Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin β1 activity, elevated cell adhesion, and migration defects of ppm1f-/- MEFs are reverted by re-expression of wildtype PPM1F. A PPM1F-/- MEFs were transduced with lentiviral particles encoding human wildtype PPM1F (hWT) or human PPM1F D360 A (hDA) in a bi-cistronic expression cassette with GFP. In addition, PPM1F-/- MEFs and PPM1F +/+ cells were transduced with a lentivirus encoding GFP alone. WCLs of sorted cells were analyzed by Western blotting with the indicated antibodies; as controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. B MEFs as in ( A ) were seeded onto 1 µg/ml FN III9-12 for 2 h. Samples were fixed and stained for talin (upper panel) or the active integrin β1 (lower panel) before analysis by confocal microscopy; scale bar: 20 µm. Insets show higher magnification of boxed areas; scale bar: 5 µm. Arrowheads point to active integrin β1 or talin enrichment. C MEFs as in ( A ) were kept in suspension for 45 min and incubated for 15 min with 10 µg/ml FN III9-12 (FN). Samples were stained for total (Hmb1-1) or active β1 integrin (9EG7) and analyzed by flow cytometry, ≥ 10 000 counts. The mean fluorescence intensity (MFI) ratio of active to total β1 integrin was calculated and normalized to the wildtype sample (= 1). Scatter blots represent mean ± SEM of 4 independent experiments; statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, ns = not significant). D MEFs were seeded in triplicates onto fibronectin-coated wells for 60 min and cell adhesion was quantified. Representative pictures from cells seeded on 10 µg/ml FN (left panel); scale bar: 150 µm. Scatter blots represent mean ± SEM of 5 independent experiments performed in technical triplicates each. Values were normalized to MEF wildtype cells (= 1). Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (** p < 0.01, * p < 0.05, ns = not significant). E MEFs were seeded onto indicated fibronectin concentrations for 45 min, fixed and stained with DAPI and Phalloidin-Cy5. Samples were imaged using confocal microscopy. Representative images from cells seeded onto 10 µg/ml FN are shown; scale bar: 10 µm (left panel). Quantification of cell spreading. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments; n ≥ 90 cells. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001, ns = not significant) (right panel). F Serum starved MEFs were stimulated by addition of 10% FCS and cell migration was monitored every 30 min for 12 h using time-lapse microscopy. Cell tracks were evaluated for velocity, covered distance and directionality. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments ( n = 30); Statistics was performed as in ( E ); *** p < 0.001, * p < 0.05, ns = not significant. See also Additional_File2

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Migration, Expressing, Transduction, Western Blot, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Suspension, Incubation, Flow Cytometry, Fluorescence, Time-lapse Microscopy

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: PPM1F contributes to the invasive phenotype of tumor cells. A WCLs from indicated cancer cell lines were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. B , C Indicated serum-starved cancer cells were seeded on top of a Matrigel basement membrane (30 µg/100 µl) in Boyden chamber cell invasion assays using 20% FCS as stimulus or 2% BSA to evaluate random invasion activity. NIH3 T3 cells served as non-invasive control cells. Representative pictures of the lower porous membrane surface (20x) are shown in (B); scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Cells were evaluated for invasion after 24 h by dye elution with 10% acetic acid and absorbance measurement at 590 nm. Graph in ( C ) shows quantified means ± SEM from three independent experiments. Statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, p ** < 0.01, ns = not significant). D MCF-7 cells were stably transduced with lentiviral particles harboring a bicistronic GFP and hPPM1F wildtype or hPPM1F D360 A expression cassette and single-cell sorted via flow cytometry for GFP positive cells to obtain a mixed population of PPM1F-overexpressing MCF-7 cells (PPM1F + + and PPM1F D360 A + +). WCL of the wildtype and modified cell lines were analyzed by Western blotting with indicated antibodies. α-tubulin antibody (lowest panel) served as loading control. E Serum-starved cells from ( D ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers. Cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Invasion was quantified by dye elution. Graph (right) shows means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C )

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Western Blot, Control, Membrane, Activity Assay, Staining, Stable Transfection, Transduction, Expressing, Flow Cytometry, Modification

