients with metastases.

G Representative ISH staining of LINC00839 in paraffin-embedded samples of CRC tumour tissues and NATs. (a) Expression of LINC00839 in tumour tissues and NATs. Scale bars, 100 μm. (b) Magnification of normal mucosal area and (c) magnification of the tumour area. Scale bars, 50 μm. (d) Weakly positive staining of LINC00839 in normal mucosa. Scale bars, 50 μm. (e) Strong positive staining of LINC00839 in tumours and (f) magnification of the local area. Scale bars, 100 μm.

H The expression score of the tumours and NATs in the ISH cohort (n = 222).

I Differential expression of LINC00839 in nonmetastatic CRC patients (nmCRC) and metastatic CRC patients (mCRC).

J Expression of LINC00839 in CRC patients with early-stage (stage I and II) and advanced-stage (stage III and IV) disease.

K, L Kaplan-Meier analysis of overall survival and disease-free survival rate of the ISH CRC cohort.

M Kaplan-Meier analysis of the disease-free survival rate of CRC patients in the LnCAR database (GSE33113).

N The expression of LINC00839 in a colon mucosa epithelial cell line (FHC) and CRC cell lines.

O The subcellular localization was determined by nucleocytoplasmic fractionation of several CRC cell lines.

Data information: In A, B, C, D and J, middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. In E, F, N and O, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. In H and I, the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
LINC00839 promotes colorectal cancer progression by recruiting RUVBL1/Tip60 complexes to activate NRF1 (2022)
Figure 2

Figure 2 - Overexpression of LINC00839 promotes CRC cell growth and metastasis in vitro and in vivo.

A Overexpression of LINC00839 in SW480 and LoVo cells was confirmed by qPCR (mean ± SD, Student's t test, n = 3 technical replicates).

B The proliferation of LINC00839-overexpressing cells was determined by CCK-8 assay (mean ± SD, ANOVA, n = 3 biological replicates).

C Colony formation assay was performed with LINC00839-overexpressing cells and vector control cells.

D Wound-healing assay was performed with LINC00839-overexpressing cells and vector control cells. Scale bars, 100 μm.

E Matrigel invasion assay was performed with LINC00839-overexpressing cells. Scale bars, 100 μm.

F The CDX model showing the effect of LINC00839 on tumour growth. Representative image of tumours harvested from the mouse model (upper panel), the quantitative analysis of tumour weight and tumour volume (medium panel), and representative images and quantification of IHC staining of Ki-67 in tumours (bottom panel) (n = 6 for each cohort, and the results are presented as the mean ± SD, Student's t test (medium left and low right panel) and ANOVA (medium right). Scale bars, 100 μm (n = 6 biological replicates).

G The CRC orthotopic mouse model revealed the effect of LINC00839 on tumour development. Representative images of primary tumours (yellow arrowheads) and liver metastasis (blue arrowheads) in the model (upper panel), analysis of tumour volume and the number of mice with liver metastasis (medium panel), and H&E staining of tumours and metastases (n = 6 for each cohort). Scale bars, 100 μm (n = 6 biological replicates).

Data information: In C, D and E, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
LINC00839 promotes colorectal cancer progression by recruiting RUVBL1/Tip60 complexes to activate NRF1 (2022)
Figure 3

Figure 3 - LINC00839 serves as a scaffold to recruit Ruvb1 to the Tip60 complex and increases its acetylase activity.

A An RNA pull-down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells.

B RNA pull-down assay and WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 cells. Dot blot of RNA-protein interactions indicating equal amounts of RNA used in the assay.

C RIP experiments were performed to confirm the binding of LINC00839 to Ruvb1. IgG was used as a negative control antibody. Primers specific for U6 and GAPDH were used as negative control primers.

D Different fragments of LINC00839 (solid line) and its antisense sequence (dotted line) were used for the RNA pull-down assay and the subsequent WB. E Full length LINC00839, antisense sequence of LINC00839, and LINC00839 sequence lacking nucleotides 1033-1290 were used for the RNA pull-down assay and the subsequent WB.

F RIP experiments were performed by incubating Tip60, INO80, SRCAP, and PIH1D1 antibodies with HCT116 cell lysates.

G The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB.

H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co-IP.

I The interaction between Ruvb1 and Tip60 in cells overexpressing wild-type LINC00839 and LINC-Δ5 was confirmed by Co-IP.

J The interaction between Ruvb1 and Tip60 in LINC00839-KD cells and cells expressing LINC-Δ5 was confirmed by Co-IP.

K Acetylase activity assays were performed with LINC00839-overexpressing and LINC00839-knockdown cells.

L Acetylase activity was measured in SW480 and LoVo cells subjected to different treatments.

Data information: In C, F, K and L, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
LINC00839 promotes colorectal cancer progression by recruiting RUVBL1/Tip60 complexes to activate NRF1 (2022)
Figure 4

Figure 4 - LINC00839 promotes NRF1-mediated OXPHOS and mitochondrial biogenesis in CRC.

A KEGG pathway analysis of the DEGs identified by RNA-seq.

B Heatmap of DEGs related to OXPHOS and mitochondrial biogenesis. Expression levels are shown in log2 scale of normalized intensity values (see colour gradient) ranging from low (blue) to high (red) expression.

