EMBO/soda-nel huggingface dataset:
K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation (2022)
Figure 1

Figure 1.

A cell-based system for conditional and selective abrogation of K27-linked ubiquitylation

A. Expression constructs (top) and two-step procedure (bottom) for generation of Doxycycline (DOX)-inducible Ub replacement cell lines.

B. Experimental setup for assessing the ability of exogenous Ub variants to rescue clonogenic survival of U2OS/shUb cells. U2OS/shUb cells were co-transfected with indicated plasmids to allow for FLP-FRT-mediated integration of an ectopic Ub expression cassette (A), which was selected with Hygromycin B (2). Expression of shUb and ectopic Ub was induced by treatment with Doxycycline (DOX) (3), and colony formation was visualized by crystal violet staining.

C. Quantification of rescue efficiency (3), normalized to FLP-FRT integration efficiency (2), for individual Ub variants (black bars, mean; n=3 independent experiments). See also Figure EV1D.

D. U2OS/shUb and derivative Ub replacement cell lines treated with DOX for the indicated times or Ub E1 inhibitor (E1i) for 1 h were fixed and immunostained with Ub conjugate-specific antibody (FK2). Levels of Ub conjugates were quantified using quantitative image-based cytometry (QIBC) (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); >5000 cells analyzed per condition).

E. Immunoblot (IB) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h.

F. As in (E), except cells were exposed to ionizing radiation (IR, 2 Gy) 1 h before fixation and co-immunostained with indicated antibodies. Scale bar, 10 μm.

G. Mass spectrometry-based quantification of native Ub(12-27) and Ub(12-27);K27R) peptides after Trypsin digestion. Data represent triplicate technical replicate samples taken over a 96-h DOX induction time course of U2OS/shUb/HA-Ub(K27R)-c21 cells.

H. As in (G), but for Ub(34-48) peptide.

I. Ubiquitin linkage abundance in Ub(WT)- and Ub(K27R)-replaced cells as indicated by Ub di-Gly peptide quantification. HA-Ub conjugates from whole cell extracts of Ub(WT) and Ub(K27R)-c21 replacement cell lines treated with DOX for 72 h were isolated by HA immunoprecipitation (IP) under denaturing conditions and analyzed by quantitative label-free mass spectrometry (Figure 3D; Figure EV3C,D) (black bars, median; n=2-3 technical replicates; ****p<0.0001, ns: not significant, unpaired t-test). See Figure EV2C for the impact of Ub(K27R) replacement on K29-linkage abundance.

Data information: Data are representative of four (D) and three (E,F) independent experiments with similar outcome.
K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation (2022)
Figure 3

Figure 3.

K27-linked ubiquitylation predominantly occurs in the nucleus and impacts the integrity of the p97 system

A. Immunoblot analysis of Ub-K27 antibody reactivity with recombinant linkage-specific di-Ub proteins.

B. Representative images of U2OS/shUb/HA-Ub(WT) or U2OS/shUb/HA-Ub(K27R) cells immunostained with antibody to K27-linked Ub (Ub-K27). Scale bar, 10 μm.

C. Intensity of nuclear signal recognized by Ub-K27 antibody in U2OS/shUb/HA-Ub(K27R) cells treated with DOX for the indicated times was quantified using QIBC (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); >3000 cells analyzed per condition).

D. Schematic depicting experimental setup for isolation and quantitative label-free mass spectrometry analysis of HA-Ub conjugates isolated from whole cell lysates of DOX-treated Ub(WT) and Ub(K27R)-c21 replacement cell lines by HA immunoprecipitation (IP) under denaturing conditions.

E. Global ubiquitylation changes induced by Ub(K27R) replacement, profiled by mass spectrometry analysis of HA-Ub conjugates isolated as shown in (D) (see also Figure EV3C,D; for full results see Dataset EV1). Volcano plot shows enrichment of individual proteins (HA-Ub(WT)/HA-Ub(K27R) ratio) plotted against the P value. Significance thresholds (FDR<0.05; s0=1) are indicated. Nuclear proteins with a known interplay with p97 are highlighted in red. See Figure 1I for Ub di-Gly peptide quantification.

