human cell lines human osteosarcoma cells  (ATCC)

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: Effects of ADAR2 overexpression in osteosarcoma cell lines. a Real-Time RT-PCR and b Western Blot analysis of ADAR2 expression in MSC, osteoblasts (OB) and osteosarcoma cell lines Saos-2 and 143B. In b upper panels : representative blots; lower panel : densitometric analysis. c FACS analysis of the proliferative rate evaluated by CMAC staining and d cell cycle analysis of Saos-2 ( left panel ) and 143B ( right panel ) cells transfected with ADAR2-pEGFP-C3 (pADAR2), ADAR2 E/A-pEGFP-C3 (pADAR2 E/A) or Empty-pEGFP-C3 (pEmpty) vectors. ADAR2 E/A vector was generated by a single mutation in the catalytic domain of ADAR2. e Migration ability of transfected Saos-2 ( left panel) and 143B ( right panel) cells. f Transwell invasion assay of transfected Saos-2 ( left panel ) and 143B cells ( right panel ). g Representative blot and h densitometric analysis of Runx2 and Osx in transfected Saos-2 ( left panel) and 143B cells ( right panel ). Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; vs pEmpty transfected cells. # P < 0.05; ## P < 0.01 vs pADAR2 transfected cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Staining, Cell Cycle Assay, Transfection, Plasmid Preparation, Generated, Mutagenesis, Migration, Transwell Invasion Assay

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: Terminal osteogenic differentiation and increased drugs susceptibility in pADAR2-transfected Saos-2 cells. a – c Mineralization assay of Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors. a Upper panels : Alizarin Red staining; lower panels : Von Kossa staining. b Absorbance analysis of Alizarin Red staining. c Densitometric analysis of Von Kossa-stained area. d – h Real-Time RT-PCR expression analysis of d COL1A2 , e DMP1 , f MEPE , g PRKCA and h NANOG . In ( b – h ) results are expressed as mean ± sd and are reported as individual data points of independent experiments. i Cell viability analysis of transfected Saos-2 cells treated for 6 days with increasing concentrations of MTX (0, 1, 5, 10, 50 and 100 nmol/L, left panel ) and of MS275 (0, 0.5, 1, 2.5, 5 and 10 μmol/L, right panel ) for 2 days. The concentration of drugs able to reduce by 50% (GI 50 ) cell viability is reported in the upper part of each graph. Results are expressed as mean ± sd of at least three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01; ### P < 0.001; #### P < 0.000 1 vs pADAR2 transfected cells. j FACS analysis of apoptosis of transfected Saos-2 cells treated with the GI 50 calculated for pEmpty transfected Saos-2, or with Vehicle. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. ** P < 0.01 vs Vehicle treated cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Transfection, Mineralization Assay, Staining, Quantitative RT-PCR, Expressing, Concentration Assay

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: In vivo experiments. Seven-weeks-old NSG male mice were intratibially injected with Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors; after 12 weeks animals were sacrificed. a Representative X-Ray pictures of primary bone tumors. b Quantification of the tumor volume. c Number of animals with metastases in liver, lungs and kidneys at sacrifice. d Hematoxylin/Eosin staining of liver, lungs and kidney metastases. Nodules were indicated by black arrowheads. Number of metastases in e liver, f lungs and g kidneys. h Representative pictures of immunohistochemistry and i quantification of Ki67 in liver, lungs and kidneys metastases. Results are expressed as mean ± sd. * P < 0.05; ** P < 0.01 vs pEmpty cells injected mice. ## P < 0.01 vs pADAR2 cells injected animals

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: In Vivo, Injection, Transfection, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: RNA-seq analysis. a Gene expression based heatmap showing the unique clusterization of ADAR2 transfected Saos-2 cells. Real-Time RT-PCR expression analysis of b COL4A1 , c SERPINH1 , d SWAP-70 and e TENM1 for transcriptional validation. f – h Editing analysis. Upper panels : Sequence chromatograms of the transcripts and editing levels of COPA , IGFBP7 and COG3 . Arrows indicate editing positions. Lower panels : percentage of editing in f COPA , g IGFBP7 and h COG3 transcripts. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; **** P < 0.000 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01; ### P < 0.001 vs pADAR2 transfected cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: RNA Sequencing, Gene Expression, Transfection, Quantitative RT-PCR, Expressing, Biomarker Discovery, Sequencing

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: IGF1R pathway analysis. a – f Investigation of IGF1R pathway in Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors. a Representative plots and b – f densitometric analysis of b p-Igf1r, c p-Irs, d p-Akt (T308), e p-Akt (S473) and f p-p70. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. ** P < 0.01; *** P < 0.00 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01 vs pADAR2 transfected cells. g – o Effects of the treatment with WT- or K95R-IGFBP7 on Saos-2 cell line. g – l Analysis of IGF1R pathway in Saos-2 cells treated with 2 μg/ml of WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. g Representative plots and h – l densitometric analysis of h p-Igf1r, i p-Irs, j p-Akt (T308), k p-Akt (S473) and l p-p70. m , n Western blot analysis of Runx2 in Saos-2 cells treated with WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. m Representative blot and n densitometric analysis. o Proliferation rate of Saos-2 treated with WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 vs Vehicle treated Saos-2 cells. # P < 0.05; ### P < 0.001 vs WT-IGFBP7 treated cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and C ) Representative microscopic images of BrdU incorporation assays in U2OS and MG63 osteosarcoma cells under normoxic (control) and CoCl 2 induced hypoxic conditions. Scale bar = 50 μm. ( B and D ) Quantification of BrdU-positive cells showing a significant decrease in proliferation in hypoxic U2OS and MG63 cells compared with their respective controls (n ≥ 20 cells per condition). ( E and G ) Representative images of colony-formation assays in control and CoCl₂-treated hypoxic U2OS and MG63 cells, respectively. (F and H) Quantification of colony numbers showing a marked reduction in the clonogenic potential of hypoxic osteosarcoma cells. ( I and K ). Representative images of cell-migration assays in U2OS and MG63 cells under control and hypoxic conditions. Scale bar = 50 µm. ( J and L) Quantification of migrated cells showed a significant reduction in the migratory capacity of hypoxic U2OS and MG63 cells, respectively. Statistical significance was calculated using two-tailed Student’s t -test and is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: BrdU Incorporation Assay, Control, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) U2OS cells, either stably expressing K294A upon doxycycline induction or uninduced controls, were biochemically fractionated into nuclear and cytoplasmic compartments. Western blot analysis confirmed fractionation quality using Lamin A/C as a nuclear marker and GAPDH as a cytoplasmic marker. Myc blotting verified the expression of K294A upon doxycycline induction. ( B ) Quantitative real-time PCR analysis revealed a significant reduction in the cytoplasmic levels of RORA and KCTD16 transcripts in K294A expressing cells compared with controls, while BNIP3 levels remained unchanged. ( C ) Western blot analysis further confirmed decreased protein levels of RORA and KCTD16 in K294A-expressing cells. Myc blotting verified stable K294A expression. ( D ) Densitometric analysis of RORA and KCTD16 protein bands from panel C was performed using ImageJ software. β-Actin was used as a loading control for normalization. Statistical analysis was calculated by performing two-tailed Student’s t -test. ( E ) Table summarizing the internal CAGE (Cap Analysis of Gene Expression) sites identified within the analysed transcripts, with the specific positions highlighted in red. ( F - J ) Bar graphs representing the genomic distribution of CAGE peaks for each gene, illustrating the relative frequency of CAGE signals across different transcript regions. ( K ) Schematic illustration of the Xrn1 susceptibility assay used to assess the stability of 5′-capped transcripts. ( L ) Relative 5′-end loss of RORA and KCTD16 was assessed using an in vitro Xrn1 susceptibility assay. In K294A-expressing cells, both transcripts exhibited a level of 5′-end loss comparable to STAT3 , a known cCE target, relative to control cells. Statistical analysis was performed using one sample Student’s t -test. ( M ) Western blot analysis showing Xrn1 protein levels in Xrn1 knockdown cells with or without doxycycline-induced K294A expression. Myc detection confirmed successful induction of the dominant-negative cCE mutant. ( N ) Quantification of Xrn1 knockdown efficiency was performed using ImageJ software, with β-Actin serving as the internal loading control. ( O ) RORA and KCTD16 exhibited the most pronounced rescue in Xrn1 knockdown cells expressing K294A, indicating their strong dependence on cytoplasmic capping for stability. Statistical significance was determined using one-way ANOVA. All the data is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Stable Transfection, Expressing, Western Blot, Fractionation, Marker, Real-time Polymerase Chain Reaction, Software, Control, Two Tailed Test, Gene Expression, Drug Susceptibility Assay, In Vitro, Knockdown, Dominant Negative Mutation, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and B ) qPCR analysis of the selected transcripts in U2OS and MG63 cells revealed reduced expression following treatment with the HIF1α inhibitor PX478, irrespective of hypoxia induction. ( C and E ) Western blot analysis of U2OS and MG63 cells demonstrated reduced protein levels of all selected targets, including HIF1α, in PX478-treated hypoxic samples. ( D and F ) Quantification of western blot band intensities corresponding to panels C and E was performed using ImageJ software. β-Actin served as the loading control for normalization. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined using one-way ANOVA. ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Expressing, Western Blot, Software, Control

