EMBO/soda-nel huggingface dataset:
An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs (2022)
Figure 4

Figure 4: Engraftment of eMPs in mdx mice.

A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm.

B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs.

C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs.

D. Representative immunostainings for the human nuclear marker lamin A/C and dystrophin in muscles of mdx mice after transplantation of eMPs. Dashed lines outline dystrophin positive fibers. Yellow arrows depict cells in the satellite cell position in the periphery of muscle fibers and white arrows show human myonuclei. Scale bars = 50 μm.

E, Schematic outlining the re-injury experiment of mdx muscles transplanted with eMPs derived from a luciferase hiPSC line.

F. Quantification and representative images of luciferase bioluminescence before (pre-injury) and after re-injury (post-injury) of eMP transplanted muscles.

G. Representative immunostainings for laminin and human lamin A/C in cross-sections of muscles of mdx mice four days after re-injury. Scale bars = 50 μm.

H. Evolution of eccentric force production of mdx muscles transplanted with eMPs or hskMPs.

I. Scheme summarizing the procedure for the generation of eMPs and potential downstream applications.

Data information: A-C. 10 days post-transplantation. D. 25 days post-transplantation. F-G. Pre-injury 25 days and post-injury 30 days after transplantation. H. 17 days post-transplantation. B,C. n=4 mice per condition. F. n=3 mice per condition. H. n=5 mice per condition. Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using ANOVA followed by a Bonferroni post hoc test for multiple comparisons or students t-test for binary comparisons. A-C,H. hiPSC donor 1. D,F-G. hiPSC donor 4.
Structural role of the T94I rhodopsin mutation in congenital stationary night blindness (2016)
Figure 1

Figure 1: Comparison of constitutively active rhodopsin mutants M257Y6.40 (left [21]), T94I2.61 (middle) and G90D2.57 (right [16]). All-trans (yellow) and cis retinal isomers (orange), the side of retinal attachment K2967.43 (blue slate), the retinal counterion E1133.28 (blue slate), GαCT peptides (cyan) and the constitutively activating mutations M257Y6.40, T94I2.61, and G90D2.57 (green) are shown as spheres. Palmitoylation at C323H8 and glycosylation at N15NT are shown as sticks. The lower panels compare the M257Y6.40, T94I2.61 and G90D2.57 retinal-binding pockets. All constructs also contain an additional thermostabilizing disulfide bridge, depicted as (c-c), to enhance the stability [18] and crystallizability [19] without changing the rhodopsin activation pathway [20].
Structural role of the T94I rhodopsin mutation in congenital stationary night blindness (2016)
Figure 2

Figure 2: Kinetic characterization of the rhodopsin dark state. Thermal decay of dark state rhodopsin: A) WT, B) T94I and C) G90D rhodopsin. The spectra are normalized to the OD280 and OD500/OD480 at t=0. D) Absorption maximum plotted as a function of time and fitted to a single exponential decay. Retinal isomerization in the rhodopsin dark state: E) WT, F) T94I and G) G90D rhodopsin, H) The fraction of 11-cis retinal is plotted as a function of time and fitted to a single exponential decay. SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.
Structural role of the T94I rhodopsin mutation in congenital stationary night blindness (2016)
Figure 3

Figure 3: Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D2.57, T94I2.61 and T94I2.61 with protonated E1133.28. In the absence of the G90D2.57 and T94I2.61 dark state structure, both aspartate and isoleucine were placed in the WT (1GZM [48]) using a favorable rotamer without introducing major clashes with the rest of the protein. However, both isoleucine and aspartate in the dark state structures introduced clashes with several amino acids and were therefore optimized using energy minimization before simulation. WT simulation was done with the protonated SB while in the G90D2.57 system, both E1133.28 and SB were protonated [36]. The protonated state of E1133.28 was unknown in T94I2.61, therefore simulation were done for both deprotonated and protonated E1133.28. The T94I2.61 dark state conformation with deprotonated E1133.28 was similar to that of the WT and was stable, whereas the dark state of G90D2.57 and T94I2.61 with protonated E1133.28 was unstable with a concomitant movement of E1133.28 residue away from the SB, thereby, causing movement of TM3 and opening of the E1133.28-SB activation switch.

In contrast to the crystal structures the orientation of the retinal -ionone ring in the simulations was variable with respect to the polyene chain. This indicates a degree of positional freedom within the hydrophobic binding pocket.
PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity (2019)
Figure 1

Figure 1: PPM1G-B56δ is a new phosphatase holoenzyme complex. (A) Interaction network of B56δ with its associated proteins identified in this study. Blue line indicates B56δ interaction with PPM1G. Red dashed line represent interaction with PP2A complex proteins. (B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB). (C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining. (D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody. (E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody. (F) Schematic representation of B56δ full length (FL), along with its various deletion and point mutants. (G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting. (H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting. (I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.
PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity (2019)
Figure 2

Figure 2: B56δ retains PPM1G in cytoplasm. (A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies. (D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively. (E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively. (F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).
PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity (2019)
Figure 3

Figure 3: α-Catenin is a new cytoplasmic substrate of PPM1G-B56δ holoenzyme. (A) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody. (B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting. (C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (D) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (E) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies. (F) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies. (G) Cells transfected with vector, SFB-PR130 and SFB-B56δ and levels of pS641 α-catenin was detected using western blotting. (H) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody. (I) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody. (J, K) Cells were transduced with shRNAs as indicated and the effect of (J) B56δ or (K) PPM1G expression on α-Catenin phosphorylation was assessed. (L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody. (M) Control shRNA and B56δ shRNA cells were transfected with vector, MYC-PPM1G and MYC-PPM1G ΔNLS in indicated combination and pS641 α-catenin levels were determined using western blotting.
PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity (2019)
Figure 4

Figure 4: PPM1G-B56δ holoenzyme is required for adherens junction assembly. (A) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting. (D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm. (E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).
PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity (2019)
Figure 5

Figure 5: PPM1G-B56δ complex controls cell migration by dephosphorylating α-Catenin (A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (I, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).

Expanded view figures
PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling (2015)
Figure 1

Figure 1: PTPRN2 and PLCβ1 promote breast cancer metastasis.

(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.

(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&E staining of representative lung sections.

(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&E staining of representative lung sections.

(E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.

(G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor’s PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors’ PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.

(H) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor’s PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients’ primary tumors’ PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.

Error bars represent S.E.M.
PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling (2015)
Figure 2

Figure 2: PLCβ1 and PTPRN2 drive metastatic migration and invasion.

(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.

(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.

(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.

(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.

(F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&E staining of representative lung sections.

(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.

(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&E staining of representative lung sections.

Error bars represent S.E.M.
PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling (2015)
Figure 3

Figure 3: PLCβ1 and PTPRN2 regulate membrane PI(4,5)P2 levels.

(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.

(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&E staining of representative lung sections. N=6 mice/group.

(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.

Error bars represent S.E.M.
PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling (2015)
Figure 4

Figure 4: Altered PI(4,5)P2 levels modulate metastatic migration and invasive capacity.

(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.

(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.

(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor’s mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients’ primary tumors’ combined PIP5K expression levels were classified as low (blue) or high (red) expression.

(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.

Error bars represent S.E.M.