Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The primer sequences used for the qRT-PCR analysis

Article Snippet: The ADSCs were plated onto 12-well plates and transduced with either 12 ml of LV particles containing shRNA targeting TWIST1 (sc-38604-V; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or LV particles containing scramble shRNA (sc-108080; Santa Cruz Biotechnology) each well in DMEM added with GlutaMAX plus 5 mg/ml polybrene for 6 h to achieve TWIST1 knockdown.

Techniques: Sequencing

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: Characterization of ADSCs and their differentiation into Schwann cells. A, Phenotypic characterization of ADSCs by flow cytometric analysis. B, The qRT-PCR analysis of TWIST1 mRNA expression levels in ADSCs 2 weeks after LV transduction. C, Immunoblotting analysis of TWIST1 protein expression levels in ADSCs 2 weeks after LV transduction. * P < 0.05 compared to the scramble siRNA by unpaired t tests. D, Representative inverted microscope images of ADSCs under Schwann cell induction for 14 days. Without induction, ADSCs showed a mesh-like structure. After 14 days, cells adopted a spindle shape with reduced volume, fewer protrusions, and a spiral growth pattern, resembling Schwann cells. Notably, the sh-TWIST1 group exhibited more pronounced Schwann cell-like features than the scramble group

Article Snippet: The ADSCs were plated onto 12-well plates and transduced with either 12 ml of LV particles containing shRNA targeting TWIST1 (sc-38604-V; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or LV particles containing scramble shRNA (sc-108080; Santa Cruz Biotechnology) each well in DMEM added with GlutaMAX plus 5 mg/ml polybrene for 6 h to achieve TWIST1 knockdown.

Techniques: Quantitative RT-PCR, Expressing, Transduction, Western Blot, Inverted Microscopy

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The sciatic nerves of rats in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were collected and subjected to histomorphological analysis. A, The sections of rat sciatic nerve stained with toluidine blue; the non-lesioned rats showed homogeneously distributed fibers, uniform myelin sheath, and normal axon diameter; the non-transplanted rats showed clear signs of nerve fiber degeneration with presence of myelin degradation, indicated by white arrows; the LV-scramble-ADSC rats showed some signs of regenerated nerve fibers but presence of degenerated axons and decreased myelin sheath thickness; the LV-sh-TWIST1-ADSC rats showed clear signs of regenerated nerve fibers surrounded by newly formed myelin sheaths. B, Morphometric analysis of axion diameter and myelin sheath thickness. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: The ADSCs were plated onto 12-well plates and transduced with either 12 ml of LV particles containing shRNA targeting TWIST1 (sc-38604-V; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or LV particles containing scramble shRNA (sc-108080; Santa Cruz Biotechnology) each well in DMEM added with GlutaMAX plus 5 mg/ml polybrene for 6 h to achieve TWIST1 knockdown.

Techniques: Transplantation Assay, Staining

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The mRNA expressions of TWIST1, NT-3, BDNF, NGF, and GDNF in the sciatic nerves of rats after sciatic repair in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were determined by the qRT-PCR analysis. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: The ADSCs were plated onto 12-well plates and transduced with either 12 ml of LV particles containing shRNA targeting TWIST1 (sc-38604-V; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or LV particles containing scramble shRNA (sc-108080; Santa Cruz Biotechnology) each well in DMEM added with GlutaMAX plus 5 mg/ml polybrene for 6 h to achieve TWIST1 knockdown.

Techniques: Transplantation Assay, Quantitative RT-PCR

Journal: Human Cell

Article Title: Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery

doi: 10.1007/s13577-024-01087-6

Figure Lengend Snippet: The protein expressions of TWIST1, NT-3, BDNF, NGF, and GDNF in the sciatic nerves of rats after sciatic repair in non-lesioned, non-transplanted, LV-scramble-ADSC, and LV-sh-TWIST1-ADSC groups 8 weeks after transplantation of ADSCs were determined by the immunoblotting analysis. * P < 0.05 compared to the non-transplanted group and # P < 0.05 compared to the LV-scramble-ADSC group by one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: The ADSCs were plated onto 12-well plates and transduced with either 12 ml of LV particles containing shRNA targeting TWIST1 (sc-38604-V; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or LV particles containing scramble shRNA (sc-108080; Santa Cruz Biotechnology) each well in DMEM added with GlutaMAX plus 5 mg/ml polybrene for 6 h to achieve TWIST1 knockdown.

