Journal: OncoTargets and Therapy

Article Title: Analysis of Lymphovascular Infiltration and Tumor-Associated Macrophages in Cervical Cancer Immunoescape

doi: 10.2147/OTT.S468484

Figure Lengend Snippet: Tumor lymphatic infiltration and tumor-associated macrophages (TAMs). ( Ai ) Lymph vessels (D2-40, brown) surrounded tumor cells and M1 TAMs (red, CD68, PGM1) in cervical cancer. ( Aii ) The count of positive M1 TAMs in cervical cancer was obviously lower than that in normal cervical tissue. ( Bi ) Lymph vessels (D2-40, brown) surrounded cervical cancer cells and M1 TAMs (red, CD68, KP1). ( Bii ) The count of positive M1 TAMs in cervical cancer was obviously lower than that in normal cervical tissue. ( Ci ) Lymph vessels (D2-40, brown) surrounded cervical cancer cells and M2 TAMs (red, CD163). ( Cii ) The count of positive M2 TAMs in cervical cancer was obviously higher than in normal cervical tissue. ( Di and Ei ) Lymph vessels (D2-40, brown) surrounded tumor cells and M2 TAMs (red, CD206) in cervical cancer tissue. ( Dii ) The count of positive M2 TAMs in cervical cancer was obviously higher than that in normal cervical tissue. ( Eii-1 ) Automatic blood cell analysis of leukocyte classification. ( Eii-2 ) Ratio of M2:M1 TAMs calculated according to double IHC staining. Count of M2 TAMs based on number of monocytes by linear regression analysis. ( Eiii-1, iii-2, iii-3, and iii-4 ) Flow cytometry: ( Eiii-1 ) scatterplot of leukocyte classification; ( Eiii-2 ) scatterplot of monocytes of classical (CD14) and intermediate (CD16) subtypes; ( Eiii-3 ) scatterplot and intensity map of M1 TAMs (CD86); ( Eiii-4 ) scatterplot and intensity map of M2 TAMs (CD206). ( Eiv-1 ) Correlation of tumor metastases with number of M2 TAMs in cervical cancer. ( Eiv-2 ) Correlation of tumor metastases with M2:M1 TAM ratio, indicating cervical cancer lymphatic infiltration accompanied by increase in M2 TAMs. ( Fi ) Protein expression for MMP2, SPARC, and GNLY in cervical cancer LVI-positive group was obviously higher than that of negative group (Western blot analysis). ( Fii-1 ) OS of patients was obviously higher in lower MMP2-expression group than in higher MMP2-expression group. ( F ii -2 ) Relapse-free survival (RFS) was obviously higher in lower MMP2-expression group than higher MMP2-expression group.

Article Snippet: The primary antibodies used in this study were MMP2 (rabbit anti-human 1:200, Abcam), SPARC (rabbit anti-human 1:200, Santa Cruz), and GNLY (mouse anti-human 1:100, Invitrogen).

Techniques: Cell Analysis, Immunohistochemistry, Flow Cytometry, Expressing, Western Blot

Journal: OncoTargets and Therapy

Article Title: Analysis of Lymphovascular Infiltration and Tumor-Associated Macrophages in Cervical Cancer Immunoescape

doi: 10.2147/OTT.S468484

Figure Lengend Snippet: Pseudotrajectory construction. ( A ) T-SNE plots of tumor-associated macrophages (TAMs) and other cell subtypes. ( B ) Scatterplots of M2 TAM markers and the related genes CD14 , CCL5 , MMP2 , SPARC , GNLY , FOSL2 , MAF , and CTLA4 that expressed higher levels in cell clusters. ( Ci ) Analyzed by BEAM function, branched heatmap of differential gene expression accompanied by pseudotime changes. ( Cii ) Dendrogram of the trajectory of main cell clusters in cervical cancer. ( Di ) Pseudotime trajectory analysis of TAM clusters in two-dimensional state. The TAM-related genes MMP2 , SPARC , GNLY , FOSL2 , MAF , and CTLA4 expressed higher levels among cell clusters. ( Dii ) Pseudotime trajectory of the main cell subtypes and state.

Article Snippet: The primary antibodies used in this study were MMP2 (rabbit anti-human 1:200, Abcam), SPARC (rabbit anti-human 1:200, Santa Cruz), and GNLY (mouse anti-human 1:100, Invitrogen).

