human sarcoma cell line u 2 os  (ATCC)

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Rationale of the genome-wide screen: loss of FA dynamics regulators by siRNA-mediated knockdown results either in an increase in FAs per cell or in an increased FA area. The green dots represent FAs, and the blue circles represent nuclei. b , Overview of the screening procedure: initial candidates were identified by screening a genome-wide siRNA pool library in 384-well format. The resulting 280 candidates were rescreened two times to identify reproducible hits. c , d , Loss of aldolase causes an increase in FAs per cell. c , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with control (siCtrl) or aldolase-specific (siALDOA) siRNAs. FA segmentation is shown below. For better visualization of the phenotype, manually drawn cell outlines were added for cells completely contained in images (red). d , A quantification of FAs per cell shown in c . The data represent mean ± s.e.m.; n = 3 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (mock versus siCtrl) = 0.8904; P (mock versus siALDOA#1) = 0.0005; P (mock versus siALDOA#2) = 0.0131). e – g , Aldolase re-expression rescues FA and cell size increase in silenced cells. e , Representative confocal images of paxillin-labelled FAs in U-2 OS cells stably expressing either HA-tagged mCherry as control or siRNA-resistant aldolase and treated with control (siCtrl) or aldolase-specific (siALDOA) siRNAs. FA segmentation and cell outlines (red) are shown below. f , g , A quantification of FAs per cell ( f ) and cell area ( g ) shown in e . The data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for f ): (siCtrl + mCherry versus siALDOA + mCherry) <0.0001; (siCtrl + mCherry versus siALDOA + aldolase wt) = 0.4083; P values (for g ): (siCtrl + mCherry versus siALDOA + mCherry) <0.0001; (siCtrl + mCherry versus siALDOA + aldolase wt) = 0.133). n.s., not significant; * P < 0.05, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Genome Wide, Knockdown, Control, Expressing, Stable Transfection

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Scheme of the plate layout for the genome-wide screen. b , Image analysis workflow for the genome-wide screen. FA number and area (top) and nuclei number (bottom) were quantified using an automated KNIME pipeline. Detailed descriptions can be found in the Methods section. Boxed areas show zooms. c, d , Validation of positive and negative controls. c , Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with siRNAs targeting either cofilin (CFL1) and destrin (DSTN) or paxillin (PXN), which served as assay quality controls. FA segmentation and cell outlines (red) are shown below. d , Quantification of FA area (in pixels) or FAs per cell shown in c . Data represent mean ± SEM; n = 8 wells of one plate. e , f , Scatter plots of all siRNAs ranked according to their Z-score in either FAs per cell or FA area. Dotted lines represent median values for each plot. Red dots indicate siRNAs that were considered hits based on their Z-scores (>4.2 for FAs per cell or >3.5 for FA area). Scale bars, 25 µm. Screen data can be found in the Supplementary Table in Tabs and .

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Genome Wide, Biomarker Discovery, Labeling

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a – c , The enzymatic activity of aldolase is essential to rescue phenotypes of aldolase knockdown cells. a , Representative confocal images of paxillin-labelled FAs in U-2 OS cells stably expressing HA-tagged mCherry or siRNA-resistant WT or catalytically inactive (D33S; K229A) aldolase and treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown. b , c , A quantification of FAs per cell ( b ) and cell area ( c ) shown in a . The data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for b ): (siCtrl + mCherry versus siALDOA + mCherry) = 0.0221; (siCtrl + mCherry versus siALDOA + aldolase WT) = 0.6234; (siCtrl + mCherry versus siALDOA + aldolase D33S) = 0.0029; (siCtrl + mCherry versus siALDOA + aldolase K229A) = 0.0102; P values (for c ): (siCtrl + mCherry versus siALDOA + mCherry) = 0.0005; (siCtrl + mCherry versus siALDOA + aldolase WT) = 0.737; (siCtrl + mCherry versus siALDOA + aldolase D33S) <0.0001; (siCtrl + mCherry versus siALDOA + aldolase K229A) = 0.0004). d , A simplified scheme of glycolysis highlighting aldolase and its neighbouring enzymes. The full arrows indicate a direct connection and the dashed arrows a substitute for missing steps. e , Efficient depletion of glycolytic enzymes. Immunoblot of U-2 OS cells treated with indicated siRNAs. β-Actin was used as loading control. N = 1 independent experiment. f – h , Loss of PFK and aldolase affect FAs per cell and cell size in opposite manner, while GAPDH depletion has no effect. f , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. g , h , A quantification of FAs per cell ( g ) and cell area ( h ) shown in f . For f – h , the data represent mean ± s.e.m; n = 6 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for g ): (siCtrl versus siPFK) = <0.0001; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siGAPDH) >0.9999; P values (for h ): (siCtrl versus siPFK) = 0.0288; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siGAPDH) = 0.3812). i , j , Decreased ATP levels are not the cause of increased FAs per cell and cell size. Relationship between ATP levels and FAs per cell ( i ) or cell area ( j ), for cells treated as indicated. The data represent mean ± s.e.m. n = 3–6 independent experiments. n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm. F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TPI, triose phosphate isomerase.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Activity Assay, Knockdown, Stable Transfection, Expressing, Western Blot, Control