Techniques: Transplantation Assay, Western Blot

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown enhances breast cancer cell sensitivity to cisplatin. ( A ) Validation of DOCK1 knockdown efficiency following the transfection of breast cancer cells with DOCK1 siRNA or negative control, respectively. ( B and C ) Cisplatin sensitivity of breast cancer cells transfected with DOCK1 siRNA or negative control, as measured by a CCK-8 assay. The IC 50 value of the cells was calculated following DOCK1 inhibition. * P < 0.05; ** P < 0.01. ( D ) Cellular proliferation was measured using EdU staining and the EdU-positive cell ratio was calculated. (Scale bar: 100 μm). * P < 0.05; ** P < 0.01. ( E ) A Western blot analysis was performed to evaluate the level of DOCK1 expression in cisplatin-treated breast cancer cells. * P < 0.05. ( F and G ) The level of DOCK1 protein and mRNA expression was detected in breast cancer cells via Western blot and qRT-PCR. ( H ) The IC 50 of cisplatin was positively correlated with the level of DOCK1 mRNA in three breast cells.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Biomarker Discovery, Transfection, Negative Control, CCK-8 Assay, Inhibition, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: The effect of DOCK1 on EMT. ( A and B ) The level of E-cadherin and Vimentin protein expression was detected in BC cells via Western blot. ( C ) DOCK1 was negatively correlated with the level of E-cadherin mRNA and was positively correlated with the level of Vimentin mRNA in BC cells. ( D and E ) Western blot analyses were performed to evaluate the level of DOCK1, Vimentin, and E-cadherin expression in BC cells transfected with DOCK1 siRNA or NC, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Western Blot, Transfection

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown promotes cell sensitivity to cisplatin via Twist. ( A ) Heatmap of EMT-associated genes in response to DOCK1 siRNA. ( B ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with Twist siRNA or in combination with DOCK1 siRNA. ( C and D ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines. ** P < 0.01; *** P < 0.001. ( E ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with the Twist plasmid or in combination with DOCK1 siRNA. ( F and G ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, CCK-8 Assay, Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown enhances breast cancer cell sensitivity to cisplatin. ( A ) Validation of DOCK1 knockdown efficiency following the transfection of breast cancer cells with DOCK1 siRNA or negative control, respectively. ( B and C ) Cisplatin sensitivity of breast cancer cells transfected with DOCK1 siRNA or negative control, as measured by a CCK-8 assay. The IC 50 value of the cells was calculated following DOCK1 inhibition. * P < 0.05; ** P < 0.01. ( D ) Cellular proliferation was measured using EdU staining and the EdU-positive cell ratio was calculated. (Scale bar: 100 μm). * P < 0.05; ** P < 0.01. ( E ) A Western blot analysis was performed to evaluate the level of DOCK1 expression in cisplatin-treated breast cancer cells. * P < 0.05. ( F and G ) The level of DOCK1 protein and mRNA expression was detected in breast cancer cells via Western blot and qRT-PCR. ( H ) The IC 50 of cisplatin was positively correlated with the level of DOCK1 mRNA in three breast cells.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Biomarker Discovery, Transfection, Negative Control, CCK-8 Assay, Inhibition, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: TBOPP can effectively enhance the effect of cisplatin on BC in vivo . ( A ) Representative images of xenograft tumors in BALB/c nude mice after different groups of treatment (Normal group, TBOPP group, Cisplatin group or TBOPP+ Cisplatin group). ( B ) The Growth curves of xenograft tumors in each group was statistically analyzed in 0 days to 14 days. ( C and D ) The Ki-67 expression and cell apoptosis in each group was detected by immunohistochemistry and a TUNEL assay, respectively. * P < 0.05. ( E ) The expression of DOCK1, Twist and Vimentin was measured by qRT-PCR in Normal group, TBOPP group, Cisplatin group or TBOPP+ Cisplatin group. * P < 0.05; ** P < 0.01.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: In Vivo, Expressing, Immunohistochemistry, TUNEL Assay, Quantitative RT-PCR