Techniques: Gene Expression

Journal: OncoTargets and Therapy

Article Title: Analysis of Lymphovascular Infiltration and Tumor-Associated Macrophages in Cervical Cancer Immunoescape

doi: 10.2147/OTT.S468484

Figure Lengend Snippet: Differentially expressed genes (DEGs) and gene set enrichment analysis (GSEA). ( Ai ) Cell-density plot showing higher density of M2 clusters (0.65) than M1 clusters (0.35). ( Aii ) Scatter- and violin plots showing that MMP2, SPARC, and GNLY expressed significantly higher levels in M2 clusters than in M1. M1 (CD68)/M2 (CCL5) clusters ratio decreased significantly. ( Aiii ) Violin plots showing M2 clusters possessed lower immunosurveillance and higher escape scores than M1. ( Aiv ) Histogram of gene-expression rankings for each cell. # Quantile and the number of genes were detected in each cell. Total number of cells and genes were 4879 and 7193, respectively. Quantile and number of genes (minimum were min(201) were 1% (243), 5% (310), 10% (339), 50% (339), and 100% (2499), respectively. ( B ) ( Bi, Genes ) Differentially expressed genes exhibited with heatmap. ( Bi,GO ) GO analysis were exhibited with heatmap. ( Bi,KEGG ) KEGG analysis exhibited with heatmap. ( Bii ) Sketch map showing M2 TAM activity increased gradually from precancerous lesions to cervical cancer. The M2 TAM–related gene MMP2 destroyed the tumor vasculature basement membrane, and SPARC antiadhesion played a key role in epithelial–mesenchymal transition and tumor invasion. M2 TAMs activated cytotoxic T cells to release GNLY, which induced the apoptosis of target cells through the formation of pores on the target-cell membrane and assisted tumor lymphovascular infiltration. ( Biii ) Adjacent gene relationships of MMP2 , SPARC , and GNLY analyzed with the protein–protein interaction (PPI) function at https://string-db.org/ .

Article Snippet: The primary antibodies used in this study were MMP2 (rabbit anti-human 1:200, Abcam), SPARC (rabbit anti-human 1:200, Santa Cruz), and GNLY (mouse anti-human 1:100, Invitrogen).

Techniques: Gene Expression, Activity Assay, Membrane

Journal: Science Advances

Article Title: 2′3′-cGAMP interactome identifies 2′3′-cGAMP/Rab18/FosB signaling in cell migration control independent of innate immunity

doi: 10.1126/sciadv.ado7024

Figure Lengend Snippet: Key reagents used in this study. ATCC, American Type Culture Collection.

Article Snippet: Anti-SPARC antibody , Santa Cruz Biotechnology , Catalog no. sc-398419.

Techniques: Reverse Transcription, SYBR Green Assay, Protease Inhibitor

Journal: ACS Applied Materials & Interfaces

Article Title: Hybrid Micro-/Nanoprotein Platform Provides Endocrine-like and Extracellular Matrix-like Cell Delivery of Growth Factors

doi: 10.1021/acsami.4c01210

Figure Lengend Snippet: MSCs differentiation in cultures over artificial hFGF2-H6 SGs PEA–FN surfaces. (A) Imaging of MSCs by fluorescence microscopy in the presence of artificial hFGF2-H6 SGs at 50 ng/mL for 14 days. Actin, vinculin, vimentin, and YAP were selected as cell markers. Merge refers to actin and vinculin combined fluorescence signals. White squares highlight cell focal adhesions. Close-up pictures of focal adhesions are displayed on the right panels. (B) Fold change on mRNA content (meaning RUNX2, OSN; osteonectin, and OPN; osteopontin gene expression) in MSCs upon incubation with soluble hFGF2-H6 (dark blue), artificial hFGF2-H6 SGs (pale blue), and free Zn 2+ (gray) at 50 ng/mL for 14 days. (C) In-cell Western (ICW) immunodetection of RUNX2, OSN, and OSP proteins in MSCs extracts upon incubation over soluble hFGF2-H6, artificial hFGF2-H6 SGs, and free Zn 2+ (gray) at 50 ng/mL for 14 days. The protein signal is displayed in green, and the cell signal is in red. (D) Statistical analysis of protein signal (green from panel C) expressed as fluorescence per cell and cm 2 in absorbance units). (E) Statistical analysis of cell signal (red from panel C) expressed as fluorescence per cm 2 in absorbance units (au). Peak numbers correspond to the increased percentage of cell growth comparing artificial hFGF2-H6 SGs (pale blue) with soluble hFGF2-H6 (dark blue), free Zn 2+ (gray), and control MSCs (black). Data are expressed as mean ± SEM, and statistical significance is achieved when p < 0.05 is represented as (*). Control refers to MSCs seeded on top of FN-PEA surfaces.

Article Snippet: Cells were incubated with monoclonal primary Abs (1:200) in blocking buffer (PBS/1% milk protein) at room temperature for 2.5 h, respectively; Runx2 (Santa Cruz Biotechnology, C1319), osteonectin (Santa Cruz Biotechnology, SC398419), and osteopontin (Santa Cruz Biotechnology, B1218).

