EMBO/soda-nel huggingface dataset:
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 4

Figure 4 - GRK2-/- cells did not respond to Hh pathway activation and failed to accumulate Smoothened in cilia.

(A,B) Defective GLI3 processing in GRK2-/- fibroblasts. Cells were serum-starved to induce cilia before treatment with the Hh pathway activator SAG (Smoothened agonist), and the whole cell lysates were immunoblotted for GLI3. Note the SAG-mediated downregulation of both full-length (FL) and repressor (R) GLI3 in control fibroblasts, demonstrating normal processing. There was no effect of SAG on GLI3 processing in GRK2-/- cells. A representative experiment of four is shown. (B) Quantitation of the GLI3FL to GLI3R ratio using densitometry. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.

(C) Defective SAG response in GRK2-/- fibroblasts. Cells were treated with SAG for 24 hours. Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT-PCR; GAPDH was used for normalization. Note the SAG-mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.

(D) Under-phosphorylation of Smoothened (SMO) in GRK2-/- fibroblasts. Lysates of SMO-transfected cells were resolved by the phospho(p)-shift PAGE and immunoblotted for SMO. The portion of pSMO analyzed by densitometry is shown. GRK2 add-back demonstrates a rescue of the pSMO in GRK2-/- fibroblasts. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.

(E-G) Defective SMO accumulation in cilia of GRK2-/- fibroblasts. Cells were treated with SAG and immunostained for SMO, ARL13B and pericentrin. Note the failure in SMO accumulation in cilia of GRK2-/- cells (arrow). Scale bar, 2 µm (E). The percentage of SMO-positive cilia was calculated and plotted. Mean ± SEM. Welch´s t-test; number of biological experiments and the total numbers of analyzed cilia are indicated (F). The intensity of ciliary SMO was analyzed and plotted. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated (G).
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 5

Figure 5 - Inhibition of GRK2 activity decreases Smoothened accumulation in cilia and GLI3 processing.

(A-D) Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) or 24 hours (C, D). (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm. (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.

(E,F) Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA. (F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 6

Figure 6 - Loss of GRK2 impairs canonical Wnt signaling.

(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.

(B,C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.

(D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 7

Figure 7 - Inhibition of GRK2 activity inhibits canonical Wnt signaling.

(A) Control (+/+) and Grk2-/- NIH3T3 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, treated with Wnt3A (100 ng/ml) for 24 hours and subjected to dual-luciferase assay. Note the impaired response to Wnt3A in Grk2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.

(B) Control (+/+) and Grk2-/- NIH3T3 cells were treated with 40 ng/ml Wnt3A for 1 hour. Note the lower LRP6 expression and inhibited Wnt3A-induced LRP6 phosphorylation in the Grk2-/- NIH3T3 cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.

(C,D) Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) and rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.

(E,F) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (E) Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in Grk2-/- cells. (F) GRK2 addback rescued the interaction. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.

Expanded View Figure legends
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 1

Figure 1. Comparison of Prrx1-Cre- and Lepr-Cre-traced cells in adult long bones.

A Schematic overview of the scRNA-seq workflow.

B t-SNE plots showing integrated analysis of Prrx1-Cre- (8-week-old mice, n=3 males) and Lepr-Cre-traced (8-week-old mice, n=4 males) cells. Cells were colored by clusters (left) or samples (right). Top 20 PCs were chosen for the clustering.

C Heatmap showing the relative expression levels (row-wide Z score) of the 20 most significant markers for each cluster (rows) across cells in the 9 clusters (columns). Bars on the top were colored as in (B).

D Violin plots showing the expression of feature genes for each cluster.

E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.

F Relative contribution to each cluster by Prrx1-Cre- and Lepr-Cre-traced cells after normalization to total cells (4170 cells in Prrx1-Cre, 3050 cells in Lepr-Cre).

G UMAP plot showing the inferred trajectories by RNA velocity analysis.
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 2

Figure 2. Integrated analysis of Lepr-Cre-traced cells under homeostatic and stress conditions.

A Schematic overview of scRNA-seq strategies in Lepr-Cre; tdTomato mice. Young adult: 8-week-old mice (4 males and 2 females); Aging: 12-month-old mice (4 females); Rosiglitazone diet: 10-week-old mice (feeding started at 5-week-old, 2 females); Irradiation: 8-week-old mice (sub-lethally irradiated at 7-week-old, 2 males and 2 females); Fracture: 8-week-old mice (femur fractured at 7-week-old, 2 males and 2 females).