C GSEA of LINC00839 in the OXPHOS pathway (GSE14333).

D, E The ATP levels and the NAD/NADH ratio were measured in LINC00839-overexpressing and LINC00839-KD cells (mean ± SD, Student's t test, n = 3 biological replicates).

F Expression of MT-ND5, MT-CYB, MT-CO1, and TFAM in LINC00839-overexpressing and LINC00839-KD cells as determined by WB.

G Analysis of NRF1 expression in CRC tissues (T) and normal tissues (N) the GEPIA database.

H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases (Pearson).

I, J Expression of NRF1 in LINC00839-overexpressin and LINC00839-KD cells was measured by qPCR and WB.

K, L The enrichment of the NRF1 promoter (P1-P10) by H4K5ac and H4K8ac antibodies in LINC00839-overexpressing and control LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control.

M, N The enrichment of the P2 and P3 fragments by H4K5ac and H4K8ac antibodies in LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control.

O The enrichment of NRF1 with a probe targeting LINC00839 relative to a NC probe in HCT116 cells, as determined by ChIRP assay. LacZ served as a negative control (NC).

Data information: In I, K, L, M, N and O, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. In G, the middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles; the data were analysed by ANOVA. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
LINC00839 promotes colorectal cancer progression by recruiting RUVBL1/Tip60 complexes to activate NRF1 (2022)
Figure 5

Figure 5 - LINC00839 promotes NRF1-mediated OXHPOS and CRC progression.

A, B ATP levels and NAD/NADH ratios were measured in SW480 and LoVo cells.

C Expression of genes related to mitochondrial biogenesis and EMT in SW480 and LoVo cells as determined by WB.

D Invasion of SW480 and LoVo cells was assessed by Matrigel invasion assay. Scale bars, 100 μm.

E Representative images of primary tumours (yellow arrowhead) and metastases in the orthotopic implantation models (n = 6 for each cohort, left panel). The number of mice with liver metastasis (blue arrowhead) was determined. The primary tumour volume in each group was calculated (right panel). H&E staining of tumours and metastases (bottom panel). Scale bars, 200 μm (n = 6 biological replicates).

F ISH staining of LINC00839 and IHC staining of NRF1, H4K5ac, and H4K8ac in paraffin-embedded samples of CRC tumour tissues and NATs. Scale bars, 200 μm.

G, H, I Correlation analysis of the expression of LINC00839 and NRF1 (G), H4K5ac (H), and H4K8ac (I) in CRC patients (n = 120).

J Correlation of NRF1 expression with the overall survival rate in patients with CRC (n = 120). NRF1 expression was stratified as high and low according to the median expression level.

K A schematic illustration of the mechanism by which LINC00839 recruits the Ruvb1/Tip60 complex to acetylate histones at the NRF1 promoter and regulates CRC tumour progression via OXPHOS.

Data information: In A, B, and D, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. In G, H and I show the Pearson correlation coefficient results. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Expanded Figure Legends
Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment (2019)
Figure 1

Figure 1 Diflunisal binds HMGB1.

A) Chemical structure of DFL.

B) DFL inhibits HMGB1-induced, but not fMLP-induced cell migration. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers, 1 nM HMGB1 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points with average ± standard deviation (avg ± sd; n=3, each point represents a biological replicate) in a representative experiment. The reproducibility of this experiment is shown in Appendix Fig. S1. Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post tests. ** p<0.01, *** p<0.001 relative to no DFL addition.

C) Histogram showing residue-specific CSPs of 15N-labelled HMGB1 (~0.1 mM) upon addition of ten-fold excess of DFL (helices are schematically represented on the top). Missing residues are prolines or are absent because of exchange with the solvent. Box A and Box B residues with CSP > avg + sd are represented in magenta and blue, respectively.

D) HADDOCK models of interaction of DFL (CPK representation) with Box A (left) and Box B (right) (color, residues with CSP > avg + sd). HMGB1 residues (sticks) involved in hydrophobic and electrostatic interactions with DFL are explicitly labelled.

E) The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown. The difference in migration towards R23A/R109A HMGB1 in the presence of increasing concentrations of DFL is statistically non-significant (one-way ANOVA plus post tests).
Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment (2019)
Figure 2

Figure 2 Diflunisal binds CXCL12.

A) Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top.

B) Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line).

C) Left: HADDOCK model of interaction of DFL (CPK representation) with CXCL12 (grey surface and cartoon). CXCL12 residues with CSP > avg + sd, located around the sY21 binding site and on the β1 strand are in red and orange, respectively. Right: Zoom in the binding site, CXCL12 residues (sticks) involved in hydrophobic and electrostatic interactions with DFL (sticks) are explicitly labelled (right).

D) DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFL is not significantly different (one-way ANOVA plus post tests).

E) DFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).
Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment (2019)
Figure 3

Figure 3 Effect of DFL on the HMGB1/CXCL12 heterocomplex.

A, B) 1H-15N HSQC HMGB1 (0.1 mM) spectrum (A) without (black) and (B) with 0.2 mM CXCL12.

C) MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1; the signal increases from ∼930 (free) to ∼950 a.u. (bound) yielding an apparent Kd of 4 ± 0.4 μM. n=3; error bars represent avg ± sd.

(D,E) H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL.

F) MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.