EMBO/SourceData huggingface dataset:
Figure 4Bar graph representing quantification of stiffness measured by atomic force microscopy (Young modulus) of ECM area generated by H1299control or H1299RASSAF1A cells in vitro. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).Bar graph representing quantification of stiffness by atomic force microscopy (Young modulus) in of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours on day 30. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.Second harmonic generation (SHG) representative Images and Quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2mg/ml), treated with siNT, siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars 10µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.
Figure 5Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.Representative immunofluorescence images show merge of NANOG (green), DAPI (blue) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass. Scale bars 10µm. Right: Quantification of nuclear co-localization (n=200cells/experiment) is represented by Pearson's coefficient.Representative immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness. Scale bars 10µm. Bottom: Quantification of n=200 cells/experiment. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.Representative images of immunofluorescence staining for the pluripotency marker NANOG and β-catenin in three-dimensional spheroids embedded in collagen matrix (2mg/ml). Scale bars 50µm.Representative images of H&E and immunohistochemical staining for NANOG and YAP1 in primary lung tumours, (day 17). Scale bars 100µm. Right: Graph bars represent quantification of NANOG and YAP1 staining based on strong (3+, 2+) nuclear intensity (%) for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.
Figure 6Relative mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three-dimensional collagen matrix (2mg/ml). Scale bars 10µm.Western blots of H1299control and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.Relative H1299control mRNA expression levels NANOG, OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4μM DPCA. Scale bars 10µm.Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.
Figure 7Western blot analyses of expression levels of TTF-1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser.Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF-1 and Mucin 5B for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF-1 based on nuclear intensity. Scale bars 100µm. Statistical significance was determined by Student's t-test (2-tailed). Error bars represent the mean ± S.E.MRepresentative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm.Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.
Figure 1A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.B Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n=24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 µm.C Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, the merged signal is white. Scale bar: 15 µm. D Quantification experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells (n=24 cells analyzed from three independent experiments; mean±SD).E Quantification experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells (n=10 cells analyzed from three independent experiments; mean±SD).F Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4 and S462 cells (n≥6 cells for each cell line; mean±SD).H HK2 displacement from HeLa MAMs after a 2 min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in (C). cl-SCRpep is used as a negative control and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n=26 cells; cl-HK2pep n=24 cells; p<0.001 with a Student's t test). Scale bar: 10 µm.Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) where both total hexokinase activity and HK1/HK2 specific activities are measured.Effect of cl-HK2pep on glucose phosphorylation in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.
Figure 5Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells (A) Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells inhibits growth in soft agar (B; colony area±SEM; n=6 replicates obtained from 3 independent experiments; Student's t test ***p<0.001; A.U., arbitrary units). Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.Effect of HK2 targeting on in vitro tumorigenicity. Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.Effect of HK2 targeting on in vitro tumorigenicity. Treatment with uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.Effect of HK2-targeting on in vivo neoplastic growth. HK2- and MMP-2-expressing CT26 cells (E, scale bar: 100 µm) are allografted in Balb/c male mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.Effect of HK2-targeting on in vivo neoplastic growth. Intratumor administration (5 injections of 2 nmol peptide every 12 h) of either cl-HK2pep or HK2pep (F; CNT n=4; cl-HK2pep n=5; HK2pep n=5 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In F, mean tumor volume±SD is reported; Student's t test ***p<0.001Effect of HK2-targeting on in vivo neoplastic growth. intraperitoneal administration of HK2pep (G; 5 injections of 60 nmol peptide every 12 h; SCRpep n=6; HK2pep n=7 mice) reduce neoplastic growth in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In G, representative ultrasound inspections and 3D reconstructions of tumor masses are shown (light grey: SCRpep treated animal; light blue: HK2pep treated animal). normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.Effect of HK2-targeting on in vivo neoplastic growth. the same effect is observed on allografts of breast carcinoma 4T1 cells (H; 5 injections of 60 nmol peptide every 12 h; SCRpep n=5; HK2pep n=7 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.
Figure 1(D) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.(F) SCD1 overexpression affects RNF145/ADIPOR2. HEK-293T cells containing an empty control vector or expressing SCD1-HA were cultured in FCS-free DMEM for 24h and whole-cell lysates analysed. * non-specific bands.
Figure 2(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.(B) ADIPOR2 regulation requires the E3 ligase activity of RNF145. HEK-293T RNF145 knockout cells (∆RNF145) were stably complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). * non-specific bands.(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.(D) The E2 ubiquitin-conjugating enzyme UBE2G2 is involved in RNF145-mediated ADIPOR2 degradation. Immunoblot analysis of HEK-293T cells depleted of RNF145 (gRNF145), UBE2G2 (gUBE2G2) or B2M (control; gCTR).(E) ADIPOR2 degradation depends on UBXD8. HEK-293T cells were depleted of B2M (gCTR) or UBXD8 (gUBXD8) and analysed by immunoblotting.
Figure 3(A) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.(B) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).(D) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.
Figure 4(A) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).(B) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.
Figure 5(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.(B) Cells were treated as in (A) and lysates analysed by immunoblotting. LE, long exposure; *non-specific bands.(C) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).
Figure 1Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples. Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice.Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.
Figure 2Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change. Cells in all wells were 100% confluent when media was changed.Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes. Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes.
Figure 3Expression of Wt1, leucine rich repeat neuronal 4 (Lrrn4), Uroplakin 3b (Upk3b), in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.Bright-field images of preadipocyte cell lines under standard culture conditions. The photographs were taken at 10x magnification.Expression of Tagln, connective tissue growth factor (Ctgf), Keratin 19 (Krt19), Mx1, caspase 1 (Casp1), and C-X-C Motif Chemokine Ligand 12 (Cxcl12) in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.
Figure 4Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice.[3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.[14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice.Extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of basal respiration. The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation. Data are shown as mean ± SEM of 3-7 cell lines per group.Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for: pJnk T183/Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes (Inflammation); pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes (Insulin Signaling); pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes (Growth Hormone).