F,G. HA-Ub conjugates isolated as in (D) were immunoblotted with indicated antibodies. See also Figure EV3F.

H. DOX-treated Ub replacement cell lines treated with p97i for the indicated times were fixed and immunostained with Ub conjugate-specific antibody (FK2). Levels of Ub conjugates in the nucleus were quantified using QIBC (solid lines, median; dashed lines, quartiles; >2500 cells analyzed per condition).

I. As in (H), except cells were subjected to stringent pre-extraction before fixation (solid lines, median; dashed lines, quartiles; >5000 cells analyzed per condition).

J. Uninduced U2OS/shUb/HA-Ub cell lines treated or not with p97i for 8 h were analyzed as in (H) (solid lines, median; dashed lines, quartiles; >5000 cells analyzed per condition).

K. As in (H), except cells were immunostained with CHOP antibody (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); >3000 cells analyzed per condition).

Data information: Data (A-C,F-K) are representative of three independent experiments with similar outcome.
K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation (2022)
Figure 4

Figure 4.

K27-linked ubiquitylation directly targets a p97 model substrate to promote its p97-dependent turnover and is functionally epistatic with p97 inhibition

A. Schematic of p97- and proteasome-dependent degradation of fluorescent Ub(G76V)-GFP reporter.

B. Representative images (upper) and QIBC analysis (lower) (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition) of Ub(G76V)-GFP expression in stable U2OS/Ub(G76V)-GFP cells treated or not with p97i for 4 h.

C. U2OS/shUb/HA-Ub replacement cell lines stably expressing shUb-resistant Ub(G76V)-GFP reporter were treated with DOX and p97i as indicated, and nuclear Ub(G76V)-GFP signal intensity was analyzed by QIBC (black bars, mean; 150-750 GFP-positive cells analyzed per condition).

D. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines were transfected with shUb-resistant Ub(G76V)-GFP construct and treated or not with p97i for 4 h. Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies.

E. Immunoblot analysis of DOX-treated U2OS/shUb/HA-Ub cell lines treated or not with MG132 for 4 h.

F. U2OS/Ub(G76V)-GFP cells left untreated or incubated with p97i or MG132 for 4 h were subjected to denaturing GFP IP and analyzed by quantitative label-free mass spectrometry. Intensity of indicated Ub di-Gly peptides was normalized to GFP peptide intensity (black bars, median; n=4 independent samples (technical replicates)).

G. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines transfected with indicated Ub(G76V)-GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies.

H. Normalized logarithmic proliferation quantification for U2OS/shUb/HA- Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines subjected or not to low-dose (1.5 μM) p97i treatment 48 h after DOX addition, as determined by Incucyte image-based confluence analysis (mean±s.e.m.; n=3 independent experiments).

I. Proportion of DOX-treated, siRNA-transfected Ub replacement cells in G2 phase, assayed by EdU incorporation, was determined by QIBC (black bars, mean; n=5-6 independent experiments; **p<0.01, ns: not significant, Mann-Whitney test).

J. Proportion of DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines, which were subjected to p97i or not for different times, in M phase was determined by staining with a H3-pS10 antibody using QIBC (black bars, mean; n=3-5 independent experiments; >4000 cells analyzed per condition, **p<0.01, ns: not significant, Mann-Whitney test).

Data information: Data are representative of four (G) and three (B-E) independent experiments with similar outcome.
K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation (2022)
Figure 5

Figure 5.

Blocking the decoding of K27-linked ubiquitylation signals impairs p97 substrate turnover
A. FLAG IPs from U2OS cells transfected with indicated FLAG- tagged expression constructs were incubated with purified K27 di-Ub and analyzed by immunoblotting.