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: The effect of DOCK1 on EMT. ( A and B ) The level of E-cadherin and Vimentin protein expression was detected in BC cells via Western blot. ( C ) DOCK1 was negatively correlated with the level of E-cadherin mRNA and was positively correlated with the level of Vimentin mRNA in BC cells. ( D and E ) Western blot analyses were performed to evaluate the level of DOCK1, Vimentin, and E-cadherin expression in BC cells transfected with DOCK1 siRNA or NC, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Western Blot, Transfection

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: The effect of DOCK1 knockdown on EMT in the cisplatin-treated breast cancer cells. ( A-D ) A Western blot was used to detect the level of DOCK1, E-cadherin, and vimentin expression. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Western Blot, Expressing

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown promotes cell sensitivity to cisplatin via Twist. ( A ) Heatmap of EMT-associated genes in response to DOCK1 siRNA. ( B ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with Twist siRNA or in combination with DOCK1 siRNA. ( C and D ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines. ** P < 0.01; *** P < 0.001. ( E ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with the Twist plasmid or in combination with DOCK1 siRNA. ( F and G ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, CCK-8 Assay, Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of Twist1 in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 (40 μM, 24 hours) treatment. ( B ) ELISA of CXCL12 secretion in culture media of control and MAOB-OE PrSC cells treated with TWIST1 siRNA or NAC (5 mM, 48 hours) ( n = 3). ( C ) qPCR of CXCL12 in indicated PrSC cells upon NAC treatment (5 mM, 48 hours) or Twist1/ TWIST1 siRNA expression ( n = 3). ( D and E ) Determination of CXCL12 mRNA (D) and 0.7-kb promoter activity (E) in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( F ) Schematic diagrams of WT and mutated CXCL12 E-box/SBE-Luc constructs and determination of their activities in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( G ) Representative proximity ligation assay staining and quantitation of indicated Twist1-Smad interactions by per-nucleus fluorescence intensity in control and MAOB-OE PrSC cells. Smad antibody incubation alone served as negative control. Numbers of nuclei included for comparisons between groups are denoted. Scale bars, 50 μm. ( H ) Co-IP assays of indicated Twist1-Smad interactions in PrSC cells with coexpression of Twist1 and individual Smads. Immunoglobulin G (IgG) was used in IP as negative control. Ten percent input was blotted as positive control. ( I ) ChIP analysis of chromatin from control and MAOB-OE PrSC cells precipitated with anti-Twist1, anti-Smad4, or a control IgG, followed by qPCR probing the E-box/SBE-centric CXCL12 promoter region ( n = 3). ( J ) ChIP analysis of chromatin from PrSC cells precipitated with anti-Smad4 antibody and then reprecipitated with anti-Twist1 or a control IgG (re-ChIP), followed by qPCR probing the E-box/SBE-encompassing CXCL12 promoter sequence ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: Human MAOB siRNA pool (sc-35849), TWIST1 siRNA pool (sc-38604), CXCR4 siRNA pool (sc-35421), CXCR7 siRNA pool (sc-94573), MAOA siRNA pool (sc-35847), FOXO1 siRNA pool (sc-35382), and nontarget control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Construct, Proximity Ligation Assay, Staining, Quantitation Assay, Fluorescence, Incubation, Negative Control, Co-Immunoprecipitation Assay, Positive Control, Sequencing

