Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet: rβig-h3 structures collagen I into thick fibers (A and B): Representative micrographs of immunofluorescence staining in pancreata from three-months-old KC mice for (A) βig-h3 (green), collagen I (red) or (B) βig-h3 (green) and F4/80 (red); (A, B) nuclear counterstaining in DAPI (blue). (C) Representative micrographs taken with polarized light of collagen I structured in the absence (top) or presence (bottom) of rβig-h3 and stained with Sirius Red. (D) Thickness topography measured by AFM of representative regions of collagen I structured in the absence (up) or presence (bottom) of rβig-h3. Two different magnifications are shown. (E) Quantification of collagen I fiber thickness measured by AFM. (F) Quantification of the Elastic modulus measured by AFM. Student’s t-test ∗∗∗∗p< 0.0001, ns - not significant. Error bars 50 μm, 25 μm.

Article Snippet: Finally, the sections were mounted in Vectashield mounting medium containing DAPI (Eurobio; Evry-France) for nuclear counterstaining.

Techniques: Immunofluorescence, Staining

Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet: rβig-h3-structured collagen I fibers modulate the phenotype and function of macrophages (A) Representative photographs of immunofluorescence staining of pancreata from three-months-old KC mice stained for βig-h3 (green) and CD206 (red). Nuclear counterstaining in DAPI (blue). (B) Representative photographs of βig-h3, F4/80, CD206, and Arg1 staining on serial section of pancreata from three-months-old KC mice. (C) FACS analysis of BMMCs cultured alone or on collagen I structured in the absence (BMMCs + Col I) or presence of rβig-h3 (BMMCs + Col I rβig-h3 ). Representative data of three independent experiments with three in vitro replicates per group are shown. ∗p<0.05, ∗∗p<0.01. Error bars 50 μm, 25 μm.

Article Snippet: Finally, the sections were mounted in Vectashield mounting medium containing DAPI (Eurobio; Evry-France) for nuclear counterstaining.

Techniques: Immunofluorescence, Staining, Cell Culture, In Vitro

Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet:

Article Snippet: Finally, the sections were mounted in Vectashield mounting medium containing DAPI (Eurobio; Evry-France) for nuclear counterstaining.

Techniques: Control, Recombinant, Saline, Modification, Plasmid Preparation, Membrane, Activation Assay, Concentration Assay, Software

Journal: EMBO Reports

Article Title: Coordinated control of adiposity and growth by anti‐anabolic kinase ERK7

doi: 10.15252/embr.201949602

Figure Lengend Snippet: A Schematic presentation showing the generation of ERK7 1 mutants by CRISPR/Cas9 targeting. B ERK7 1 mutants have elevated triacylglycerol (TAG) levels ( N = 4 replicates of ≥ 10 larvae/ replicate for each genotype). C Fat body‐specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels ( N = 4 replicates of 10 larvae/replicate for each genotype). D Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK7 1 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E ERK7 1 mutant fat body cells contain more lipid droplets than control cells ( N = 30 cells for each genotype). F After fed [ 13 C]glucose, the ERK7 1 mutant fat bodies display elevated [ 13 C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry‐based lipidomics, N = 3 replicates of 15 fat bodies/replicate, TAG = triacylglycerol, PE = phosphatidylethanolamine) G ERK7 overexpression in the fat body results in reduced TAG levels, while kinase‐dead (K54R) and activation loop phosphorylation‐deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage ( N = 4 replicates of ≥ 10 larvae/replicate for each genotype). H–J ERK7 expressing, GFP‐marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) contains less (I) and smaller (J) lipid droplets than control cells (marked by white dotted line), visualized by LipidTOX staining ( N = 11 for control and 4 for ERK7 overexpression). K qRT–PCR‐based expression analysis of ERK7 from fat bodies of w 1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies/replicate for each genotype). Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t ‐test).

Article Snippet: Immunostainings were performed as described previously (Hasygar & Hietakangas, ); samples were mounted using Vectashield Mounting Medium with DAPI (Mediq) and imaged using a Leica TCS SP5 MP SMD FLIM and Leica SP8 Up‐right microscopes.