Techniques: Imaging, Fluorescence, Microscopy, Gene Expression, Incubation, In-Cell ELISA, Immunodetection, Control

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Co-culture of desmin-overexpressing EBs ( des +/+ D ect ) with wild-type ( des +/+ ), or desmin-null ( des -/- ) EBs on permeable tissue culture sieve inserts promoted cardiomyogenesis in wild-type ( des +/+ ) and desmin null ( des -/- ) EBs. The onset of cardiomyogenesis in EBs was monitored from day 6 to day 10 and the percentage of EBs with rhythmically contracting cardiomyocytes was determined. B , Desmin promotes SPARC expression in ESCs. Quantification of SPARC mRNA in ESCs of different genotypes by RT-sqPCR analysis. Number of experiments, n=3. GAPDH was used as reference gene. Error bars, standard deviation. C , Desmin promotes the secretion of SPARC from EBs. The concentration of secreted SPARC was calculated from dot blot analysis of media, used to culture EBs, with anti-SPARC antibodies and known concentrations of recombinant SPARC n=3. D ; Sequestration of SPARC in the media with neutralizing antibodies attenuates the positive paracrine effect of des +/+ D ect EBs on cardiomyogenesis in des +/+ and des -/- EBs, respectively. EBs of the indicated genotypes (observed EBs) were cultured under sieve inserts with EBs used to secrete desmin-induced paracrine factors promoting cardiomyogenesis (secreting EBs), anti-SPARC antibodies were added from day 4.8 to day 7 of the in vitro differentiation experiment, and cardiomyogenesis was quantified between days 7 and 13 and normalized to the values obtained from des +/+ EBs. E , Paracrine SPARC increases Smad2 phosphorylation in des -/- EBs, within 2 hours. Quantification of pSmad2 relative to total protein by Western blots. n=2. F , Promotion of cardiomyogenesis in des -/- EBs by SPARC is mediated by the TGF-ß receptor ALK5. ALK5 activity was inhibited by 3.5μmol/l SB431542in the presence or absence of 3mg/l SPARC for 48 hours. Cardiomyogenesis was normalized to that of des -/- EBs. G , Attenuation of nuclear import of pSmad2 by AcSDKP in des +/+ EBs for 3 days partially suppressed cardiomyogenesis. ( A , D , F and G ), n=2; N (EBs)=20 per measurement; error bars, standard deviation; *, Student′s t-test p values p<0.05; **, p<0.01; n.s., not significant.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against SPARC (#sc-25574, Santa Cruz, 1:500) for 60 minutes.

Techniques: Co-Culture Assay, Expressing, Standard Deviation, Concentration Assay, Dot Blot, Recombinant, Cell Culture, In Vitro, Phospho-proteomics, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Localization of SPARC on the surface of CSCs. Confocal immunofluorescence images of CSCs incubated with anti-SPARC antibodies (green) and DAPI-stained DNA (blue). B , RT-qPCR analysis of SPARC mRNA expression in CSCs compared to ESCs relative to RPL32 expression when cultured for 24 hours without feeder cells. n=5; error bars, standard deviation; **, Student′s t-test p value p=0.01. C , SPARC protein levels in CSCs and ESCs determined by quantitative Western blotting. N=2; p<0.001. D , Immunofluorescence images of mouse heart sections incubated with anti-SPARC antibodies (red), DAPI (blue) and anti-Sca1 (green) antibodies; Arrowheads in merged images, SPARC-positive cells at the edge of muscle fibers. E , Expression of SPARC-mCherry fusion protein in self-renewing CSCs. Fluorescence images showing expression of SPARC-mCherry (red), DAPI-stained DNA (blue). Representative images from 2 out of 10 transgenic cell lines showing cytoplasmic and nuclear localization of SPARC-mCherry protein (arrowheads). F , Perinuclear and nuclear localization of SPARC-mCherry in self-renewing CSCs. G , Cytoplasmic, vesicular and nuclear localization of SPARC-mCherry in differentiated CSC progeny cells. H , SPARC-mCherry fluorescence in media conditioned by SPARC-mCherry expressing CBs and wild-type CBs (control). Data are from 10 measurements. n=3; error bars, standard deviation; p=0.0001. I , SPARC-mCherry secreted by CBs but not mCherry, also secreted by CBs, binds to an ECM produced by wild-type CBs. Control, medium conditioned with wild-type CBs. Data are from n=3 independent experiments (SPARC-mCherry and control) and n=2 experiments (mCherry); error bars, standard deviation; p<10 -8 . Note, control levels in H and I represent autofluorescence of the medium and CSC derived ECM, respectively. J , Rate of SPARC-mCherry fusion protein uptake into differentiating CSCs, perinuclear vesicles and nuclei, respectively, between 0 and 24 hours and 24 and 72 hours, respectively, after initiation of differentiation by withdrawal of LIF. Data are from 4 measurements each. n=2; error bars, standard deviation; *, Student′s t-test p value p<0.05.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against SPARC (#sc-25574, Santa Cruz, 1:500) for 60 minutes.