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 cell proliferation in monoculture and coculture with control and MAOB-OE PrSC cells upon selegiline treatment (10 nM, 72 hours) ( n = 3). ( B ) BLI-based growth curves of Luc-tagged PC-3 tumors grown in the prostates of male NSG mice treated with selegiline at various doses (0.5, 2, and 10 mg/kg) or saline as a vehicle ( n = 5 per group). ( C ) BLI images of mice from each group at the end point. ( D ) Determination of tumor weights ( n = 5). ( E ) Determination of MAOA and MAOB enzymatic activities in mouse liver tissue from each group at the end point ( n = 3). ( F ) Representative images of H&E and IHC staining of tumor-expressed Ki-67, p-Src, and p-JNK and stroma-expressed αSMA and CXCL12 and their quantitation in tumor samples from each group ( n = 5). Scale bars, 100 μm. ( G ) Mouse body weights determined weekly ( n = 5). ( H ) Representative H&E images of mouse liver and kidney tissue from each group. Scale bars, 100 μm. ( I to L ) ELISA of ALT (I), AST (J), BUN (K), and creatinine (L) in mouse sera at the end point ( n = 5). ( M ) Schematic depicting stromal-derived MAOB activation of paracrine CXCL12-CXCR4/Src/JNK signaling through interplay between ROS-dependent Twist1 (via a HIF1α/VEGF-A/AKT/FOXO1 pathway) and TGFβ1/Smads to promote stromal-epithelial interactions for PC growth and progression. Statistical analysis was performed using one-way ANOVA with Tukey’s test in (A), (D) to (F), and (I) to (L) and two-way ANOVA with Tukey’s test in (B) and (G). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: Human MAOB siRNA pool (sc-35849), TWIST1 siRNA pool (sc-38604), CXCR4 siRNA pool (sc-35421), CXCR7 siRNA pool (sc-94573), MAOA siRNA pool (sc-35847), FOXO1 siRNA pool (sc-35382), and nontarget control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Quantitation Assay, Control, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay

Journal: Cell stem cell

Article Title: Skull progenitor cell-driven meningeal lymphatic restoration improves neurocognitive functions in craniosynostosis.

doi: 10.1016/j.stem.2023.09.012

Figure Lengend Snippet: Figure 1. Twist1+/– mice with craniosynostosis exhibit impaired meningeal lymphatics, CSF influx, and ISF efflux (A) Bright-field representative images of mouse head before (top) and after (bottom) uDISCO clearing. Scale bars, 2 mm. The mouse heads are outlined by dashed lines. The orange box indicates the zoomed imaging areas shown in (B). (B) The low-magnification light-sheet microscopy images of a Prox1-eGFP mouse head after clearing. The skulls are outlined by dashed lines, and the orange boxes indicate the zoomed imaging areas shown in the lower panel (Bʹ and B00). Scale bars, 2 and 1 mm (inset). (C) Representative images of meningeal whole-mount staining from Prox1-eGFP and Prox1-eGFP;Twist1+/ mice with antibody against LYVE1 (red). Nuclei were counterstained with DAPI (blue). The lymphatic vessels were outlined with dashed lines. Scale bars, 1 mm and 100 mm (inset). (D) Quantification of area fraction of PROX1+ (left, Prox1-eGFP, n = 5; Prox1-eGFP;Twist1+/, n = 5 mice) and LYVE1+ (right, Prox1-eGFP, n = 4; Prox1- eGFP;Twist1+/, n = 4 mice) lymphatic vessels. (E) Diagram showing that WT or MUT mice were injected (i.c.m.) with OVA-647, and after 2 h, the brain and dCLNs samples were collected for analysis. (F) The representative images of dCLN sections collected from mice injected (i.c.m.) with OVA-647 (green). Sections were counterstained with DAPI (blue). Scale bars, 200 mm. (G) Quantification of OVA-647+ volume fraction of dCLNs (WT, n = 5 and MUT, n = 4 mice). (H) The representative images of brain coronal sections with OVA-647 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 2 and 1 mm (inset). (I) Quantification of OVA-647+ area fraction of brain sections (WT, n = 3 and MUT, n = 3 mice). (J) Diagrams showing that mouse corpus striatum (CPu) was intracranially injected with OVA-647 or Ab42-488, and brain samples were collected after 60 min.

Article Snippet: Images were acquired using Keyence Fluorescence microscope (Keyence, Cat #BZ-X810). e5 Cell Stem Cell 30, 1472–1485.e1–e7, November 2, 2023 Twist1 knockdown in suture progenitor cells (SPCs) using shRNA lentivirus Twist1 knockdown in SPCs was performed using TWIST1 shRNA lentivirus particles according to manufacturer’s protocol (Santa Cruz Biotechnology, Cat #sc-38604-V).