B. U2OS/Ub(G76V)-GFP cells were transfected with indicated FLAG-tagged expression constructs, and levels of Ub(G76V)-GFP in cells gated for FLAG expression were quantified using QIBC (solid lines, median; dashed lines, quartiles; >2000 transfected cells analyzed per condition).

C. Cells transfected as in (B) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting.

D. Cells transfected as in (B) were treated with p97i (4h) and harvested at the indicated times after removal of the drug. Levels of Ub(G76V)-GFP in cells gated as in (B) were quantified using QIBC (solid lines, median; dashed lines, quartiles; >1000 transfected cells analyzed per condition).

E. Schematic of fluorescent Ub(G76V)-GFP and Ub(G76V)-GFP-20AA reporters and their dependency on p97 and/or proteasome activity for their turnover.

F. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-GFP-20AA cell lines were treated or not with p97i (5 μΜ) for the indicated periods of time, and levels of GFP signal were quantified using QIBC (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition).

G. As in (F), except cells were treated with MG132 for the indicated times (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition).

H. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP cell lines were transfected with indicated FLAG-tagged expression constructs, and GFP levels in cells gated for FLAG expression were quantified using QIBC (solid lines, median; dashed lines, quartiles; >2000 transfected cells analyzed per condition).

I. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP cell lines were transfected or not with FLAG-UCHL3 C95S expression construct, treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting.

Data information: Data are representative of six (B) and three (A,C,D,F-I) independent experiments with similar outcome.

Expanded View figure legends
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 1

Figure 1. Dectin-3-/- mice have increased tumor load upon AOM-DSS treatment than WT mice.

Single-housed WT mice and Dectin-3-/- mice (n = 8 for each group) were injected intraperitoneally with one dose of AOM (10 mg/kg), followed by three cycles of feeding water with 2% DSS. After induction of tumorigenesis, mice were euthanized on day 100.

A Representative images of colon tumors were shown. Scale bars, 10 mm.

B Tumor number, tumor size and tumor load in each mouse was measured.

C,D Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.

E,F Tumor tissues were stained for Ki67 and p-STAT3. The percentage of Ki67-positive and p-STAT3-positive cells was quantified. Scale bars, 25 µm.

G Proportion of CD11b+F4/80+ and RORγt+/CD45+lin- cells was analyzed in colonic LP cells and mLNs among tumor-bearing WT and Dectin-3-/- mice by flow cytometry.

H Relative expression of Il-6, Il-22, Cxcl1 and Il-17a in LP cells from tumor-bearing WT and Dectin-3-/- mice were detected using qPCR.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by unpaired Student's t test. See also Figures EV1.
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 2

Figure 2. Feces from tumor-bearing Dectin-3-/- mice promote CAC.

Germ free (GF) mice were oral transferred with feces from tumor-bearing WT and Dectin-3-/- mice during administrated with AOM-DSS (n=8, each group). After induction of tumorigenesis (100 days), mice were euthanized.

A Tumor number, tumor size, and tumor load in colons were measured.

B Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist.

C Tumor tissues were stained for Ki67. The percentages of Ki67-positive tumor cells were quantified. Scale bars, 25 µm.

D Mice were treated as described in Fig 1A and Fig 2A. Feces were collected from colons in each mouse. Total fungal burden in feces were quantified using qPCR.

E Mice were treated as described in Fig 1A. Fungal ITS2 rDNA gene sequence was performed in each group. Fungal-taxon-based analysis at the phylum level in feces of mice. Color from blue to red indicates enrichment of fungus.

F Fungal-taxon-based analysis at the genus level in feces of mice. 1 Candidan 2 Fusarium 3 Kazachstania 4 Thermomyces 5 Phaeococcomyces 6 Mycosphaerella 7 Penicillium 8 Aspergillus 9 Coprinellus 10 Cryptococcus 11Mortierella 12 Guehomyces 13 Preussia 14 Chaetomium 15 others

G Three-dimensional principal component analysis (PCA) based on fungal ITS2 rDNA gene sequence abundance in the feces. WT: samples are from WT mice, De: samples are from Dectin-3-/- mice.