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Genetic deletion of PPM1F in tumor cells diminishes matrix invasion and integrin phosphorylation. A WCLs from A172 wildtype cells and two clonal PPM1F KO cell lines (1 and 2) were analyzed by Western blotting using the indicated antibodies. α-Tubulin antibody was used as loading control. B Serum starved A172 wildtype cells and PPM1F KO cell lines (clone 1 and clone 2) were seeded in triplicate onto fibronectin-, vitronectin-, or 2% BSA-coated wells for 60 min either in presence of 50 µM cilengitide or DMSO as control. Wells were washed and adherent cells were stained with crystal violet. Representative pictures are shown; scale bar: 150 µm. C Adherent crystal violett stained cells from ( B ) were quantified by dye elution. Graph depicts individual values as well as mean ± SEM of 4 independent experiments performed in technical triplicates. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001; ** p < 0.01; p * < 0.05; ns = not significant) and shown for the PPM1F knock-out clones in relation to the A172 wildtype cells. D Serum-starved cells as in ( C ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers and cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Cells were evaluated for invasion after 24 h and representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores (left). Invasion assays were quantified by dye elution. Graph depicts individual values as well as means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C ). See also Additional_File4 and Additional_File5

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Phospho-proteomics, Western Blot, Control, Staining, Knock-Out, Clone Assay, Membrane

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin-based cell adhesion in PPM1F-deficient cells prohibits cell spreading despite elevated PAK activity. A Serum-starved A172 wildtype, sgRNA control and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 45 min and WCLs were subjected to Western blotting with indicated antibodies (left panel). Graphs (right panel) show densitometric quantification of band intensities from pThr402PAK2 versus PAK antibody signal for the indicated samples from 5 independent experiments; wildtype was set to 1. Statistics were performed using one-way ANOVA, followed by Bonferroni post-hoc test (* p < 0.05, ns = not significant). B Serum-starved A172 wildtype and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 1.5 h, fixed and F-actin was stained. Samples were imaged using confocal microscopy. Representative pictures are shown; scale bar: 20 µm. C Cells as in ( B ) were seeded for 2 h on surfaces coated with 10 µg/ml fibronectin or poly-L-lysine, before fixation, F-actin staining and analysis by confocal microscopy; scale bar: 10 µm. D Spreading assays were performed with serum-starved A172 wildtype and PPM1F KO cells re-expressing mKate2 or re-expressing PPM1F-mKate2 cells, pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeding onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed, stained for F-actin and the covered area was quantified in ImageJ. Boxes and whiskers indicate median with 95% confidence intervals from two independent experiments; n ≥ 30 cells; dots indicate outliers. Statistics was performed using one-way ANOVA, followed by post-hoc Bonferroni test, (*** p < 0.001, ns = not significant). E Serum-starved cells as in ( D ) were pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeded onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed and stained for active integrin β1. Cells were imaged by confocal microscopy. Representative pictures are shown; scale bar: 10 µm. See also Additional_File6 and Additional_File7

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Control, Western Blot, Staining, Confocal Microscopy, Expressing, Suspension

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin β1 activity, elevated cell adhesion, and migration defects of ppm1f-/- MEFs are reverted by re-expression of wildtype PPM1F. A PPM1F-/- MEFs were transduced with lentiviral particles encoding human wildtype PPM1F (hWT) or human PPM1F D360 A (hDA) in a bi-cistronic expression cassette with GFP. In addition, PPM1F-/- MEFs and PPM1F +/+ cells were transduced with a lentivirus encoding GFP alone. WCLs of sorted cells were analyzed by Western blotting with the indicated antibodies; as controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. B MEFs as in ( A ) were seeded onto 1 µg/ml FN III9-12 for 2 h. Samples were fixed and stained for talin (upper panel) or the active integrin β1 (lower panel) before analysis by confocal microscopy; scale bar: 20 µm. Insets show higher magnification of boxed areas; scale bar: 5 µm. Arrowheads point to active integrin β1 or talin enrichment. C MEFs as in ( A ) were kept in suspension for 45 min and incubated for 15 min with 10 µg/ml FN III9-12 (FN). Samples were stained for total (Hmb1-1) or active β1 integrin (9EG7) and analyzed by flow cytometry, ≥ 10 000 counts. The mean fluorescence intensity (MFI) ratio of active to total β1 integrin was calculated and normalized to the wildtype sample (= 1). Scatter blots represent mean ± SEM of 4 independent experiments; statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, ns = not significant). D MEFs were seeded in triplicates onto fibronectin-coated wells for 60 min and cell adhesion was quantified. Representative pictures from cells seeded on 10 µg/ml FN (left panel); scale bar: 150 µm. Scatter blots represent mean ± SEM of 5 independent experiments performed in technical triplicates each. Values were normalized to MEF wildtype cells (= 1). Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (** p < 0.01, * p < 0.05, ns = not significant). E MEFs were seeded onto indicated fibronectin concentrations for 45 min, fixed and stained with DAPI and Phalloidin-Cy5. Samples were imaged using confocal microscopy. Representative images from cells seeded onto 10 µg/ml FN are shown; scale bar: 10 µm (left panel). Quantification of cell spreading. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments; n ≥ 90 cells. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001, ns = not significant) (right panel). F Serum starved MEFs were stimulated by addition of 10% FCS and cell migration was monitored every 30 min for 12 h using time-lapse microscopy. Cell tracks were evaluated for velocity, covered distance and directionality. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments ( n = 30); Statistics was performed as in ( E ); *** p < 0.001, * p < 0.05, ns = not significant. See also Additional_File2