Techniques: Imaging, Microscopy, Staining, Injection

Journal: Cell stem cell

Article Title: Skull progenitor cell-driven meningeal lymphatic restoration improves neurocognitive functions in craniosynostosis.

doi: 10.1016/j.stem.2023.09.012

Figure Lengend Snippet: Figure 2. Meningeal lymphatic defects are restored by SPC implantation in Twist1+/– mice (A) The schematic of Gli1+ SPC implantation on Twist1+/ mouse with bilateral craniosynostosis. (B) 3D reconstructed microCT images of the calvaria of Twist1+/ mice with SPC implantation at one month post-surgery (upper panel) and immunofluorescent imaging of a coronal section of the Gli1+ SPC implantation site (lower panel). White arrows indicate regenerating sutures, and white arrowheads indicate dura. Scale bars, 1 mm (upper) and 50 mm (lower). (C) A schematic of the ICP measurement setup. (D and E) Representative ICP traces and quantification of ICP values (WT, n = 6; MUT, n = 5; REG, n = 5; Ves- [Vessel ablation], n = 6 mice). (F) Representative images of meningeal whole-mount staining with antibody against LYVE1 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 500 mm. (G) Representative images of dCLN sections of mice injected (i.c.m.) with OVA-647 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 100 mm. (H) Quantification of area fraction of LYVE1+ lymphatic vessels (WT, n = 6; MUT, n = 6; REG, n = 6 mice). (I) Quantification of OVA-647+ volume fraction of dCLNs (WT, n = 5; MUT, n = 4; REG, n = 4 mice). (J) Representative images of brain coronal sections collected from mice injected (i.c.m.) with OVA-647 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 2 mm. (K) The representative images of brain coronal sections collected from mice intracranially injected with Ab42-488. Nuclei were counterstained with DAPI (blue). Scale bars, 2 mm. (L) Quantification of OVA-647+ area fraction of brain sections (WT, n = 6; MUT, n = 6; REG, n = 6 mice). (M) Quantification of Ab42-488+ area fraction of brain sections (WT, n = 3; MUT, n = 3; REG, n = 3 mice). Data are mean ± SEM calculated by one-way ANOVA with Tukey post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Images were acquired using Keyence Fluorescence microscope (Keyence, Cat #BZ-X810). e5 Cell Stem Cell 30, 1472–1485.e1–e7, November 2, 2023 Twist1 knockdown in suture progenitor cells (SPCs) using shRNA lentivirus Twist1 knockdown in SPCs was performed using TWIST1 shRNA lentivirus particles according to manufacturer’s protocol (Santa Cruz Biotechnology, Cat #sc-38604-V).

Techniques: Imaging, Staining, Injection

Journal: Cell stem cell

Article Title: Skull progenitor cell-driven meningeal lymphatic restoration improves neurocognitive functions in craniosynostosis.

doi: 10.1016/j.stem.2023.09.012

Figure Lengend Snippet: Figure 3. Meningeal lymphatic vessel ablation blocks SPC implantation-mediated beneficial effects on neurocognitive function in Twist1+/–

Article Snippet: Images were acquired using Keyence Fluorescence microscope (Keyence, Cat #BZ-X810). e5 Cell Stem Cell 30, 1472–1485.e1–e7, November 2, 2023 Twist1 knockdown in suture progenitor cells (SPCs) using shRNA lentivirus Twist1 knockdown in SPCs was performed using TWIST1 shRNA lentivirus particles according to manufacturer’s protocol (Santa Cruz Biotechnology, Cat #sc-38604-V).

Techniques:

Journal: Cell stem cell

Article Title: Skull progenitor cell-driven meningeal lymphatic restoration improves neurocognitive functions in craniosynostosis.

