EMBO/soda-nel huggingface dataset:
Downregulation of basal myosin-II is required for cell shape changes and tissue invagination (2018)
Figure 6

Figure 6. Stabilization of basal myosin-II after the cell elongation phase has terminated and initial tissue invagination has started inhibits deep invagination and closure of the furrow.

(A) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized.

(B-I) Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C) or the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square). (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I).

Data information: Scale bars, 20 µm.
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 1

Figure 1. CARM1 dimethylates LSD1 at arginine 838.

A Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838.

B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively.

C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells.

D Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).

E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).

F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.

G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.

H Sequence alignments of LSD1 among mammals. LSD1 residue at R838 is denoted in the protein sequences.
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 2

Figure 2. LSD1 R838 methylation promotes the stabilization of LSD1.

A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.

B, C Protein and relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) and qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).

D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.

E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.

F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.

G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.

H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.

I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.

J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.

K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.

L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 3

Figure 3. CARM1-dependent methylation of LSD1 promotes deubiquitylation of LSD1 through USP7.

A Immunoprecipitation assay was performed using anti-Flag antibody in MM-231-shLSD1-WT/R838A cells. The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies.

B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.

C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.

D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.

E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.

F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.

G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.

H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 4

Figure 4. LSD1 R838 methylation promotes breast cancer invasion and metastasis.

A, C Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) and MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).

B, D invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) and MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).

E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).

F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 5

Figure 5. CARM1 and USP7 regulate the expression of E-cadherin and vimentin depending on LSD1 R838 methylation.

A, B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) and vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).

C, D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) and vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).

E, F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) and vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).
Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer (2019)
Figure 6

Figure 6. LSD1R838me2a/CARM1/LSD1 level is associated with grade of breast carcinoma.

A Typical pictures of the immunohistochemical staining of LSD1 R838me2a, LSD1 and CARM1 in breast cancer samples. Scale bars, 100 μm.

B, C, D The percentage of samples with high level of LSD1 R838me2a (B), LSD1 (C) and CARM1 (D) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).

E, F, G The correlation of LSD1 R838me2a with LSD1 (E), CARM1 with LSD1 R838me2a (F) and CARM1 with LSD1 (G) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al. 19[].
Cryo-EM reveals mechanisms of angiotensin I-converting enzyme allostery and dimerization (2022)
Figure 2

Figure 2. Biochemical characterization of purified sACES1211.

A. SDS-PAGE analysis of purified sACES1211 showed a single band of ~170 kDa and thus indicates extensive N-glycosylation (protein-only molecular weight is 138 kDa).

B. SEC-HPLC analysis of the respective samples of purified sACES1211, truncated N-domain, or truncated C-domain showed a single symmetric peak for each protein with calibrated molecular weights of 244 kDa, 147 kDa, and 75 kDa, respectively. The sACES1211 sample did not show any peaks corresponding to single-domain or homodimer molecular weights.

C. Native PAGE analysis showed a major diffuse band of ~260 kDa and a minor band of ~500 kDa for purified sACES1211, representing monomeric and dimeric forms of the protein. Commercial gel filtration standards and bovine serum albumin (BSA) were used to estimate the molecular weight. Purified truncated N-domain was used as an additional molecular weight marker for sACES1211 and showed a single diffuse band of ~160 kDa.
Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue (2019)
Figure 1

Figure 1. Afadin is phosphorylated at Serine 1795/1718

(A) Phosphorylation of Afadin at Serine1795 (marked with asterisk) revealed by quantitative phosphoproteomic analysis in brown preadipocytes stimulated with IGF-1 for 5 min. Table shows 8 identified phosphopeptides belonging to Afadin, where the phosphorylated residue is underlined and highlighted in bold. (B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform. (C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4). (D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3). (E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3). (F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6). (G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6). (H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6). (I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue (2019)
Figure 2

Figure 2. Insulin action is improved in adipose tissue of mice with adipose tissue specific ablation of Afadin.

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (A+B) BAT; Immunoblotting and quantification of Afadin S1795 phosphorylation (n=4-5). (C+D) sWAT; Immunoblotting and quantification of Afadin S1795 phosphorylation (n=4-5). (E+F) Liver; Immunoblotting and quantification of Afadin S1795 phosphorylation (n=4-5). (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue (2019)
Figure 3

Figure 3. Cell-intrinsic modulation of insulin action by Afadin depends on S1795 phosphorylation.

(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO. (B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). (C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting. (D) Quantification of insulin-induced Akt phosphorylation from immunoblots. (E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue (2019)
Figure 4

Figure 4. Afadin interacts with HDAC6 and modulates its activity to regulate IR levels.

(A) Experimental strategy for the identification of insulin- and S1795-dependent Afadin binding partners. (B) Venn diagrams illustrating the number of proteins interacting with WT Afadin or S1705A Afadin upon insulin stimulation for 20 min (n=4). (C) Acetylated α-tubulin levels in cell lines (n=3). (D) Acetylated α-tubulin levels in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=4). (E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). (F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). (G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.
Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue (2019)
Figure 5

Figure 5. Ablation of Afadin in adipose tissue improves diet-induced insulin resistance despite no changes in body weight or fat mass.

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. (B) Body composition. (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). (D) IPGTT after 12 weeks of diet and AUC. (E) ITT after 13 weeks of diet and AUC and (F) normalized to basal insulin. (G) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 1.

SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A–C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1. (D–F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 2.

The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A–E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top). (B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction. (C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3. (D) Survival data. n = 4. (E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species–mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 3.

The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial. (C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.