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a-d , Loss of PFK (isoform PFKP) in U-2 OS cells by an alternative siRNA also decreases FA numbers and cell size. a , Efficient depletion of PFK in U-2 OS cells with an siRNA targeting PFKP. Immunoblot of U-2 OS cells treated with indicated siRNAs. CHC was used as loading control. N = 1 independent experiment. b , Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. Scale bars, 25 µm. c , d , Quantification of FAs per cell and cell area shown in b . Data represent mean ± SEM; n = 3 independent experiments; two-sided paired t-test ( P value ( c ) = 0.0175; P value ( d ) = 0.0429). e-f , U-2 OS cells depleted of aldolase, PFK or GAPDH do not show increased cell death. Cells treated with the indicated siRNAs were stained with the cell-permeable DNA stain SiR-Hoechst and the membrane-impermeable DNA dye SYTOX Green which can only enter cells with compromised cell membrane integrity. Cells treated with the detergent Triton X-100 to impair membrane integrity were used as positive control. e , Representative epifluorescence images of SiR-Hoechst- and SYTOX-labeled U-2 OS cells treated with the indicated siRNAs. Scale bars, 25 µm. f , Quantification of percentage of SYTOX-positive cells. Data represent mean ± SEM; n = 3 independent experiments; RM one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl vs. 0.5% Triton) = 0.0039; (siCtrl vs. siPFK) = 0.5294; (siCtrl vs. siALDOA) = 0.2328; (siCtrl vs. siGAPDH) = 0.798). g , U-2 OS cells depleted of aldolase are to a lower extent in S phase. Cells treated with the indicated siRNAs were analyzed by FACS for their cell cycle distribution after 30 min incubation with 10 µM EdU to label cells in S phase (gating strategy illustrated in Supplementary Figure 1). Quantification of the percentage of cells in the indicated cell cycle phases. Data represent mean ± SEM; n = 3; two-way ANOVA with Dunnett’s post-test ( P values: G0/G1 phase: (siScr vs. siPFK) = 0.0002; (siScr vs. siALDOA) = <0,0001; (siScr vs. siGAPDH) = 0.3297; S phase: (siScr vs. siPFK) = 0.0015; (siScr vs. siALDOA) = <0,0001; (siScr vs. siGAPDH) = 0.6219; G2/M phase: (siScr vs. siPFK) = 0.7589; (siScr vs. siALDOA) = 0,0012; (siScr vs. siGAPDH) = 0.9552). Ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Western Blot, Control, Labeling, Staining, Membrane, Positive Control, Incubation

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Loss of PFK, aldolase or GAPDH results in lower ATP levels. Relative ATP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± SEM; n = 5 independent experiments; two-sided One sample t-test ( P value (siPFK) = 0.014; (siALDOA) = 0.0024; (siGAPDH) = 0.0136). b-e , Lower ATP levels due to inhibition of oxidative phosphorylation do not affect FA numbers or cell area. b , Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with DMSO or 1 µM Antimycin A for 48 h. FA segmentation and cell outlines (red) are shown below. c , d , Quantification of FAs per cell and cell area shown in b . Data represent mean ± SEM; n = 3 independent experiments; two-sided unpaired Student’s t-test ( P value ( c ) = 0.3716; P value ( d ) = 0.8734). e , Relative ATP levels measured in U-2 OS cells treated with DMSO or 1 µM Antimycin A for 48 h. Data were normalized to DMSO and represent mean ± SEM. n = 3 independent experiments; two-sided One sample t-test ( P value = 0.0014). f-i , Lower ATP levels due to inhibition of glycolysis phenocopy loss of PFK. f , Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with PBS or 25 mM 2-deoxy-D-glucose (2-DG) for 48 h. FA segmentation and cell outlines (red) are shown below. g , h , Quantification of FAs per cell and cell area shown in f . Data represent mean ± SEM; n = 3 independent experiments; two-sided unpaired Student’s t-test ( P value ( g ) = 0.002; P value ( h ) = 0.0069). i , Relative ATP levels measured in U-2 OS cells treated with PBS or 25 mM 2-DG for 48 h. Data were normalized to PBS and represent mean ± SEM; n = 3 independent experiments; two-sided One sample t-test ( P value = 0.0216). Ns, not significant; *p < 0.05, **p < 0.01. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Inhibition, Phospho-proteomics, Labeling

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Loss of aldolase results in severely increased FBP levels, whereas PFK depletion lowers FBP. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t -test ( P values: (siPFK) = 0.0345; (siALDOA) = 0.0425; (siGAPDH) = 0.2188). b , Efficient codepletion of PFK and aldolase. Immunoblot of U-2 OS cells treated with indicated siRNAs. Clathrin heavy chain (CHC) was used as loading control; N = 1 independent experiment. c , Codepletion of PFK restores normal FBP levels in aldolase-knockdown cells. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 4 independent experiments; two-sided one-sample t -test ( P values: (siCtrl + siPFK) = 0.0017; (siCtrl + siALDOA) = 0.0491; (siPFK + siALDOA) = 0.16). d – f , Co-depletion of PFK restores FA numbers and cell size in aldolase knockdown cells. d , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown on the right. e , f , A quantification of FAs per cell ( e ) and cell area ( f ) shown in d . For e , f , data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for e ): (siCtrl + siCtrl versus siCtrl + siPFK) = 0.0015; (siCtrl + siCtrl versus siCtrl + siALDOA) = 0.0002; (siCtrl + siCtrl versus siPFK + siALDOA) = 0.1021; P values (for f ): (siCtrl + siCtrl versus siCtrl + siPFK) = 0.0044; (siCtrl + siCtrl versus siCtrl + siALDOA) <0.0001; (siCtrl + siCtrl versus siPFK + siALDOA) = 0.8423). g , h , Inhibiting glycolytic flux rescues FAs per cell ( g ) and cell area ( h ) in aldolase-knockdown cells. A quantification of FAs per cell and cell area of U-2 OS cells treated with control (−) or ALDOA-specific siRNA (+) followed by 48 h treatment with PBS (−) or 25 mM 2-DG (+). Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for g ): (siCtrl + PBS versus siALDOA + PBS) = 0.0056; (siCtrl + PBS versus siCtrl + 2-DG) = 0.0567; (siCtrl + PBS versus siALDOA + 2-DG) = 0.1605; P values (for h ): (siCtrl+PBS versus siALDOA+PBS) <0.0001; (siCtrl + PBS versus siCtrl + 2-DG) = 0.2411; (siCtrl + PBS versus siALDOA + 2-DG) = 0.9888). Corresponding images are shown in Extended Data Fig. . n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Western Blot, Control, Knockdown

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Inhibiting glycolysis lowers FA number and cell size to normal levels in aldolase knockdown cells. Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with indicated siRNAs followed by 48 h treatment with PBS or 25 mM 2-deoxy-D-glucose (2-DG). FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . b , Inhibiting glycolysis rescues elevated FBP level in aldolase-depleted cells. Relative FBP levels measured in U-2 OS cells treated with control (-) or aldolase-specific siRNA (ALDOA, +) followed by 48 h treatment with PBS (-) or 25 mM 2-DG. Data were normalized to siCtrl + PBS and represent mean ± SEM; n = 4 independent experiments; two-sided One sample t-test ( P values: (siALDOA) = 0.0227; (siCtrl+2DG) = 0.005; (siALDOA+2-DG) = 0.007). Ns, not significant; *p < 0.05, **p < 0.01. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Knockdown, Labeling, Control

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , e , Reduced FBP levels associated with PFK depletion decrease FA assembly ( a ), whereas increased FBP levels upon aldolase knockdown increase the FA assembly rate ( e ). Representative TIRF microscopy time-lapse series of U-2 OS cells stably expressing eGFP–paxillin and treated with indicated siRNAs. Zoom-ins of the orange boxes are shown. b , c , f , g , A quantification of FA assembly rate ( b , f ) and disassembly rate ( c , g ). Data represent median and interquartile ranges. For b and c , n (siCtrl) = 34 and n (siPFK) = 39 cells from five independent experiments; for f and g , n (siCtrl) = 37 and n (siALDOA) = 35 cells from five independent experiments; two-sided Mann–Whitney test ( P values (for b ) = 0.0047; (for c ) = 0.2941; (for f ) = 0.0178; (for g ) = 0.0492). d , h , A quantification of novel FAs formed over 4 h for siPFK ( d ) and siALDOA ( h ). Data represent median and interquartile ranges. For d , n (siCtrl) = 35 and n (siPFK) = 40 cells from five independent experiments; for h , n (siCtrl) = 37 and n (siALDOA) = 35 cells from five independent experiments; two-sided Mann-Whitney test ( P values: (for d ) <0.0001; (for h ) = 0.0061). n.s., not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001. Scale bars, 25 µm and 10 µm for zoomed-in images.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Knockdown, Microscopy, Stable Transfection, Expressing, MANN-WHITNEY

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Increased FBP levels upon aldolase knockdown (KD) alter the organization of the actin cytoskeleton and elevate cellular protrusion. a , Representative confocal images of the phalloidin-labelled F-actin cytoskeleton in U-2 OS cells treated with indicated siRNAs. Zoom-ins of the orange boxes are shown. b , A quantification of the protrusive area of U-2 OS cells treated with indicated siRNAs. For a and b , data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siPFK) = 0.5203; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siGAPDH) = 0.5458). Corresponding images are shown in Extended Data Fig. . c , d , Aldolase depletion results in elevated cell spreading. c , Representative confocal images of phalloidin-labelled U-2 OS cells treated with indicated siRNAs. The cells were seeded and fixed after indicated time points. d , A quantification of cell spreading shown in c . For c and d , data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siPFK) = 0.2822; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siGAPDH) = 0.9188). e – h , Less pronounced elevations in FBP levels also increase cell size. e , Efficient depletion of aldolase and GAPDH. Immunoblot of U-2 OS cells treated 1× or 2× with indicated siRNAs. Clathrin heavy chain (CHC) was used as loading control. The cropped lanes are from the same blot; N = 1 independent experiment. f , LC–MS/MS-based FBP measurements of U-2 OS cells treated 1× or 2× with indicated siRNAs. Data represent mean ± s.e.m.; n = 3 independent experiments; one-way ANOVA with Tukey’s post-test ( P values: (1xkd-siCtrl versus 1xkd-siALDOA) = 0.0186; (1xkd-siCtrl versus 2xkd-siCtrl) >0.9999; (1xkd-siCtrl versus 2xkd-siALDOA) <0.0001; (1xkd-siCtrl versus 2xkd-siGAPDH) >0.9999; (1xkd-siALDOA versus 2xkd-siCtrl) = 0.018; (1xkd-siALDOA versus 2xkd-siALDOA) = 0.0036; (1xkd-siALDOA versus 2xkd-siGAPDH) = 0.0209; (2xkd-siCtrl versus 2xkd-siALDOA) <0.0001; (2xkd-siCtrl versus 2xkd-siGAPDH) >0.9999; (2xkd-siALDOA versus 2xkd-siGAPDH) <0.0001). g , A quantification of cell area shown in h . Data represent mean ± s.e.m.; n = 3 independent experiments; repeated-measures one-way ANOVA with Tukey’s post-test ( P values: (1xkd-siCtrl versus 1xkd-siALDOA) = 0.0063; (1xkd-siCtrl versus 2xkd-siCtrl) = 0.2089; (1xkd-siCtrl versus 2xkd-siALDOA) = 0.0161; (1xkd-siCtrl versus 2xkd-siGAPDH) = 0.0105; (1xkd-siALDOA versus 2xkd-siCtrl) = 0.0018; (1xkd-siALDOA versus 2xkd-siALDOA) = 0.0267; (1xkd-siALDOA versus 2xkd-siGAPDH) = 0.0313; (2xkd-siCtrl versus 2xkd-siALDOA) = 0.0137; (2xkd-siCtrl versus 2xkd-siGAPDH) = 0.0172; (2xkd-siALDOA versus 2xkd-siGAPDH) = 0.0242). h , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated 1× or 2× with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. i , Protruding cells display strongly elevated FBP levels. LC–MS/MS-based FBP measurements in confluent (24 h after dense plating), migrating (6 h after sparse plating) or spreading (1 h of sparse plating) U-2 OS cells. Data represent mean ± s.e.m.; n = 3 independent experiments; one-way ANOVA with Tukey´s post-test ( P values: (immobile versus migrating) = 0.0046; (immobile versus spreading) <0.0001; (migrating versus spreading) <0.0001). n.s., not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Knockdown, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Example of the image analysis workflow used to quantify protrusive area using an automated Fiji macro. Total cell area was visualized via the plasma membrane dye CellMask. Elevated cell regions were detected by their greater accumulation of the cytoplasmic fluorescent CellTracker dye in comparison to the very thin cellular protrusions. A detailed description of the image analysis procedure can be found in the Methods section. b , Representative phase contrast and epifluorescent images of U-2 OS cells treated with indicated siRNAs. Segmentation is shown below. Corresponding quantification is shown in Fig. . Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Clinical Proteomics, Membrane, Comparison

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Addition of FBP to whole cell lysate causes a conformational change in Rac1. a , Whole cell lysates of U-2 OS cells were subjected to LiP–MS. Peptide intensity (amino acids (aa) 6–16) of Rac1 in response to increasing FBP concentrations. b , Responding peptide (red, aa 6–16) highlighted in the GTP-bound structure of Rac1 (PDB ID: 1MH1 ). c , Increased Rac1 activity upon loss of aldolase. Left: representative immunoblot of pull-down assay using GST-PAK-PBD beads to detect active Rac1 in lysates from siRNA treated U-2 OS cells. α-Tubulin was used as a loading control. Right: a quantification of Rac1-GTP over total Rac1. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 5 independent experiments; two-sided one-sample t -test ( P value = 0.0326). d , Efficient Rac1 depletion. Representative immunoblot of U-2 OS cells treated with the indicated siRNAs. β-actin and clathrin heavy chain (CHC) were used as loading controls; N = 3 independent experiments. e , Codepletion of Rac1 does not significantly lower FBP levels in aldolase-knockdown cells. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t -test ( P values: (siRAC1) = 0.1494; (siALDOA) = 0.027; (siRAC1 + siALDOA) = 0.105). f – h , Codepletion of Rac1 restores FA numbers and cell size in aldolase knockdown cells. f , Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. g , h , A quantification of FAs per cell ( f ) and cell area ( g ) shown in f . For f – h , data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for g ): (siCtrl versus siRAC1) = 0.0481; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.9979; P values (for h ): (siCtrl versus siRAC1) = 0.0003; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.2799). i , j , Expression of dominant-negative Rac1 in aldolase-depleted cells rescues increased FA numbers ( i ) and cell size ( j ). A quantification of FAs per cell and cell area of U-2 OS cells transfected with control (−) or ALDOA-specific (+) siRNAs in combination with either eGFP or myc-Rac1-T17N. Corresponding images are shown in Extended Data Fig. . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for i ): (siCtrl + GFP versus siCtrl + T17N) = 0.1497; (siCtrl + GFP versus siALDOA + GFP) <0.0001; (siCtrl + GFP versus siALDOA + T17N) = 0.0633; P values (for j ): (siCtrl + GFP versus siCtrl + T17N) = 0.4683; (siCtrl + GFP versus siALDOA + GFP) <0.0001; (siCtrl + GFP versus siALDOA + T17N) = 0.0981). k , l , Expression of constitutively active Rac1 in PFK-depleted cells rescues reduced FA numbers and cell size. Quantifications of FAs per cell ( k ) and cell area ( l ) of U-2 OS cells transfected with control (−) or PFK-specific (+) siRNAs in combination with either eGFP or myc-Rac1-Q61L. Corresponding images are shown in Extended Data Fig. . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for k ): (siCtrl + GFP versus siCtrl + Rac1-Q61L) = 0.0342; (siCtrl + GFP versus siPFK + GFP) = 0.0063; (siCtrl + GFP versus siPFK + Rac1-Q61L) = 0.8904; P values (for l ): (siCtrl + GFP versus siCtrl + Rac1-Q61L) <0.0001; (siCtrl + GFP versus siPFK + GFP) = 0.0116; (siCtrl + GFP versus siPFK + Rac1-Q61L) = 0.0004). n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Activity Assay, Western Blot, Pull Down Assay, Control, Knockdown, Expressing, Dominant Negative Mutation, Transfection