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) Western blot analysis of c-Myc protein levels in U2OS cells under normoxic and CoCl₂-induced hypoxic conditions. ( B ) Densitometric quantification of c-Myc expression from panel A using ImageJ, showing reduced c-Myc levels in hypoxic U2OS cells. β-Actin served as the loading control. Statistical significance was determined using two-tailed Student’s t -test. ( C and E ) Western blots showing siRNA-mediated depletion of RORA ( C ) and KCTD16 ( I ) in U2OS and MG63 cells under hypoxic conditions. HIF1α blot confirms hypoxia induction. c-Myc levels were elevated upon depletion of either gene. ( D and J ) ImageJ-based densitometric quantification of blots from panels C and I , normalized to β-actin. Statistical analysis was performed using one-way ANOVA. ( E and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 depleted hypoxic U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( F and L ) Quantification of BrdU-positive cells showing increased proliferation upon RORA or KCTD16 depletion under hypoxic conditions (n ≥ 20 cells per condition). ( G and M ) Representative images of colony formation assays in RORA-and KCTD16-depleted hypoxic osteosarcoma cells. ( H and N ) Quantitative analysis showing enhanced clonogenic potential following RORA or KCTD16 depletion in hypoxic cells. Statistical significance was calculated using one-way ANOVA. All the data is represented as ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Expressing, Control, Two Tailed Test, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and I ) Western blots showing RORA, KCTD16, and c-Myc levels in U2OS and MG63 cells, respectively. ( B and J ) Densitometric quantification of the blots using ImageJ demonstrated that overexpression of RORA or KCTD16 led to reduced c-Myc expression in both cell lines. β-Actin was used as a loading control for normalization. ( C and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 overexpressing U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( D and L ) Quantification of BrdU-positive cells showing significantly decreased proliferative capacity in RORA and KCTD16-overexpressing cells (n ≥ 20 cells per condition). ( E and M ) Representative images of colony formation assays in RORA and KCTD16 overexpressing osteosarcoma cells. ( F and N ) Quantification of colonies demonstrating a marked reduction in clonogenic potential upon RORA or KCTD16 overexpression. ( G and O ) Representative images of migration assays in RORA and KCTD16-overexpressing cells. Scale bar = 50 μm. ( H and P ) Quantification of migrated cells showing significantly impaired migratory capacity in RORA and KCTD16 overexpressing osteosarcoma cells. Statistical analysis was performed using one-way ANOVA, and all data are presented as mean ± SD from three independent biological replicates. Significance is indicated as follows: ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Over Expression, Expressing, Control, BrdU Incorporation Assay, Migration

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