B, C t-SNE plots showing 17224 CD45- Ter119- Tie2- tdTomato+ single cells from all 5 conditions. Cells are colored by conditions (B) or clustering (C). The adipogenic, osteogenic, periosteal, and cycling cells were highlighted by dotted lines in (C). Top 20 PCs were chosen for the clustering.

D Heatmap showing the relative expression levels (row-wide Z score) of the 20 most significant markers for each cluster (rows) across cells in the 8 clusters (columns). Bars on the top were colored as in (C).

E Violin plots showing the expression of feature genes for each cluster.

F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.

G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 3

Figure 3. Identification of a Notch3+ subpopulation within the adipogenic lineage cells.

A t-SNE plots showing the original (left) and re-clustered (right) adipogenic lineage cells by integrated analysis of homeostatic and stress conditions. Top 15 PCs were chosen for the clustering.

B Dot plots showing the gene expression patterns of selected genes in each cluster.

C Stacked bar charts showing the cell cycle status in all clusters.

D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.

E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm.

F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent.

G Graphical illustration of the 3D co-culture system.

H Volcano plot showing DEGs in the bulk RNA-seq analysis (n=3 biological replicates). Green dots represent down-regulated genes. Red dots represent up-regulated genes.

I qPCR analysis of Notch3 and Cdk6 expression (n=3 independent experiments).

J Flow cytometry analysis of BrdU incorporation in Lepr-Cre+Notch3+/- BMSCs after 3D co-cultured with bone marrow ECs (n=3 independent experiments). BrdU was administered at a final concentration of 10 µM from culture day 1 to day 5.

K, L GO terms (K) and KEGG pathways (L) enriched in up-regulated genes.

M Representative confocal images of BMSCs 3D co-cultured with bone marrow ECs. Z-stack projection was shown. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Arrowheads indicated Notch3+tdTomato+ adventitial cells. Arrows indicated bone marrow ECs. Scale bar was 50 µm.

N Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs after 3D co-culture (n=3 biological replicates from three independent experiments). Cells within 25 µm diameter of ECs were considered adjacent.

O Heatmap showing the average expression levels (column-wide Z score) of secreted factors with clustering.

Data information: All data represented Mean ± SD. The statistical significance of differences was analyzed by two-tailed unpaired Student's t test. *p<0.05, ***p<0.001.
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 4

Figure 4. Identification of candidate osteo-chondrogenic regulators promoting fracture repair.

A t-SNE plots showing the original (left) and re-clustered (right) osteogenic lineage cells by integrated analysis of homeostatic and stress conditions. Top 10 PCs were chosen for the clustering.

B Pseudotime analysis by Monocle 2. Arrow indicated inferred direction of the differentiation trajectory.

C Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides.

D Frequencies of different osteogenic clusters under homeostatic and stress conditions.

E Heatmap showing the AUC score of regulons enriched in each stress. Z-score (row scaling) was calculated. Representative regulons were highlighted on the right.

F Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm.

G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 5

Figure 5. Delineating the periosteal lineage cells.

A t-SNE plots showing the original (left) and re-clustered (right) periosteal lineage cells by integrated analysis of homeostatic and stress conditions. Top 10 PCs were chosen for the clustering.

B Violin plots showing the expression of feature genes for each cluster.

C Pseudotime trajectories of 4 clusters. Cells were colored as in (A).

D Dot plots showing the top5 GO terms enriched for each cluster.

E Heatmap showing the AUC score of regulons enriched in each cluster. Z-score (column scaling) was calculated. Representative regulons were highlighted on the top.

F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.

G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.

H Representative CFU-F colony images. Two hundred DAPI- CD45- Ter119- Tie2- tdTomato+ (Lepr-Cre+), DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+ (Lepr-Cre+Sca-1+) or DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1- (Lepr-Cre+ Sca-1-) cells were sorted from the limb bone periosteum of Lepr-Cre; tdTomato mice and cultured for 8 days, followed by crystal violet staining.

I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools (2021)
Figure 6

Figure 6. Functional analysis of the Sca-1+ subpopulation in the periosteum.

A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.

C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).

E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).

Data information: The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01.