H Specific fungal burden of C.albicans in the feces were quantified using qPCR.

I GF mice were orally gavaged with C.albicans (twice a week, 1×107) during administration with AOM-DSS (n=8, each group). Mice were euthanized on Day 100, tumor load in each mouse were measured.

J ELISA results for binding assays of Dectin-3 with C. albicans. Plates were coated with C.albicans and then were added with 100 ml/well recombinant Dectin-3 at indicated concentrations.

K, L GFP-C. albicans (5×106) were added onto 1×106 BMDMs, and incubated at 37°C for 1h. Wells were washed and fresh media containing fluconazole (300 μg/ml) was added. At 6 hours and 18 hours, C. albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar. Scale bars, 25 µm.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student's t test. See also Fig EV2 and Appendix Figure S1.
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 3

Figure 3. Up-regulation of IL-22 in Dectin-3-/- mice contributes to CAC development.

A,B Mice were treated as described in Fig.2A. LP cells were isolated from each mouse and were culture for 48h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression.

C mRNA expressions of IL-22, β-defensin, Reg3g, and Cxcl1 in LP cells were detected by qPCR.

D LP cells and mLNs were isolated from each mouse and were culture for 48h. Production of IL-22 in LP cells and mLNs were detected by ELISA.

E LP cells were isolated from each mouse and were culture for 48h. Expression of Il-17 in LP cells were detected by qPCR. Production of IL-17 in LP cells were detected by ELISA.

F Protein level of p-STAT3 and STAT3 in colonic epithelial cells was detected by using Western Blot (one mouse per lane). The relative expression of p-STAT3 was calculated.

G WT and Dectin-3-/- mice were intraperitoneally treated with anti-IL22 antibody or anti-IgG antibody as control during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student's t test. See also Fig EV3.
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 4

Figure 4. IL-22 is mainly produced by RORγt+ ILC3.

A, B Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in CD3-RORγt+, CD3-RORγt-, CD3+RORγt- and CD3+RORγt+ were determined by flow cytometry.

C, D Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in NKp46-RORγt+, NKp46+RORγt- and NKp46+RORγt+ were determined by flow cytometry.

E WT and Dectin-3-/- mice were intraperitoneally treated with anti-CD90 antibody or anti-IgG antibody during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, feces were collected in colon from each mouse. Total fungal burden in feces were quantified using 18S rDNA qPCR.

F Mice were treated as described in Fig.4E and colonic LP cells were isolated. Numbers of IL-22+ cells in LP cells were determined by flow cytometry.

G Mice were treated as described in Fig.4E. Tumor number, tumor size, tumor load and histological score in colons were measured.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01 as determined by Student's t test. See also Fig EV4.
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 5

Figure 5. C.albicans induce glycolysis and IL-7 production in macrophages.

A Mice were treated as described in Fig.2A and Fig.2I. Colonic LP cells were isolated. Proportion and absolute number of IL-22+ cells in LP cells were detected by flow cytometry.

B Primary macrophages were isolated from LP cells in WT and Dectin-3-/- tumor-bearing mice. mRNA expression of glycolysis-relative genes detected by using qPCR.

C BMDM cells were stimulated with C.albicans (5×106), Curdlan, and α-mannan for 24h. Glucose uptake, pyruvate level, lactate production, and ATP level were determined using assay kit.

D BMDM cells were stimulated with C.albicans (5×106) for 24h, and ECAR and OCR were examined.

E BMDMs acquired from WT and Dectin-3-/- mice were stimulated with C.albicans for 24h. Cytokine and chemokine production of BMDMs were detected using multiplex cytokine assay.

F WT-derived BMDMs were stimulated with C.albicans, curldan, or α-mannan in combination with or without 2-DG (2.5mM) for 24h. mRNA expression of Il-7 were detected by using qPCR.