Techniques: Immunofluorescence, Incubation, Staining, Quantitative RT-PCR, Expressing, Cell Culture, Standard Deviation, Western Blot, Fluorescence, Transgenic Assay, Control, Produced, Derivative Assay

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Expression of SPARC and B , nanog mRNA in developing A5 wild-type CSC-derived CBs and W4 wild-type ESC-derived EBs after withdrawal of feeder cells and aggregation at d0. RT-qPCR analysis using primer pairs for SPARC and nanog and RPL32, as internal reference. Data from 3 biological replicates and 2 technical replicates each; number of experiments n=3. Error bars, standard deviation; Student′s t-test p values for all SPARC and the nanog day 7 data, p<0.05. C , Inhibition of extracellular autocrine SPARC negatively affects cardiomyogenesis in A5 wild-type CBs between days 0 and 7 before cardiomyocytes start to contract at day 11. Anti-SPARC antibodies were added to CBs for time intervals as indicated on the x-axis. Cardiomyogenesis was assessed by counting the number of beating clusters of cardiomyocytes per CB between days 13 and 20. Data are normalized to control without anti-SPARC antibodies. Error bars, standard deviation; Student′s t-test p values for day 0-5 and 5-7 data, p<0.05. D , Surplus paracrine SPARC accelerates cardiomyogenesis between days 10 and 14 in A5 CBs, whereas neutralizing antibodies against SPARC significantly delay cardiomyogenesis. Time course of myocardial differentiation in CBs. Data represent the percentage of CBs with at least one rhythmically contracting cluster of cardiomyocytes. Paracrine recombinant SPARC (2mg/l) (pSPARC) and neutralizing amounts of anti-SPARC antibodies, respectively, were added between days 4.8 and 7. E , Paracrine SPARC increases the number of cardiomyocyte clusters per CB, and inhibition of SPARC by neutralizing antibodies reduces the number of cardiomyocyte clusters. SPARC was added to CBs between days 4.8 and 7 and the number of cardiomyocyte clusters in CBs was determined between days 9 and 13. ( D and E ) n=2; 3 biological replicates each. Error bars, standard deviation; *, p<0.05. F , Overexpression of SPARC increases the onset of cardiomyogenesis and the percentage of CBs with contracting cardiomyocytes, whereas mono-allelic expression delayed and attenuated cardiomyogenesis in CBs. Development of contracting cardiomyocytes in CBs over time. sparc +/+ , wild-type CSCs; sparc +/+ S ect , SPARC-overexpressing CSCs (mean of 2 cell lines A5K4 and A5K10); sparc +/- , heterozygous knockout cell lines (mean of 2 cell lines A5K11 and A5K17). Data from each cell line from 3 technical replicates; n=2; N (CBs)=60 each. G , Overexpression of SPARC-mCherry fusion protein increases the onset of cardiomyogenesis, the number of contracting cardiomyocytes, and the longevity of contracting cardiomyocytes compared to mCherry-expressing CBs. Development of contracting cardiomyocytes in CBs over time. sparc +/+ S:mC ect , SPARC-mCherry-expressing CBs; sparc +/+ mC ect mCherry-expressing CSCs. Data from 3 technical replicates; n=2; N (CBs)=60 each. H , Reducing the time intervals for monitoring the onset of myocardial contraction in CBs between day 10 and day 13 and the density of CBs on a plate demonstrates a significant acceleration of cardiomyogenesis by SPARC-mCherry fusion protein. Other experimental conditions as in 3G. I , Expression of SPARC mRNA in A5 CSCs and He2 CSCs. RT-qPCR analysis with primer pairs for SPARC, nanog, and sdha, the latter two as internal controls. Data from 3 biological replicates with each 2 technical replicates. Error bars, standard deviation; *, p<0.05. J , Mono-allelic expression of SPARC ( sparc +/- ) in He2K2 CSC-derived CBs with a Balb/c background delays and reduces cardiomyogenesis more severely than in A5 wild-type CBs. Experimental conditions as in 3G. Note that the onset of rhythmic contraction of cardiomyocytes in He2 CBs with a Balb/c background is delayed by 1 day and by 2 days in He2K2 compared to A5 CBs. K , Paracrine addition of secreted SPARC-mCherry fusion protein to wild-type He2 CBs in co-cultures with A5 sparc +/+ S:mC ect CBs (He2 + paracrine SmC) also increases the rate of cardiomyogenesis, affects the onset of cardiomyogenesis, and increases the longevity of rhythmically contracting cardiomyocytes compared to He2 CBs co-cultured with A5 sparc +/+ mC ect control CBs (He2 + paracrine mC). Experimental conditions as in 3G. L , Mean number of contracting cardiomyocyte clusters per CB monitored from day 13 to day 26. Data are from experiments shown in 3C – 3H . Error bars, standard deviation; ***, p<0.001; *, p<0.05. M , Longevity of rhythmically contracting cardiomyocytes in CBs ectopically expressing SPARC and with mono-allelic expression of SPARC compared to wild-type cardiac bodies. Data are from n=3 or more experiments ( 3C - 3H ) and n=2 (3J and 3K) independent experiments with 3 technical replicates each. N , Addition of 3mg/l paracrine recombinant SPARC from day 4.8 to 7 rescues cardiomyogenesis in sparc +/- CBs and, O , increases the number of clusters with contracting cardiomyocytes. Data are from 3 technical replicates; n=2; N (CBs)=60 each. Mean number of individual contracting cardiomyocyte clusters per CB monitored from day 16 to day 19. Error bars, standard deviation; *, Student′s t-test p value p<0.05.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against SPARC (#sc-25574, Santa Cruz, 1:500) for 60 minutes.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Standard Deviation, Inhibition, Control, Recombinant, Over Expression, Knock-Out, Cell Culture