doi: 10.1016/j.stem.2023.09.012

Figure Lengend Snippet: Figure 6. VEGF-C treatment restores meningeal lymphatics, CSF influx, and ISF efflux in Twist1+/– mice with craniosynostosis (A) Representative images of meningeal whole-mount staining with antibodies against VEGF-C (green) and LYVE1 (blue). Gli1+ SPCs (red) were labeled with Gli1- CreERT2;Rosa26-tdTomato with Tamoxifen induction. Scale bars, 50 mm. (B) ELISA measurement of VEGF-C concentrations in meningeal lysates (WT, n = 3; MUT, n = 3). (C) Diagrams showing that WT or MUT mice were injected (i.c.m.) with AAV1-eGFP or AAV1-VEGF-C, and 4 weeks later injected (i.c.m.) with Ab42-488 or OVA- 647. Brain and dCLN samples were collected 2 h after tracer injection. (D) Quantification of the ICP (WTGFP, n = 4; WTVEGF-C, n = 6; MUTGFP, n = 4; MUTVEGF-C, n = 6 mice). (E) Representative images of meningeal whole-mount staining with antibody against LYVE1 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 500 mm. (F) The representative images of dCLN sections of mice injected (i.c.m.) with OVA-647. Sections were counterstained with DAPI. Scale bars, 100 mm. (G) Quantification of area fraction of LYVE1+ lymphatic vessels (WTGFP, n = 6; WTVEGF-C, n = 6; MUTGFP, n = 6; MUTVEGF-C, n = 6 mice). (H) Quantification of OVA-647+ volume fraction of dCLNs (WTGFP, n = 4; WTVEGF-C, n = 4; MUTGFP, n = 4; MUTVEGF-C, n = 4 mice). (I) Representative images of brain coronal sections of a mouse injected (i.c.m.) with OVA-647. Sections were counterstained with DAPI. Scale bars, 2 mm. (J) The representative images of brain coronal sections collected from mice intracranially injected with Ab42-488 after 60 min. Nuclei were counterstained with DAPI (blue). Scale bars, 2 mm. (K) Quantification of OVA-647+ area fraction of brain sections (WTGFP, n = 3; WTVEGF-C, n = 3; MUTGFP, n = 3; MUTVEGF-C, n = 3 mice). (L) Quantification of Ab42-488+ area fraction of brain sections (WTGFP, n = 3; WTVEGF-C, n = 3; MUTGFP, n = 3; MUTVEGF-C, n = 3 mice). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, calculated by one-way ANOVA with Tukey post hoc tests.

Article Snippet: Images were acquired using Keyence Fluorescence microscope (Keyence, Cat #BZ-X810). e5 Cell Stem Cell 30, 1472–1485.e1–e7, November 2, 2023 Twist1 knockdown in suture progenitor cells (SPCs) using shRNA lentivirus Twist1 knockdown in SPCs was performed using TWIST1 shRNA lentivirus particles according to manufacturer’s protocol (Santa Cruz Biotechnology, Cat #sc-38604-V).

Techniques: Staining, Labeling, Enzyme-linked Immunosorbent Assay, Injection

Journal: Cell stem cell

Article Title: Skull progenitor cell-driven meningeal lymphatic restoration improves neurocognitive functions in craniosynostosis.

doi: 10.1016/j.stem.2023.09.012

Figure Lengend Snippet: Figure 7. VEGF-C treatment rescues the neu- rocognitive and motor behavior deficits of Twist1+/– mice with craniosynostosis (A) Representative animal tracks of sociability (upper panels) and social memory (lower panels) in the three-chamber test. (B) Representative animal tracks of novel object test. (C and D) Quantification of the preference index in sociability (C) and social memory (D) of three- chamber test (WTGFP, n = 10; WTVEGF-C, n = 10; MUTGFP, n = 10; MUTVEGF-C, n = 10 mice). (E) Quantification of the preference index in the novel object test (WTGFP, n = 10; WTVEGF-C, n = 10; MUTGFP, n = 10; MUTVEGF-C, n = 10 mice). (F) Schematics of the rotarod test. (G) Rotarod performance scored as time (seconds) on the rotarod (WTGFP, n = 10; WTVEGF-C, n = 10; MUTGFP, n = 10; MUTVEGF-C, n = 10 mice). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, calculated by one-way ANOVA with Tukey post hoc tests.

Article Snippet: Images were acquired using Keyence Fluorescence microscope (Keyence, Cat #BZ-X810). e5 Cell Stem Cell 30, 1472–1485.e1–e7, November 2, 2023 Twist1 knockdown in suture progenitor cells (SPCs) using shRNA lentivirus Twist1 knockdown in SPCs was performed using TWIST1 shRNA lentivirus particles according to manufacturer’s protocol (Santa Cruz Biotechnology, Cat #sc-38604-V).

Techniques: