EMBO/soda-nel huggingface dataset:
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 5

Figure 5. Irgm2 interferes with caspase-11-dependent but not caspase-11-independent NLRP3 activation upstream of cytosolic LPS accessibility

(A) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured.

(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured.

(C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured.

(D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt.

(E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt.

Data information: Shown are means ± SEM from n = 5 (A) or n = 3 (B, C, D, E) independent experiments. **p < 0.01, ***p < 0.001 for indicated comparisons by two-way ANOVA with Tukey's multiple comparisons test. N.S, non significant.
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 6

Figure 6. Irgm2 protects against caspase-11-driven septic shock during endotoxemia

(A) WT (n = 9), Irgm2-/- (n = 10), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 8) mice were injected i.p. with LPS (8 mg/kg). Morbidity and mortality were observed for 48 h at 3 hour intervals.

(B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, and IL-18 was measured by ELISA. Data shown are from 2 pooled experiments.

Data information: Graphs (B) show means ± SEM. *p < 0.05, ***p < 0.001 for indicated comparisons by log-rank Mantel-Cox test (A) or one-way ANOVA with Tukey's multiple comparisons test (B). N.S, non significant.
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 7

Figure 7. Restoring expression of endogenous Irgm1 is sufficient to hypersensitize panIrgm-/-, macrophages for caspase-11 activation

(A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments).

(B) IFNγ-primed WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 8 hpt/ hpi (n = 3 independent experiments).

(C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments.

(D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals.

Data information: Graphs show means ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 for indicated comparisons by two-way ANOVA (A, B) or one-way ANOVA (C) with Tukey's multiple comparison test, or log-rank Mantel-Cox test (D). N.S, non significant.

EXPANDED VIEW FIGURE LEGENDS:
Curcumin prevents obesity by targeting TRAF4-induced ubiquitylation in a m6A dependent manner (2021)
Figure 2

Figure 2. Curcumin inhibits adipogenesis through attenuating ALKBH5 expression

A ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8. (Scale bar=500μm)

B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice.

C ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8. (Scale bar=500μm)

D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin.

E LC-MS/MS quantification of the m6A/A in mRNA of iWAT of mice fed with HFD and HFD+CUR groups.

F, G mRNA and protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups.

H LC-MS/MS quantification of the m6A/A in mRNA of 3T3-L1 cells.

I, J mRNA and protein expression levels of m6A regulator in 3T3-L1 cells were analyzed by qPCR.

K Oil Red O staining of Vector or OE-ALKBH5 transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8. (Scale bar=500μm)

L mRNA expression of PPARγ, C/EBPα and FABP4 expression of cells described in (K).

Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.
Curcumin prevents obesity by targeting TRAF4-induced ubiquitylation in a m6A dependent manner (2021)
Figure 3

Figure 3 ALKBH5 modulates adipogenesis via regulating TRAF4

A The relationship between ALKBH5 and TRAF4 was analyzed in GEO data base. Pearson's correlation analysis indicated TRAF4 and ALKBH5 had negative relationship (Cor = -0.70)

B Western blot analysis of TRAF4 protein levels in control and ALKBH5-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression.

C Western blot analysis of TRAF4 protein levels in control and ALKBH5-overexpressing cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression.

D Western blot analysis of PPARγ and C/EBPα protein levels in control and TRAF4-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression.

E Oil Red O staining of control, ALKBH5-depleted and ALKBH5+TRAF4 depleted cells after induced for 8 days. (Scale bar=500μm)

F qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell described in (E). β-Actin was used as an internal control.

G Western blot analysis of PPARγ and C/EBPα protein levels in cells described in (E), and quantification of protein levels normalized to β-actin expression.

Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.
Curcumin prevents obesity by targeting TRAF4-induced ubiquitylation in a m6A dependent manner (2021)
Figure 4

Figure 4 ALKBH5 regulates the protein expression of TRAF4 via m6A-YTHDF1-dependent manner

A LC-MS/MS quantification of the m6A/A in mRNA of control, wild-type (WT)- and mutant (MUT)-ALKBH5 overexpressing cells.