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Co-culture of desmin-overexpressing EBs ( des +/+ D ect ) with wild-type ( des +/+ ), or desmin-null ( des -/- ) EBs on permeable tissue culture sieve inserts promoted cardiomyogenesis in wild-type ( des +/+ ) and desmin null ( des -/- ) EBs. The onset of cardiomyogenesis in EBs was monitored from day 6 to day 10 and the percentage of EBs with rhythmically contracting cardiomyocytes was determined. B , Desmin promotes SPARC expression in ESCs. Quantification of SPARC mRNA in ESCs of different genotypes by RT-sqPCR analysis. Number of experiments, n=3. GAPDH was used as reference gene. Error bars, standard deviation. C , Desmin promotes the secretion of SPARC from EBs. The concentration of secreted SPARC was calculated from dot blot analysis of media, used to culture EBs, with anti-SPARC antibodies and known concentrations of recombinant SPARC n=3. D ; Sequestration of SPARC in the media with neutralizing antibodies attenuates the positive paracrine effect of des +/+ D ect EBs on cardiomyogenesis in des +/+ and des -/- EBs, respectively. EBs of the indicated genotypes (observed EBs) were cultured under sieve inserts with EBs used to secrete desmin-induced paracrine factors promoting cardiomyogenesis (secreting EBs), anti-SPARC antibodies were added from day 4.8 to day 7 of the in vitro differentiation experiment, and cardiomyogenesis was quantified between days 7 and 13 and normalized to the values obtained from des +/+ EBs. E , Paracrine SPARC increases Smad2 phosphorylation in des -/- EBs, within 2 hours. Quantification of pSmad2 relative to total protein by Western blots. n=2. F , Promotion of cardiomyogenesis in des -/- EBs by SPARC is mediated by the TGF-ß receptor ALK5. ALK5 activity was inhibited by 3.5μmol/l SB431542in the presence or absence of 3mg/l SPARC for 48 hours. Cardiomyogenesis was normalized to that of des -/- EBs. G , Attenuation of nuclear import of pSmad2 by AcSDKP in des +/+ EBs for 3 days partially suppressed cardiomyogenesis. ( A , D , F and G ), n=2; N (EBs)=20 per measurement; error bars, standard deviation; *, Student′s t-test p values p<0.05; **, p<0.01; n.s., not significant.

Article Snippet: EBs and CBs were cultured in the presence of either 3mg/l recombinant SPARC (S5174-25UG, Sigma-Aldrich; 942-SP-050, R&D Systems), various amounts of anti-SPARC antibodies (Santa Cruz, (H-90): sc-25574 Sigma, for EBs; PA5-89864, Thermo Fischer Scientific, for CBs), conditioned media from STO cells, various CSC lines, EBs or CBs (see EB and CB co-cultures and media conditioning), 3.5μmol/l SB431242 (S4317, Sigma-Aldrich), or AcSDKP (350461, Abbiotec) for different time periods, as indicated in the figure legends.