Expanded View Figures and Legends
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 1

Figure 1 LncRNA LINC00115 is upregulated by TGF-β1 in GSCs and is clinical relevant in GBM

Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.05) in mesenchymal (MES) 1123 and proneural (PN) 528 GSCs. GSCs were pre-cultured for 16 h in DMEM/F12 medium with EGF (2 ng/ml) and bFGF (2 ng/ml) and then followed by co-culturing with or without 20-μg/ml TGF-β1 for 3 h.

Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis. Images are representative of two independent experiments. Scale bars: 50 μm.

Kaplan-Meier analysis of patients with high versus low LINC00115-expressing GBM tumors from B. Median survival (in months): low, 13.9; high, 8.1. Black bars, censored data.

Expression levels of LINC00115 are higher in LGG (low grade gliomas) versus normal brain tissues and GBM versus LGG. Expression data of LINC00115 were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM. **, p < 0.01, ***, p < 0.001, by two-tailed t test.

E to G. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing Grade III and GBM tumors from the REMBRANDT (E) or the TCGA (G) 26[], and GBM from GSE83300 50[] (F) datasets. Median survival (in months) in E: low, 20.2; high, 15.9; in F: low, 19.4; high, 13.0; in G: low, 33.2; high, 25.4. Black bars, censored data.

Data information: In C, E, F, and G, *, p < 0.05, by log-rank test.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 2

Figure 2 LINC00115 is important for GSC growth, neuro-like sphere forming frequency, and tumorigenicity

qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs.

B and C. Effects of LINC00115 knockdown on GSC proliferation (B) and neuro-like sphere formation (C).

D. Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC, shL115-1, or shL115-2 at 45 days after implantation. Images represent results of 5 mice per group of 2 independent experiments.

E. Quantification of the bioluminescence activity in D.

F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm.

G. Quantification of tumor volume in F.

H. Immunofluorescence (IF) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F. Images are representative of two independent experiments. Scale bars: 50 μm.

I. Kaplan-Meier analysis of animal implantation with 1123/shC, 1123/shL115-1 or 1123/shL115-2 GSCs. n = 10.

Data information: In A to C, data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA in A and C, by two-tailed t-test in B. In I, **, p < 0.01, by log-rank test.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 3

Figure 3 LINC00115 physically associates with miR-200s

A. The prediction for miR-200s binding sites in the LINC00115 transcript sequence.

B and C. MS2-RIP followed by qRT-PCR analysis of endogenous microRNAs association with LINC00115. 12XMS2 empty vector, LINC00115-12XMS2, or LINC00115-mut- 12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs.

D. Schematics of the predicted binding sites of miR-200s on LINC00115 and LINC00115 mutants.

E. RNA pull-down and qRT-PCR assays of LINC00115 binding with miR-200s. GSC1123 cell lysates were incubated with biotin-labeled LINC00115.

F. Luciferase activity analysis of 1123 GSCs co-transfected with miR-200s and luciferase reporters containing LINC00115 WT, mutant, or empty vector (EV).

Data information: In C, E and F, data are representative of three independent experiments. Error bars, ± SD. In C, ***, p < 0.001, by two-tailed t-test. In E and F, p < 0.05, **, p < 0.01, by One-way ANOVA.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 4

Figure 4 LINC00115 upregulates ZEB1 signaling and promotes GSC self-renewal through competitively binding miR-200s

A and B. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (A) and REMBRANDT GBM array dataset (B). ***, p < 0.001, by two-tailed t-test.

C. LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 and 528 GSCs.

D. Effects of LINC00115 knockdown on ZEB1, vimentin, and E-cadherin protein expression.

E. Effects of re-expression of shRNA-resistant LINC00115 WT (L115-WT), miR-200 binding mutant (L115-mut) with or without miR-200b on ZEB1 signaling activation.

F. Neuro-like sphere formation assay of effects of re-expression of shRNA-resistant LINC00115 WT and mutant with or without miR-200b.

G. Kaplan-Meier analysis of animal implantation with 1123 GSCs with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.

H and I. GSEA of EMT signature genes using ranked gene expression changes in TGF-β1-treated 1123/shLINC00115 GSCs versus the control (H) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector (EV) (I). NES, normalized enrichment score.

Data information: In C to F, data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by One-way ANOVA in C, by two-tailed t-test in F.