B Western blot analysis of ALKBH5 and TRAF4 in control, WT- and MUT-ALKBH5 overexpressing cells and quantification of protein levels normalized to β-actin expression.

C Integrative genomics viewer (IGV) plots of m6A peaks at TRAF4 mRNAs. The y-axis shows sequence read number, blue boxes represent exons, and blue lines represent introns.

D MeRIP-qPCR analysis of m6A levels of TRAF4 mRNA in control and ALKBH5-depleted cells.

E MeRIP-qPCR analysis of m6A levels of TRAF4 mRNA in in control, WT and MUT-ALKBH5 overexpressing cells.

F Left panel: Schematic diagram of dual luciferase reporter constructs. Right panel: Relative luciferase activity of WT or MUT (A-to-T mutation) TRAF4 3ʹUTR luciferase reporter in control, WT and MUT-ALKBH5 overexpressing cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity.

G Western blot analysis of TRAF4 protein levels in control and YTHDF1 overexpressing cells and quantification of protein levels normalized to β-actin expression.

H Polysome profiling assays. The fractionation of lysates from 3T3-L1 cells transfected with or without YTHDF1 plasmid is shown on the top. RNAs in different ribosome fractions were extracted and subjected to qPCR analysis.

I RIP-qPCR analysis of the interaction of TRAF4 with FLAG in cells overexpressing FLAG-YTHDF1. Enrichment of TRAF4 with FLAG was measured by qPCR and normalized to input.

J Relative luciferase activity of WT or MUT TRAF4-3ʹUTR luciferase reporter in cells transfected with control or YTHDF1 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity.

K Western blot analysis of YTHDF1, ALKBH5 and TRAF4 in control, ALKBH5-depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β-actin expression.

L Oil Red O staining of control, ALKBH5-depleted and ALKBH5+YTHDF1 depleted cells on day 8 of differentiation. (Scale bar=500μm)

M qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell described in (L). β-Actin was used as an internal control.

Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.
Curcumin prevents obesity by targeting TRAF4-induced ubiquitylation in a m6A dependent manner (2021)
Figure 5

Figure 5. TRAF4 suppresses adipogenesis through ubiquitination of PPARγ

A Western blot analysis of PPARγ protein levels in control and TRAF4-depleted cells and quantification of protein levels normalized to β-actin expression.

B Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells and quantification of protein levels normalized to β-actin expression.

C, D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β-actin expression.

E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI. Cells were treated with MG132 for 6 h.

Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey's test. **P < 0.01, ***P < 0.001 compared to control group.
PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis (2021)
Figure 3

Figure 3

Involvement of the PRMT1-targeted arginines in p14ARF's nucleolar localization.

A, B: U2OS cells were transfected with EGFP-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. p14ARF-EGFP (green), endogenous NPM as a nucleolar marker (red, using α-NPM antibody), DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin) were visualized by fluorescence microscopy, for which representative results are shown in (A). Scale bars: 10 μm. The subcellular distribution of p14ARF-EGFP (exclusively nucleolar, not-exclusively nucleolar but additionally nucleo-/cytoplasmic or exclusively cytoplasmic) was quantified by cell counting (percentage of cells) for three independent experiments in (B) (mean ± SD, **: p-value ≤ 0.005 using Welch´s t-test).

C: U2OS cells were transfected with EGFP-tagged wild type (wt) or mutant (RK) p14ARF, either alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. Cells with predominant nucleo-/cytoplasmic localization of p14ARF-EGFP (for both wt and RK) were quantified in the absence or presence of PRMT1 overexpression, i.e. in p14ARF-positive as well as p14ARF/Myc-PRMT1-double positive cells, by immunofluorescence staining and cell counting. Relocalization of p14ARF wt and RK into the nucleo/cytoplasm upon overexpression of PRMT1 was defined in percentage of cells for five independent experiments (mean ± SD).

D: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunoprecipitation (IP) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut.

E: Indicated NLS/NoLS p14ARF peptides (aa 91-99 or aa 92-103) either unmodified or premodified (asymmetric dimethylation of R96 in peptide aa 91-99 and of R96/R99 in peptide aa 92-103) were covalently coupled to Sulfolink-beads and incubated with recombinant, baculoviral purified Flag-tagged NPM. Pulldown reactions and input of NPM protein were resolved by SDS-PAGE and analyzed by α-NPM immunoblotting.
PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis (2021)
Figure 6

Figure 6

Protection from apoptosis by PRMT1-mediated arginine methylation of p14ARF.

A-C: HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) DNA staining for a representative experiment ((A), Appendix Fig. S8). The subG1 fraction of the siControl_4 and siPRMT1_4 condition was quantified for three independent experiments in (B) (mean ± SD, **: p-value ≤ 0.005 using Welch´s t-test). PRMT1 depletion and p14ARF methylation of the samples (from (A)) were monitored by immunoblotting using the indicated antibodies (C). The p14ARF and the methylated p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with siControl_4 condition set to 1.

D: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC. After 0, 2, and 4 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The percentage of PARP cleavage was densitometrically quantified and is specified below the blot.

E: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC. After 4 hours, the apoptotic cell fraction was analyzed by flow cytometry using FITC-labeled AnnexinV and propidium iodide (PI). The increase in the apoptotic cell fraction upon UVC irradiation was quantified for three independent experiments (mean ± SD, **: p-value ≤ 0.005 and *: p-value ≤ 0.05 using Welch´s t-test).

F: HeLa CRISPR/Cas9 PRMT1 deleted (KO_1) cells were transfected with Flag-tagged mutant RK or RF p14ARF-containing plasmid and subsequently irradiated at 150 J/cm2 UVC. After 4 hours, the apoptotic cell fraction was quantified by flow cytometry using FITC-labeled AnnexinV and propidium iodide (PI) for seven independent experiments (mean ± SD, *: p-value ≤ 0.05 using the paired t-test).

G: GST alone, GST-tagged wild type (wt) and mutant (RF) p14ARF proteins were coupled to Glutathione beads and incubated with baculoviral expressed, purified Flag-tagged p32. Pulldown reactions and input of p32 protein were resolved by SDS-PAGE and analyzed by immunoblotting using α-Flag as well as α-p14ARF antibodies. Short and long exposure times are specified. Staining results derive from the same blot and exposure times with white lines indicating where tracks were cut.

H: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC or not irradiated. After 4 hours, immunoprecipitations (IP) of endogenous p14ARF were performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are specified.
PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis (2021)
Figure 7

Figure 7

Prognostic relevance of PRMT1 for clinical PDAC outcome.

A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot).

B: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm.

C: The pie chart depicts the percentage of patients of the PDAC cohort (n=75) belonging to the three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression.

D: Patients of the PDAC cohort were grouped according to their survival time in months into three survival groups: short-term (≤12 months), medium-term (13-23 months) and long-term survivors (≥24 months). Subsequently, the percentage of patients belonging to the three different PRMT1 expression scores (legend as in (C)) are displayed for each survival group.

E: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm.

F: p14ARF-positive patients of the PDAC cohort were grouped according to their survival time in month into two survival groups: short-term (≤ 12 months) and long-term survivors (≥ 24 month). Subsequently, the percentages of patients with either not/moderately elevated PRMT1 expression or highly elevated PRMT1 expression are displayed for the two survival groups.
IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment (2015)
Figure 1

Figure 1. Characterization of TEMs in CRC liver metastasis mouse model.

A. Schematic representation of the experimental procedure.

B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.

C. Representative images (top panels) and corresponding H&E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.

D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.

E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.
IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment (2015)
Figure 3

Figure 3. CT26-RFP+ CRC cell colonization of the liver.

A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7 within the liver of Tie2-GFP or Tie2-IFNα mice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105 CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFP mice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).

B. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.

C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).