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Migration, Expressing, Transduction, Western Blot, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Suspension, Incubation, Flow Cytometry, Fluorescence, Time-lapse Microscopy

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: PPM1F contributes to the invasive phenotype of tumor cells. A WCLs from indicated cancer cell lines were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. B , C Indicated serum-starved cancer cells were seeded on top of a Matrigel basement membrane (30 µg/100 µl) in Boyden chamber cell invasion assays using 20% FCS as stimulus or 2% BSA to evaluate random invasion activity. NIH3 T3 cells served as non-invasive control cells. Representative pictures of the lower porous membrane surface (20x) are shown in (B); scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Cells were evaluated for invasion after 24 h by dye elution with 10% acetic acid and absorbance measurement at 590 nm. Graph in ( C ) shows quantified means ± SEM from three independent experiments. Statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, p ** < 0.01, ns = not significant). D MCF-7 cells were stably transduced with lentiviral particles harboring a bicistronic GFP and hPPM1F wildtype or hPPM1F D360 A expression cassette and single-cell sorted via flow cytometry for GFP positive cells to obtain a mixed population of PPM1F-overexpressing MCF-7 cells (PPM1F + + and PPM1F D360 A + +). WCL of the wildtype and modified cell lines were analyzed by Western blotting with indicated antibodies. α-tubulin antibody (lowest panel) served as loading control. E Serum-starved cells from ( D ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers. Cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Invasion was quantified by dye elution. Graph (right) shows means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C )

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Western Blot, Control, Membrane, Activity Assay, Staining, Stable Transfection, Transduction, Expressing, Flow Cytometry, Modification

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Genetic deletion of PPM1F in tumor cells diminishes matrix invasion and integrin phosphorylation. A WCLs from A172 wildtype cells and two clonal PPM1F KO cell lines (1 and 2) were analyzed by Western blotting using the indicated antibodies. α-Tubulin antibody was used as loading control. B Serum starved A172 wildtype cells and PPM1F KO cell lines (clone 1 and clone 2) were seeded in triplicate onto fibronectin-, vitronectin-, or 2% BSA-coated wells for 60 min either in presence of 50 µM cilengitide or DMSO as control. Wells were washed and adherent cells were stained with crystal violet. Representative pictures are shown; scale bar: 150 µm. C Adherent crystal violett stained cells from ( B ) were quantified by dye elution. Graph depicts individual values as well as mean ± SEM of 4 independent experiments performed in technical triplicates. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001; ** p < 0.01; p * < 0.05; ns = not significant) and shown for the PPM1F knock-out clones in relation to the A172 wildtype cells. D Serum-starved cells as in ( C ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers and cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Cells were evaluated for invasion after 24 h and representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores (left). Invasion assays were quantified by dye elution. Graph depicts individual values as well as means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C ). See also Additional_File4 and Additional_File5

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Phospho-proteomics, Western Blot, Control, Staining, Knock-Out, Clone Assay, Membrane

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin-based cell adhesion in PPM1F-deficient cells prohibits cell spreading despite elevated PAK activity. A Serum-starved A172 wildtype, sgRNA control and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 45 min and WCLs were subjected to Western blotting with indicated antibodies (left panel). Graphs (right panel) show densitometric quantification of band intensities from pThr402PAK2 versus PAK antibody signal for the indicated samples from 5 independent experiments; wildtype was set to 1. Statistics were performed using one-way ANOVA, followed by Bonferroni post-hoc test (* p < 0.05, ns = not significant). B Serum-starved A172 wildtype and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 1.5 h, fixed and F-actin was stained. Samples were imaged using confocal microscopy. Representative pictures are shown; scale bar: 20 µm. C Cells as in ( B ) were seeded for 2 h on surfaces coated with 10 µg/ml fibronectin or poly-L-lysine, before fixation, F-actin staining and analysis by confocal microscopy; scale bar: 10 µm. D Spreading assays were performed with serum-starved A172 wildtype and PPM1F KO cells re-expressing mKate2 or re-expressing PPM1F-mKate2 cells, pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeding onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed, stained for F-actin and the covered area was quantified in ImageJ. Boxes and whiskers indicate median with 95% confidence intervals from two independent experiments; n ≥ 30 cells; dots indicate outliers. Statistics was performed using one-way ANOVA, followed by post-hoc Bonferroni test, (*** p < 0.001, ns = not significant). E Serum-starved cells as in ( D ) were pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeded onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed and stained for active integrin β1. Cells were imaged by confocal microscopy. Representative pictures are shown; scale bar: 10 µm. See also Additional_File6 and Additional_File7