Techniques: Co-Culture Assay, Expressing, Standard Deviation, Concentration Assay, Dot Blot, Recombinant, Cell Culture, In Vitro, Phospho-proteomics, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Localization of SPARC on the surface of CSCs. Confocal immunofluorescence images of CSCs incubated with anti-SPARC antibodies (green) and DAPI-stained DNA (blue). B , RT-qPCR analysis of SPARC mRNA expression in CSCs compared to ESCs relative to RPL32 expression when cultured for 24 hours without feeder cells. n=5; error bars, standard deviation; **, Student′s t-test p value p=0.01. C , SPARC protein levels in CSCs and ESCs determined by quantitative Western blotting. N=2; p<0.001. D , Immunofluorescence images of mouse heart sections incubated with anti-SPARC antibodies (red), DAPI (blue) and anti-Sca1 (green) antibodies; Arrowheads in merged images, SPARC-positive cells at the edge of muscle fibers. E , Expression of SPARC-mCherry fusion protein in self-renewing CSCs. Fluorescence images showing expression of SPARC-mCherry (red), DAPI-stained DNA (blue). Representative images from 2 out of 10 transgenic cell lines showing cytoplasmic and nuclear localization of SPARC-mCherry protein (arrowheads). F , Perinuclear and nuclear localization of SPARC-mCherry in self-renewing CSCs. G , Cytoplasmic, vesicular and nuclear localization of SPARC-mCherry in differentiated CSC progeny cells. H , SPARC-mCherry fluorescence in media conditioned by SPARC-mCherry expressing CBs and wild-type CBs (control). Data are from 10 measurements. n=3; error bars, standard deviation; p=0.0001. I , SPARC-mCherry secreted by CBs but not mCherry, also secreted by CBs, binds to an ECM produced by wild-type CBs. Control, medium conditioned with wild-type CBs. Data are from n=3 independent experiments (SPARC-mCherry and control) and n=2 experiments (mCherry); error bars, standard deviation; p<10 -8 . Note, control levels in H and I represent autofluorescence of the medium and CSC derived ECM, respectively. J , Rate of SPARC-mCherry fusion protein uptake into differentiating CSCs, perinuclear vesicles and nuclei, respectively, between 0 and 24 hours and 24 and 72 hours, respectively, after initiation of differentiation by withdrawal of LIF. Data are from 4 measurements each. n=2; error bars, standard deviation; *, Student′s t-test p value p<0.05.

Article Snippet: EBs and CBs were cultured in the presence of either 3mg/l recombinant SPARC (S5174-25UG, Sigma-Aldrich; 942-SP-050, R&D Systems), various amounts of anti-SPARC antibodies (Santa Cruz, (H-90): sc-25574 Sigma, for EBs; PA5-89864, Thermo Fischer Scientific, for CBs), conditioned media from STO cells, various CSC lines, EBs or CBs (see EB and CB co-cultures and media conditioning), 3.5μmol/l SB431242 (S4317, Sigma-Aldrich), or AcSDKP (350461, Abbiotec) for different time periods, as indicated in the figure legends.

Techniques: Immunofluorescence, Incubation, Staining, Quantitative RT-PCR, Expressing, Cell Culture, Standard Deviation, Western Blot, Fluorescence, Transgenic Assay, Control, Produced, Derivative Assay