Techniques: CRISPR, Staining, Control, Mutagenesis, Mass Spectrometry, Over Expression, Activation Assay, Phospho-proteomics, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: EMBO Reports

Article Title: Coordinated control of adiposity and growth by anti‐anabolic kinase ERK7

doi: 10.15252/embr.201949602

Figure Lengend Snippet: A Representative immunofluorescent images of control and ERK7 1 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C ERK7 1 mutant fat bodies display increased nuclear area (B, N > 160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N > 80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies. D–F ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti‐fibrillarin antibodies ( N = 10). G, H Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT–PCR. Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies/replicate for each genotype). Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, ns—not significant (Student’s t ‐test).

Article Snippet: Immunostainings were performed as described previously (Hasygar & Hietakangas, ); samples were mounted using Vectashield Mounting Medium with DAPI (Mediq) and imaged using a Leica TCS SP5 MP SMD FLIM and Leica SP8 Up‐right microscopes.

Techniques: Control, Mutagenesis, Staining, Expressing, Clone Assay, Quantitative RT-PCR, Standard Deviation

Journal: EMBO Reports

Article Title: Coordinated control of adiposity and growth by anti‐anabolic kinase ERK7

doi: 10.15252/embr.201949602

Figure Lengend Snippet: A ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos‐tag Western blot (marked with red arrow). Insulin‐inducible bands (dark blue arrows) remain unresponsive to ERK7. B Representative immunofluorescent images of PWP1 localization in fat bodies of ERK7 1 mutant and control third instar larvae. Scale bar: 20 µm. C PWP1 localization displays increased nucleolar/nuclear ratio in ERK7 1 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype). D Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP‐marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype). F Fat body‐specific depletion of PWP1 by RNAi (NIG‐Fly 6751R‐3) leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae/replicate for each genotype). G Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG‐GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I Fat body‐specific knockdown of PWP1 (NIG‐Fly 6751R‐1) suppresses increased lipid droplet number (H; N > 30 cells per genotype) and increased nuclear area (I; N > 45 cells per genotype) caused by ERK7 knockdown (BDSC 56939). Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). ** P < 0.01, *** P < 0.001 (Student’s t ‐test).

Article Snippet: Immunostainings were performed as described previously (Hasygar & Hietakangas, ); samples were mounted using Vectashield Mounting Medium with DAPI (Mediq) and imaged using a Leica TCS SP5 MP SMD FLIM and Leica SP8 Up‐right microscopes.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Mutagenesis, Control, Clone Assay, Knockdown, Staining, Standard Deviation

Journal: EMBO Reports

Article Title: Coordinated control of adiposity and growth by anti‐anabolic kinase ERK7

doi: 10.15252/embr.201949602

Figure Lengend Snippet: A, B Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) ( N = 3 replicates of 10 fat bodies/replicate for each genotype). C ERK7 1 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae/replicate for each genotype). D Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 1 and sug mutants ( sug 17Δ /Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E sug mutant ( sug 17Δ /Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK7 1 mutants ( N > 20). F Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae ( N = 4 replicates of ≥10 larvae/replicate for each genotype). G sug mutant ( sug 17Δ /Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK7 1 mutant fat body cells ( N ≥ 25 cells per genotype). H, I Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) ( P < 0.001 for the rescue, analyzed by Log‐rank test, N = 4 replicates of 30 larvae/replicate for each genotype) and pupal volume of CG>ERK7 larvae (I) ( N = 4 replicates of 10 pupae/replicate for each genotype). J Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet ( P < 0.0001 for the rescue, analyzed by Log‐rank test, N = 4 replicates of 30 larvae/replicate for each genotype and diet). Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t ‐test). Expression of RP49 was used for normalization of (A–C).

Article Snippet: Immunostainings were performed as described previously (Hasygar & Hietakangas, ); samples were mounted using Vectashield Mounting Medium with DAPI (Mediq) and imaged using a Leica TCS SP5 MP SMD FLIM and Leica SP8 Up‐right microscopes.

Techniques: Expressing, Knockdown, Staining, Mutagenesis, Standard Deviation