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , PFK depletion decreases the level of active Rac1. Left: Representative immunoblot of pulldown assay using GST-PAK-PBD beads to detect active Rac1 in lysates from U-2 OS cells treated with indicated siRNAs. β-actin was used as loading control. Right: Quantification of GTP-Rac1 over β-actin. Data were normalized to siCtrl and represent mean ± SEM; n = 5 independent biochemical experiments; two-sided One sample t-test ( P value = 0.0043). b-e , Co-depletion of Rac1 restores FA numbers and cell size upon aldolase knockdown also based on alternative Rac1-targeting siRNAs in RPE-1 cells. b , Representative confocal images of paxillin-labeled FAs in RPE-1 cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. c, d , Quantification of FAs per cell and cell area shown in b . Data represent mean ± SEM; n = 3 (c: siRAC1#6, siRAC1#6 + siALDOA), 4 (c, all others), 5 (d: siRAC1#6, siRAC1#6 + siALDOA) or 6 (d, all others) independent experiments; statistical testing by mixed-effects model (REML) with Dunnett’s multiple comparison test ( P values ( c ): (siCtrl vs. siRAC1#5) = 0.6235; (siCtrl vs. siRAC1#6) = 0.4587; (siCtrl vs. siALDOA) = 0.0332; (siCtrl vs. siRAC1#5 + siALDOA) = 0.3327; (siCtrl vs. siRAC1#6 + siALDOA) = 0.9142; P values ( d ): (siCtrl vs. siRAC1#5) = 0.3583; (siCtrl vs. siRAC1#6) = 0.1119; (siCtrl vs. siALDOA) = 0.0006; (siCtrl vs. siRAC1#5 + siALDOA) = 0.8081; (siCtrl vs. siRAC1#6 + siALDOA) = 0.2114). Ns, not significant. e , Efficient Rac1 and aldolase depletion in RPE-1 cells. Immunoblot of RPE-1 cells treated with indicated siRNAs. CHC was used as loading control. N = 1 independent experiment. f , Expression of dominant-negative Rac1 in aldolase-depleted cells rescues FA number and cell size. Representative confocal images of paxillin-labeled FAs in U-2 OS cells transfected with control or ALDOA-specific siRNAs in combination with either eGFP or myc-Rac1-T17N. FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . g , Expression of constitutively active Rac1 in PFK depleted cells elevates FA number and cell size to normal levels. Representative confocal images of paxillin-labeled FAs in U-2 OS cells transfected with control or PFK-specific siRNAs in combination with either eGFP or myc-Rac1-Q61L. FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . Ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Western Blot, Control, Knockdown, Labeling, Comparison, Expressing, Dominant Negative Mutation, Transfection

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Codepletion of Rac1 in aldolase-treated cells normalizes F-actin organization and cell protrusion. a , Representative confocal images of the phalloidin-labelled F-actin cytoskeleton in U-2 OS cells treated with indicated siRNAs. Zoom-ins of orange boxes are shown. b , A quantification of the protrusive area of U-2 OS cells treated with indicated siRNAs. Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siRAC1) = 0.5925; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.0825). Corresponding images are shown in Extended Data Fig. . c , d , Codepletion of Rac1 in aldolase-depleted cells restores cell spreading to normal levels. c , Representative confocal images of phalloidin-labelled U-2 OS cells treated with indicated siRNAs. The cells were seeded and fixed after indicated time points. d , A quantification of cell spreading shown in c . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values: (siCtrl versus siRAC1) = 0.7944; (siCtrl versus siALDOA) <0.0001; (siCtrl versus siRAC1 + siALDOA) = 0.6024). e , f , Glucose triggers cell spreading. e , Representative images of phalloidin-labelled RPE-1 cells incubated with the indicated glucose concentrations. The cells were seeded and fixed after 2 h. f , A quantification of cell spreading shown in e . Data represent mean ± s.e.m.; n = 3 independent experiments; two-sided unpaired t -test ( P value = 0.0012). g , h , Increased Rac1 activity upon glucose. g , Representative immunoblot of a pull-down assay using GST-PAK-PBD beads to detect active GTP-Rac1 in lysates from untreated or glucose exposed RPE-1 cells. β-actin was used as a loading control. h , A quantification of GTP-Rac1 over total Rac1. The data were normalized to 0 mM glucose condition and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t -test ( P value = 0.0228). n.s., not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001. Scale bars, 25 µm for a and c ; 10 µm for e .

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Incubation, Activity Assay, Western Blot, Pull Down Assay, Control

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: Co-depletion of Rac1 in aldolase knockdown cells decreases cell protrusion to normal levels. Representative phase contrast and epifluorescent images of U-2 OS cells treated with indicated siRNAs and labeled with CellTracker and CellMask (see also Extended Data Fig. ). Segmentation is shown below. Corresponding quantification is shown in Fig. . Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Knockdown, Labeling

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , b , Addition of FBP to whole cell lysate causes a conformational change in RCC2. Whole cell lysates of U-2 OS cells were subjected to LiP–MS. a , Peptide intensity of RCC2 (amino acids (aa) 110–120) in response to increasing FBP concentrations. b , FBP-responsive peptides of Rac1 (red, aa 6–16) and RCC2 (pink, aa 110–120) highlighted in a putative Rac1-RCC2 complex using the structurally related Ran-RCC1 complex as a template (Rac1 PBD ID: 1MH1, RCC2 aa 89-522 PDB ID: 5GWN , Ran-RCC1 complex PDB ID: 1I2M ). c , FBP directly acts on RCC2. Purified eGFP–RCC2 in combination with increasing FBP levels was subjected to LiP–MS. Peptide intensity of RCC2 aa 454–470, 57–68 and 55–67 in response to increasing FBP concentrations. d , Efficient depletion of RCC2. Representative immunoblot of U-2 OS cells treated with the indicated siRNAs. β-actin and clathrin heavy chain (CHC) were used as loading controls, N = 2 independent experiments. e – g , RCC2 deletion phenocopies loss of aldolase. e , Representative confocal images of paxillin-stained FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. f , g , A quantification of FAs per cell ( f ) and cell area ( g ) shown in e . Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for f ): (siCtrl versus siRCC2#1) <0.0001; (siCtrl versus siRCC2#2) = 0.0092; P values (for g ): (siCtrl versus siRCC2#1) <0.0001; (siCtrl versus siRCC2#2) <0.0001). h , Efficient codepletion of RCC2 and Rac1. Representative immunoblot of U-2 OS cells treated with the indicated siRNAs. β-actin and CHC were used as loading controls; N = 2 independent experiments. i , j , Codepletion of Rac1 in RCC2-knockdown cells restores FA number ( i ) and cell area ( j ). A quantification of FAs per cell and cell area of U-2 OS cells treated with indicated siRNAs. Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test ( P values (for i ): (siCtrl versus siRAC1) = 0.0025; (siCtrl versus siRCC2#2) <0.0001; (siCtrl versus siRAC1 + siRCC2#2) = 0.0548; P values (for j ): (siCtrl versus siRAC1) = 0.007; (siCtrl versus siRCC2#2) <0.0001; (siCtrl versus siRAC1 + siRCC2#2) = 0.6939). Corresponding images are shown in Extended Data Fig. . k , l , Expression of RCC2 in aldolase-depleted cells rescues FA numbers ( k ) and cell size ( l ). A quantification of FAs per cell and cell area of U-2 OS cells transfected with control (−) or ALDOA-specific (+) siRNAs in combination with indicated plasmids. Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Tukey’s post-test ( P values (for k ): (siCtrl + eGFP versus siCtrl + eGFP–RCC2) = 0.6828; (siCtrl + eGFP versus siALDOA + eGFP) = 0.0006; (siCtrl + eGFP versus siALDOA + eGFP–RCC2) = 0.261; (siCtrl + eGFP–RCC2 versus siALDOA + eGFP) <0.0001; (siCtrl + eGFP–RCC2 versus siALDOA + eGFP–RCC2) = 0.0357; (siALDOA + eGFP versus siALDOA + eGFP–RCC2) = 0.0263; P values (for l ): (siCtrl + eGFP versus siCtrl + eGFP–RCC2) = 0.7046; (siCtrl + eGFP versus siALDOA + eGFP) <0.0001; (siCtrl + eGFP vesus siALDOA + eGFP–RCC2) = 0.1491; (siCtrl + eGFP–RCC2 versus siALDOA + eGFP) <0.0001; (siCtrl + eGFP–RCC2 versus siALDOA + eGFP–RCC2) = 0.0193; (siALDOA + eGFP versus siALDOA + eGFP–RCC2) = 0.001). m , n , FBP impairs complex formation between RCC2 and Rac1. eGFP-RCC2 or eGFP as control were coupled to eGFP-trap beads and incubated in the absence or presence of FBP (10 mM) with purified GST-Rac1. m , Eluates from washed beads were analysed by immunoblotting with GFP-, Rac1- and CHC-specific antibodies. n , A quantification of Rac1 amount bound to RCC2 in the presence or absence of FBP. Data represent mean ± s.e.m.; n = 5 independent experiments; two-sided one-sample t -test ( P value = 0.0029). n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Purification, Western Blot, Staining, Knockdown, Expressing, Transfection, Control, Incubation

Journal: Nature Cell Biology

Article Title: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion

doi: 10.1038/s41556-026-01911-1

Figure Lengend Snippet: a , Additional views of FBP-responsive peptide (pink, aa 110-120) highlighted in the structure of RCC2 (aa 89-522, PDB ID: 5GWN) (see also Fig. ). b , Overview of FBP-responsive peptides identified from whole cell lysate (orange, aa 110-120) and purified RCC2 (pink, aa 55-67, aa 57-68 and aa 454-470) highlighted in the predicted structure of full-length RCC2 by AlphaFold (AF- Q9P258 -F1). c , Overview of the F2,6BP-responsive peptide identified from purified RCC2 (pink, aa 52-67). d , Co-depletion of Rac1 in RCC2 knockdown cells restores FA number and cell area. Representative confocal images of paxillin-labeled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown below. Corresponding quantification is shown in Fig. . e , Model for the regulation of cell adhesion and protrusion by FBP: under low FBP concentrations, the inhibitory RCC2–Rac1 complex is intact, preventing Rac1 activation and thus resulting in decreased cell protrusion and adhesion. Increased FBP levels destabilize the RCC2–Rac1 complex, likely by direct binding of FBP to RCC2, leading to Rac1 activation, cell spreading and adhesion. Green dots represent FAs, red lines represent F-actin, and blue circles represent nuclei. Scale bars, 25 µm.

Article Snippet: The following cell lines were obtained from the American Type Culture Collection (ATCC), which authenticated them via short tandem repeat profiling: the human sarcoma cell line U-2 OS (ATCC: HTB-96; female), the human embryonic kidney cell line HEK293T (ATCC: CRL-3216; sex not specified), the human breast cancer cell line MDA-MB-231 (ATCC: HTB-26; female) and the telomerase-immortalized human retinal pigment epithelium cell line hTERT RPE-1 (ATTC: CRL-4000; female).

Techniques: Purification, Knockdown, Labeling, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.Lollipop chart for subcellular location of the 67 versus 40 proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 (top) and I-BET151 (middle), respectively. Subcellular location of each protein, based on immunohistochemistry and confocal microscopy, was retrieved from the Human Protein Atlas ( proteinatlas.org ). When a protein had more than one subcellular location assigned, that protein was included once per subcellular location in the graph. Bottom graph show a Venn diagram of the overlap between proteins stabilized/destabilized in (+)-JQ1 and I-BET151. Only 11 proteins were found to overlap between the two compounds. B. Physical protein-protein interaction (PPI) networks for the proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 and I-BET151, respectively. PPI networks were retrieved from the STRING db. Nodes are colored by subcellular location (from Human Protein Atlas). Large nodes correspond to proteins detected by TPP, where small nodes are additionally added nodes from the STRING db during network retrieval. C. Top: Physical PPI networks for (+)-JQ1 (same as B), except green nodes here indicate proteins found to be stabilized by (+)-JQ1 in cell extract. Bottom: Venn diagram for overlap of proteins detected in whole cells versus cell extract after perturbation with (+)-JQ1. D. Radar chart for features in cell painting data for (+)-JQ1 and I-BET151. Features were grouped into categories based on two criteria: (i) Cell Profiler module, i.e. Intensity (I), Correlation (C), Granularity (G), Location (L) and RadialDistribution (RD); and (ii) stains, i.e. Nucleus (Hoechst), ER (Concanavalin A), Nucleoli and cytoplasmic RNA (SYTO14), Golgi apparatus and F-actin cytoskeleton (WGA and Phalloidin) and Mitochondria (Mitotracker). Features were only consider for the object Cell, except for features from the stain for Nucleus, which was only considered in the object Nucleus. Additionally, area-shape related features were grouped by cell compartment, i.e. Cell (C), Cytoplasm (Cy) and Nucleus (N). E. t-SNE for the morphological features in the SPECS cell painting data on U2OS cells. Blue dots show the location of all cells treated with BET bromodomain inhibitors sharing at least one target with either (+)-JQ1 or I-BET151. Despite several BET bromodomain inhibitors clustering close to (+)-JQ1 or I-BET151, there are also several compounds with distinctly different morphological changes.

Article Snippet: For the purpose of this study, U2OS human bone sarcoma cell line (sourced from ATCC U2OS #HTB-96) was expanded and painted at passage number +8.

Techniques: Immunohistochemistry, Confocal Microscopy, Staining

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: UFL1 and C53 interact with γTuRC proteins. ( A ) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1 301–389 , C53, γ-tubulin (γ-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ). ( B ) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, or GCP4. Load ( lane 1 ), immobilized Abs not incubated with cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and carriers without Abs incubated with cell extracts ( lane 4 ). ( C ) The size distribution of UFL1, C53, γ-tubulin (γ-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. ( D ) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel ( C ) were precipitated with Ab to γ-tubulin. The blots were probed with Abs to UFL1, C53, and γ-tubulin (γ-Tb). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ).