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A , Expression of SPARC and B , nanog mRNA in developing A5 wild-type CSC-derived CBs and W4 wild-type ESC-derived EBs after withdrawal of feeder cells and aggregation at d0. RT-qPCR analysis using primer pairs for SPARC and nanog and RPL32, as internal reference. Data from 3 biological replicates and 2 technical replicates each; number of experiments n=3. Error bars, standard deviation; Student′s t-test p values for all SPARC and the nanog day 7 data, p<0.05. C , Inhibition of extracellular autocrine SPARC negatively affects cardiomyogenesis in A5 wild-type CBs between days 0 and 7 before cardiomyocytes start to contract at day 11. Anti-SPARC antibodies were added to CBs for time intervals as indicated on the x-axis. Cardiomyogenesis was assessed by counting the number of beating clusters of cardiomyocytes per CB between days 13 and 20. Data are normalized to control without anti-SPARC antibodies. Error bars, standard deviation; Student′s t-test p values for day 0-5 and 5-7 data, p<0.05. D , Surplus paracrine SPARC accelerates cardiomyogenesis between days 10 and 14 in A5 CBs, whereas neutralizing antibodies against SPARC significantly delay cardiomyogenesis. Time course of myocardial differentiation in CBs. Data represent the percentage of CBs with at least one rhythmically contracting cluster of cardiomyocytes. Paracrine recombinant SPARC (2mg/l) (pSPARC) and neutralizing amounts of anti-SPARC antibodies, respectively, were added between days 4.8 and 7. E , Paracrine SPARC increases the number of cardiomyocyte clusters per CB, and inhibition of SPARC by neutralizing antibodies reduces the number of cardiomyocyte clusters. SPARC was added to CBs between days 4.8 and 7 and the number of cardiomyocyte clusters in CBs was determined between days 9 and 13. ( D and E ) n=2; 3 biological replicates each. Error bars, standard deviation; *, p<0.05. F , Overexpression of SPARC increases the onset of cardiomyogenesis and the percentage of CBs with contracting cardiomyocytes, whereas mono-allelic expression delayed and attenuated cardiomyogenesis in CBs. Development of contracting cardiomyocytes in CBs over time. sparc +/+ , wild-type CSCs; sparc +/+ S ect , SPARC-overexpressing CSCs (mean of 2 cell lines A5K4 and A5K10); sparc +/- , heterozygous knockout cell lines (mean of 2 cell lines A5K11 and A5K17). Data from each cell line from 3 technical replicates; n=2; N (CBs)=60 each. G , Overexpression of SPARC-mCherry fusion protein increases the onset of cardiomyogenesis, the number of contracting cardiomyocytes, and the longevity of contracting cardiomyocytes compared to mCherry-expressing CBs. Development of contracting cardiomyocytes in CBs over time. sparc +/+ S:mC ect , SPARC-mCherry-expressing CBs; sparc +/+ mC ect mCherry-expressing CSCs. Data from 3 technical replicates; n=2; N (CBs)=60 each. H , Reducing the time intervals for monitoring the onset of myocardial contraction in CBs between day 10 and day 13 and the density of CBs on a plate demonstrates a significant acceleration of cardiomyogenesis by SPARC-mCherry fusion protein. Other experimental conditions as in 3G. I , Expression of SPARC mRNA in A5 CSCs and He2 CSCs. RT-qPCR analysis with primer pairs for SPARC, nanog, and sdha, the latter two as internal controls. Data from 3 biological replicates with each 2 technical replicates. Error bars, standard deviation; *, p<0.05. J , Mono-allelic expression of SPARC ( sparc +/- ) in He2K2 CSC-derived CBs with a Balb/c background delays and reduces cardiomyogenesis more severely than in A5 wild-type CBs. Experimental conditions as in 3G. Note that the onset of rhythmic contraction of cardiomyocytes in He2 CBs with a Balb/c background is delayed by 1 day and by 2 days in He2K2 compared to A5 CBs. K , Paracrine addition of secreted SPARC-mCherry fusion protein to wild-type He2 CBs in co-cultures with A5 sparc +/+ S:mC ect CBs (He2 + paracrine SmC) also increases the rate of cardiomyogenesis, affects the onset of cardiomyogenesis, and increases the longevity of rhythmically contracting cardiomyocytes compared to He2 CBs co-cultured with A5 sparc +/+ mC ect control CBs (He2 + paracrine mC). Experimental conditions as in 3G. L , Mean number of contracting cardiomyocyte clusters per CB monitored from day 13 to day 26. Data are from experiments shown in 3C – 3H . Error bars, standard deviation; ***, p<0.001; *, p<0.05. M , Longevity of rhythmically contracting cardiomyocytes in CBs ectopically expressing SPARC and with mono-allelic expression of SPARC compared to wild-type cardiac bodies. Data are from n=3 or more experiments ( 3C - 3H ) and n=2 (3J and 3K) independent experiments with 3 technical replicates each. N , Addition of 3mg/l paracrine recombinant SPARC from day 4.8 to 7 rescues cardiomyogenesis in sparc +/- CBs and, O , increases the number of clusters with contracting cardiomyocytes. Data are from 3 technical replicates; n=2; N (CBs)=60 each. Mean number of individual contracting cardiomyocyte clusters per CB monitored from day 16 to day 19. Error bars, standard deviation; *, Student′s t-test p value p<0.05.