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Control, Western Blot, Staining, Confocal Microscopy, Expressing, Suspension

Journal: Advanced Science

Article Title: NaBC1 Boron Transporter Enables Myoblast Response to Substrate Rigidity via Fibronectin‐Binding Integrins

doi: 10.1002/advs.202407548

Figure Lengend Snippet: AlphaFold 3 predictions and experimental validation of NaBC1 interaction with β 1 integrin and vinculin. A) AlphaFold 3 models of the interaction between a NaBC1 dimer (both monomers shown in orange, with monomer A slightly lighter than monomer B for clarity) and the transmembrane domain along with the intracellular C‐terminal tail of β 1 integrin (residues 722–798, both monomers shown in green, with monomer C lighter than monomer D for clarity). The models, viewed from the side, front, and bottom (intracellular side), predict the transmembrane helix of β 1 integrin to sit in a groove on the side of the interacting face of the NaBC1 dimer. The C‐terminal tail of β 1 integrin is predicted to wrap along the underside of the intracellular domain of NaBC1. The model of NaBC1 and vinculin (both monomers shown in purple, with monomer C lighter than monomer D for clarity) suggests that vinculin interacts with both the transmembrane domain (TMD) and the N‐terminal intracellular domain (NTD) of NaBC1. B) Detailed view of the interactions. The C‐terminal tail of each β 1 integrin monomer is predicted to interact with the intracellular domains of both NaBC1 monomers. A section of vinculin's proline‐rich hinge region (residues 839–873) is predicted to insert between the NaBC1 monomers, reaching from the intracellular domains up to the transmembrane domains and interacting with the 10 β1 and 10 β2 β‐sheets of NaBC1. Red asterisk represents the presence of B ion. pLDDT confidence scores: 68.6 for NaBC1/β 1 integrin; 76.1 for NaBC1/vinculin. C) Immunoblots validating the interaction of NaBC1 with β 1 integrin. β 1 integrin was immunoprecipitated from cells cultured on rigid hydrogels in the absence or presence of B (1.47 m m ) for 24 h. Immunodetection was performed using antibodies specific for β 1 integrin and NaBC1. An increase in the interaction of NaBC1 with β 1 integrin was observed in the presence of B. D) Immunoblots showing interaction between NaBC1 and vinculin (VCL). Vinculin was immunoprecipitated using protein‐specific antibodies, and NaBC1 and vinculin were immunodetected. An increase in the interaction of NaBC1 with β 1 integrin was observed in the presence of B. IP, Immunoprecipitated protein; IB, Immunodetected protein; Un, untreated cells; L, protein ladder; B, B‐treated cells. Cell lysate is referred to as input to confirm protein‐specific bands. All immunoblot experiments were duplicated with identical results. Uncropped immunoblots are presented in Figure (Supporting Information).

Article Snippet: Antibodies specific for β 1 integrin (Proteintech) and vinculin (Proteintech) were conjugated to Dynabeads beads coated with protein A or G (ThermoFisher) and then incubated with equal amounts of protein lysates (2 mg) to allow formation of protein–antibody complexes which was then eluted from the beads by using reducing agent (ThermoFisher) and sample buffer (ThermoFisher) at final concentration of 1×, followed by heating at 95 °C for 10 min. Electrophoresis was then performed at 190 V for 50 min at room temperature, and proteins were transferred to PVDF membrane at 20 V and 160 mA for 70 min in a cooled environment, followed by blocking using 5% non‐fat dry milk (NFDM) and immunodetection using protein‐specific antibodies for 1.5 h at room temperature.