G BMDMs were acquired from WT and HIF-1-/- mice and were stimulated with C.albicans, curldan, or α-mannan for 24h. mRNA expression of Il-7 were detected by using qPCR.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student's t test. See also Fig EV5. and Appendix Figure S2
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 6

Figure 6. IL-7 induces IL-22 production in ILC3s through AhR and STAT3.

A Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 were detected by qPCR.

B BMDMs were stimulated with C.albicans for 24h and the supernatant was isolated and added to primary ILC3 cells. (1) Supernatant from BMDMs without stimulation. (2) Supernatant from BMDMs with stimulation of C.albicans. (3) Supernatant from BMDMs with stimulation of C.albicans and IL-7 antibody. Productions of IL-22 by ILC3 cells were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.

C, D Primary ILC3 cells were sorted from LPs and were stimulated with (IL-7 group) or without IL-7 (None group). mRNA expressions of relative genes were detected by qPCR.

E Primary ILC3 cells were sorted in LPs from STAT3fl/fl and STAT3fl/fl VillinCre mice and were stimulated with IL-7. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.

F Primary ILC3 cells were sorted from LPs and were stimulated with IL-7 in combined with FIZC and AhR Inh. Cells without stimulation were used as control (None). Production of IL-22 was detected by ELISA.

G AhR, STAT3, ROR binding sites in the il-22 promoter.

H HEK293T cells were transiently transfected with the indicated expression constructs (Ctl or AhR) and were stimulated with (IL-7 group) or without IL-7 (none group). ChIP analysis of the interaction of AhR and STAT3 to binding sites in the il22 promoter with or without IL-7, respectively.

I HEK293T cells were transiently transfected with the indicated expression constructs. Whole cell extracts were subjected to anti-HA immunoprecipitation, and were then immunoblotted with HA antibody and anti-STAT3 antibody.

J EL4 cells were transduced with retroviruses encoding AhR and STAT3. IL-22 mRNA expression was analyzed by qPCR.

Data information: Error bars represented SD of triplicate samples. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by unpaired Student's t test.
Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22 (2021)
Figure 7

Figure 7. IL-22 is correlated with fungal burden in patients with colon cancer.

A Patients with colon cancer were divided into four groups based on tumor stage. mRNA expressions of Dectin-3 in tumor tissues were detected by qPCR. The box represents the 25th to 75th percentile, and the whisker plots represent the minimum and maximum percentiles.

B Patients with colon cancer were divided into two groups based on fungal burden in the feces. mRNA expressions of Dectin-3 was compared between these two groups.

C Overall survival rate was shown by K-M survival curve.

D, E Tumor tissues were stained for IL-22 and p-STAT3. The percentages of IL-22-positive and p-STAT3-positive cells were quantified. mRNA expressions of IL22 and STAT3 were detected by qPCR. Scale bars, 25 µm.

Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student's t test.
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 1

Figure 1- Identification of E4 as a modulator of retrograde axonal transport

A) Small-molecule kinase inhibitor screen. Data are shown as an XY plot of normalized mean staining intensity of AlexaFluor555-HcT versus α-p75NTR. The α-p75NTR was detected using an AlexaFluor 647-conjugated donkey anti-rabbit secondary antibody [12]. Compounds that increased the accumulation of HcT or α-p75NTR by at least three standard deviations (yellow box) were classified as active compounds (A1-light blue, C3-purple, E4-dark blue). The negative control (EHNA) is shown in red (≥ 25 cell bodies were imaged per condition, N= 3 independent experiments).

B) Speed distribution profile of PMN treated with 0.5 µM E4 or DMSO for 30 min. Graph shows an increase in the rate of axonal transport upon E4 treatment (DMSO, 153 endosomes, 13 axons; E4, 165 endosomes, 13 axons; N=4 independent experiments, data shown are mean ± SEM).

C) PMN treated with 0.5 µM E4 for 30 min showed a significant decrease in phospho-IGF1R levels compared to controls. Protein levels were normalised to β-actin (*** P< 0.001, Students t-test, N=3 independent experiments, data shown are mean ± SEM).