Article Snippet: EBs and CBs were cultured in the presence of either 3mg/l recombinant SPARC (S5174-25UG, Sigma-Aldrich; 942-SP-050, R&D Systems), various amounts of anti-SPARC antibodies (Santa Cruz, (H-90): sc-25574 Sigma, for EBs; PA5-89864, Thermo Fischer Scientific, for CBs), conditioned media from STO cells, various CSC lines, EBs or CBs (see EB and CB co-cultures and media conditioning), 3.5μmol/l SB431242 (S4317, Sigma-Aldrich), or AcSDKP (350461, Abbiotec) for different time periods, as indicated in the figure legends.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Standard Deviation, Inhibition, Control, Recombinant, Over Expression, Knock-Out, Cell Culture

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: A and B , RT-qPCR analysis of mRNA expression of indicated genes in CSCs in the absence of LIF and 3mg/l recombinant SPARC. n=2, 2 biological and 2 technical replicates each. mRNA levels were normalized to RPL32 mRNA. C , Fluorescence activated cell sorting analysis of the percentage of eGFP-positive CSCs expressing a brachyury , mesp1 , nkx2.5 , or a desmin eGFP reporter gene in the presence of conditioned medium from STO and 3T3 fibroblasts (control) or STO and 3T3 fibroblasts secreting SPARC from an expression cassette, after 3 hours of incubation. Box plots show the median and the second and third quartiles; whiskers indicate maximum and minimum values, respectively. n=3; D , Expression of an nxk2.5::eGFP knock-in allele in CSC after incubation with medium from SPARC-secreting STO cells for 3 and 24 hours. Whiskers blot as in B. E , Expression of an nkx2.5::luciferase-reporter gene in transiently transfected, CSC-derived, differentiated cardiac cells from 9-day-old CBs. Fluorescence measurements were performed 24 hours after transfection plus 4 or 24 additional hours of incubation with medium from SPARC-secreting STO cells. n=3; error bars, standard deviation; *, all Student′s t-test p values p<0.05.

Article Snippet: EBs and CBs were cultured in the presence of either 3mg/l recombinant SPARC (S5174-25UG, Sigma-Aldrich; 942-SP-050, R&D Systems), various amounts of anti-SPARC antibodies (Santa Cruz, (H-90): sc-25574 Sigma, for EBs; PA5-89864, Thermo Fischer Scientific, for CBs), conditioned media from STO cells, various CSC lines, EBs or CBs (see EB and CB co-cultures and media conditioning), 3.5μmol/l SB431242 (S4317, Sigma-Aldrich), or AcSDKP (350461, Abbiotec) for different time periods, as indicated in the figure legends.

Techniques: Quantitative RT-PCR, Expressing, Recombinant, Fluorescence, FACS, Control, Incubation, Knock-In, Luciferase, Transfection, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

doi: 10.1101/2024.03.28.587296

Figure Lengend Snippet: SPARC and desmin mRNA expression levels and cytoplasmic protein concentrations were quantified in CSC lines with mono-allelic expression of either desmin or SPARC, and in CSC lines ectopically expressing desmin or SPARC in addition to the bi-allelic expression. mRNA and protein quantities were compared to the parental wild-type sparc +/+ des +/+ CSC line. mRNA levels were normalized to RPL32 mRNA levels and protein levels were normalized to total protein found in the same number of cells of each CSC line. A , SPARC mRNA levels and B , SPARC protein levels in 2 sparc +/- and 2 sparc +/+ S ect CSC lines. C , Desmin mRNA levels and D , desmin protein levels in the same sparc +/- and sparc +/+ S ect CSC lines. E , Desmin mRNA levels and F , Desmin protein levels in each 3 des +/- and 2 des +/+ D ect CSC lines, respectively. G , SPARC mRNA levels and H , SPARC protein levels in the same des +/- and des +/+ D ect CSC lines. Number of independent experiments, n≥3; all Student′s t-test p values p≤0.001. I , Quantification of SPARC protein secreted into the cell culture supernatant of sparc and desmin mutant CSC lines, respectively. Protein concentration in the culture medium was normalized to that of total protein found in CSC cultures with the same number of cells after 24 hours of culture. n≥3; all Student′s t-test p values p<0.05. J , SPARC mRNA expression in wild-type, sparc +/- and des +/- CSCs 3 hours after addition of 3mg/l SPARC and in CBs 3 days after SPARC addition. K , SPARC protein levels (arbitrary units) in CSCs after addition of SPARC for 3 hours; conditions as in J. Number of independent experiments, n=2; with 2 technical replicates each; all Student′s t-test p values p≤0.003.

Article Snippet: EBs and CBs were cultured in the presence of either 3mg/l recombinant SPARC (S5174-25UG, Sigma-Aldrich; 942-SP-050, R&D Systems), various amounts of anti-SPARC antibodies (Santa Cruz, (H-90): sc-25574 Sigma, for EBs; PA5-89864, Thermo Fischer Scientific, for CBs), conditioned media from STO cells, various CSC lines, EBs or CBs (see EB and CB co-cultures and media conditioning), 3.5μmol/l SB431242 (S4317, Sigma-Aldrich), or AcSDKP (350461, Abbiotec) for different time periods, as indicated in the figure legends.

Techniques: Expressing, Cell Culture, Mutagenesis, Protein Concentration