Techniques: Biomarker Discovery, Western Blot, Immunoprecipitation, Cell Culture, Immunodetection

Journal: iScience

Article Title: Mechanical control of the alternative splicing factor PTBP1 regulates extracellular matrix stiffness induced proliferation and cell spreading

doi: 10.1016/j.isci.2025.112273

Figure Lengend Snippet: PTBP1 is important for cellular mechanoresponses (A) Mesenchymal stem cells were cultured on plastic (stiff) and transfected with the indicated siRNAs. Osteoblastic differentiation was quantified using alkaline phosphatase staining. Bar represents 100 μm and refers to the bottom images. Data were analyzed using ordinary one-way ANOVA. (B) Mesenchymal stem cells were cultured on collagen coated stiff PAA gels (25 kPa), transfected with the indicated siRNAs and osteoblastic differentiation was quantified as described in (A). Bar represents 100 μm. (C) MCF10A cells transfected with the indicated siRNAs and seeded on stiff (25 kPa) or soft (0.2 kPa) collagen coated PAA gels. Cells were stained with F-actin for measurement of spreading area. Knockdown was confirmed by PTBP1 immunofluorescence. Bar represents 10 μm. Data were analyzed using ordinary one-way ANOVA. (D) MCF10A cells were transfected with the indicated siRNAs followed by a surface biotinylation assay. Total proteins and cell surface proteins were analyzed by western blot using indicated antibodies. Bar diagrams represent the quantification of the experiments. Data were analyzed by ordinary one-way ANOVA. (E) MCF10A cells were transfected with the indicated siRNAs and the surface amount of integrin β1 (CD29) was quantified using flow cytometry. Data were analyzed by ordinary one-way ANOVA. (F) MCF10A cells were cultured on stiff (25 kPa) or soft (0.2 kPa) collagen coated PAA gels and transfected with the indicated siRNAs. Proliferation rate and knockdown was determined by immunofluorescence. Bar diagram shows the quantification of BrdU incorporation. Bar represents 20 μm. Data were analyzed by ordinary one-way ANOVA. (G) MCF10A cells were cultured on stiff (25 kPa) collagen coated PAA gels and treated with 50 μM ATN161 inhibitor overnight. Proliferation rate was quantified using the BrdU assay. Bar represents 50 μm. Data were analyzed by unpaired t-test. (H) MCF10A cells were seeded on stiff (25 kPa) collagen coated PAA gels and transfected with the indicated siRNAs. Knockdown was confirmed by western blot and proliferation rate was quantified by BrdU assay. Bar represents 50 μm. Data were analyzed by unpaired t-test. Three replicates in all experiments. n = numbers of cells analyzed or in the case of BrdU assays refers to the number of analyzed microscopic fields. All values are means ± s.d. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.

Article Snippet: Primary antibodies were used as follows: YAP (1:200; Santa Cruz, sc-101199), GFP (1:2000; Invitrogen, A-11122), PTBP1 (1:500; Thermo Fisher Scientific, 32–4800), Pitx2 (1:1000; Capra Science, PA-1020), CCT2 (1:200; Santa Cruz, sc-374152), Nucleolin (1:300; Santa Cruz, sc-8031), Numb (1:1000; Cell Signaling Technology, 2756), Integrin β-1 (1:1000; Santa Cruz, sc-374429), Transferrin receptor 1 (TfR1) (1:1000; Cell Signaling Technology, 13113), β-tubulin (1:5000; Sigma-Aldrich, T4026), γ-adaptin (1:1000; gift from Dr Andrew Peden, University of Sheffield), GAPDH (1:20000; Proteintech, 60004-1-IG), β-catenin (1:2000; BD Biosciences, 610153).

Techniques: Cell Culture, Transfection, Staining, Knockdown, Immunofluorescence, Surface Biotinylation Assay, Western Blot, Flow Cytometry, BrdU Incorporation Assay, BrdU Staining

Journal: iScience

Article Title: Mechanical control of the alternative splicing factor PTBP1 regulates extracellular matrix stiffness induced proliferation and cell spreading

doi: 10.1016/j.isci.2025.112273

Figure Lengend Snippet:

Article Snippet: Primary antibodies were used as follows: YAP (1:200; Santa Cruz, sc-101199), GFP (1:2000; Invitrogen, A-11122), PTBP1 (1:500; Thermo Fisher Scientific, 32–4800), Pitx2 (1:1000; Capra Science, PA-1020), CCT2 (1:200; Santa Cruz, sc-374152), Nucleolin (1:300; Santa Cruz, sc-8031), Numb (1:1000; Cell Signaling Technology, 2756), Integrin β-1 (1:1000; Santa Cruz, sc-374429), Transferrin receptor 1 (TfR1) (1:1000; Cell Signaling Technology, 13113), β-tubulin (1:5000; Sigma-Aldrich, T4026), γ-adaptin (1:1000; gift from Dr Andrew Peden, University of Sheffield), GAPDH (1:20000; Proteintech, 60004-1-IG), β-catenin (1:2000; BD Biosciences, 610153).

Techniques: Recombinant, Selection, Staining, Negative Control, Software