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Immunoprecipitation, Negative Control, Incubation, Fractionation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Subcellular localization of UFL1 and C53. ( A ) U2OS cells expressing TagRFP-tagged proteins were fixed and stained with Ab to γ-tubulin. Localization of C53-TagRFP ( a , d ) and γ-tubulin ( b , e ). Superposition of images ( c , f ) C53-TagRFP, red; γ-tubulin, green; DAPI, blue). Localization of UFL1-TagRFP ( g ) and γ-tubulin ( h ). Superposition of images ( i , UFL1-TagRFP, red; γ-tubulin, green; DAPI, blue). Arrows indicate the same positions. Fixation D/F/M. Scale bar, 20 μm ( a – c , g – i ), 5 μm ( d – f ). ( B ) Association of proteins with purified centrosomes. Centrosomes enriched by centrifugation onto a Ficoll cushion were further purified by sucrose gradient centrifugation. The gradient was fractionated from the bottom. Individual fractions are indicated on the top, sucrose density in the fractions is shown at the bottom. Blots were probed with Abs to pericentrin, CDK5RAP2, ODF2, γ-tubulin (γ-Tb), C53, UFL1, PKCα, histone H1.4, and calnexin.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Staining, Purification, Centrifugation, Gradient Centrifugation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Deletion of UFL1 or C53 induces UPR and expansion of ER. ( A ) Immunoblot analysis of calnexin and PDI in whole-cell lysates of U2OS cells lacking UFL1 or C53. GAPDH served as a loading control. Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4 for calnexin; n = 3 for PDI). ( B ) Immunofluorescence microscopy. ( a – d ) Control U2OS cells, ( e – h ) UFL1-deficient cells (UFL1_KO) and ( i – l ) C53-deficient cells (C53_KO). Cells were fixed and double-labeled for calnexin ( a , e , i ) and β-tubulin ( b , f , j ; Microtubules). Higher magnification views of the regions delimited by rectangles are shown on the right of images from control ( c , d ), UFL1_KO ( g , h ), and C53_KO ( k , l ) cells. The images ( a , e , i ) and ( c , g , k ) were collected and processed in the same manner. Fixation F/Tx. Scale bars, 20 μm ( j ), and 5 µm ( l ). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. ER area coefficients (area occupied by ER/area free of ER) were calculated from fluorescence intensities for calnexin. The distributions of ER area coefficients (arbitrary units [AU]) are shown as box plots (four independent experiments, ≥16 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO ( n = 111) and C53_KO cells ( n = 127) relative to control cells ( n = 149). The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( A , C ) One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Labeling, Staining, Fluorescence

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Generation of ER stress by tunicamycin increases centrosomal microtubule nucleation. U2OS cells were treated with 1 µg/mL tunicamycin (+Tunicam.) or DMSO carrier (Control) for 24 h. ( A ). Effect of tunicamycin on ER expansion and subcellular distribution of calnexin, PDI, and ER stress-induced transcription factor DDIT3. ( a , b ) Visualization of ER in live cells by ER-Tracker. ( c – h ) Fixed cells stained for calnexin ( c , d ), PDI ( e , f ), and DDIT3 ( g , h ). Insets represent an enlargement of the boxed area. Fixation F/Tx. Pairs of images ( a , b ), ( c , d ), ( e , f ), and ( g , h ) were collected and processed in the same manner. Scale bars, 20 μm ( h ) and 5 µm (inset in f ). ( B ) ER area quantification in live cells stained with ER-Tracker. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥23 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 81) relative to control cells ( n = 108). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥19 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 80) relative to control cells ( n = 65). ( D , E ) The distributions of α-tubulin or γ-tubulin fluorescence intensities (arbitrary units [AU]) in 2-μm ROIs at 3.0 min of microtubule regrowth in control and tunicamycin-treated cells are shown as box plots (four independent experiments, >27 cells counted for each experimental condition). Box plot of α-tubulin ( D ) and γ-tubulin ( E ) fluorescence intensities in tunicamycin-treated cells ( n = 234) relative to control cells ( n = 181). ( F ) Time-lapse imaging of control and tunicamycin-treated cells expressing EB3-mNeonGreen. Still images of EB3 (Single frame) and tracks of EB3 comets over 10 s (10 frames project.). Scale bar, 5 µm. ( G ) Microtubule nucleation rate (EB3 comets/min) in tunicamycin-treated cells relative to control cells. Three independent experiments (at least 9 cells counted in each experiment). Control ( n = 31), tunicamycin-treated cells ( n = 31). The bold and thin lines within the dot plot represent mean ± SD. ( B – E ) The bold and thin lines within the box represent mean and median (the 50th percentile), respectively. The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( B – E , G ) Two-tailed, unpaired Student’s t -test was performed to determine statistical significance. **** p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Staining, Fluorescence, Imaging, Expressing, Two Tailed Test