EMBO/soda-nel huggingface dataset:
MET Mutation Causes Muscular Dysplasia and Arthrogryposis (2019)
Figure 3

Figure 3. Met mutation led to the defects in the migration of muscle progenitor cells and impaired proliferation of secondary myoblasts. (A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm. (B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm. (C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm. (D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm. (E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm. (F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.

Expanded View Figures
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 1

Figure 1: Hh is localized to the LDs within cortex glial cells

Images in this and following figures are of larval central brains (CBs) at 96ALH.

A) Schematic showing NBs that undergo asymmetric division to self-renew and produce GMCs, which terminally differentiate to generate post-mitotic neurons or glial cells (left). Each NB is surrounded by a microenvironment, composed of glial cells (right).

B-D'') Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB (yellow arrows), quantified in (I) (n=20 LDs). Glial cells are marked by repo-GAL4>GFP and CB is circled in (B).

E-G'') In the posterior compartment of the developing wing disc (WD) pouch region where Hh is expressed, LDs and Hh are not tightly associated (white arrows), quantified in (J) (n = 17 LDs).

H-J) Hh-LD association is quantified by plotting the pixel intensities of both Hh (cyan) and LDs (red) along a line across LDs. Y axis represents gray intensity values, and X axis represents relative LD position.

K-L'') Hh-LD associations are observed in the cortex glia (yellow arrows, NP2222-GAL4>mGFP).

Data information: Hh is detected with a Hh antibody and LDs are visualized with LipidTOX unless otherwise stated. (D-D''), (G-G''), (L-L'') are zoomed in images of (C, F, K), respectively. Scale bar=50 μm in (C and F), scale bar=20 μm in (D-D'', K-L''), scale bar=10 μm in (G-G''). Error bar represents SEM. In (I): Welch's t test, (****) p<0.0001. In (J): unpaired t test, (ns) p=0.7113.
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 2

Figure 2: Hh autonomously regulates cortex gliogenesis and non-autonomously regulates NB proliferation

A-D) Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4>mGFP with UAS-dcr2), cortex glial membrane and overall Repo+ glial cell number are significantly reduced, quantified in (C) (n=7, 8 brain lobes) and (D) (n=7, 8 brain lobes), respectively.

E-G) Hh knockdown in glia (repo-GAL4>GFP) results in niche disruption and clustering of NBs (circled with yellow dashed line), as well as an increase in the percentage of NBs in M phase (pH3+), quantified in (E) (n=12, 10 brain lobes).

H-K) Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex-glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n=16, 20 brain lobes) and (K) (n=11, 14 brain lobes), respectively.

L-M'') Representative images showing ci-lacZ is expressed in NBs (yellow arrows). (M-M'') are zoomed in images of (L).

N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).

Data information: NBs are marked with Mira and EdU+ NBs are circled by yellow dashed line. Scale bar=50 μm. Error bar represents SEM. In (C): Mann-Whitney test, (**) p=0.0059. In (D): unpaired t test, (*) p=0.0176. In (E): unpaired t test, (*) p=0.0215. In (J): unpaired t test, (***) p=0.0002. In (K): unpaired t test, (***) p=0.0001. In (P): unpaired t test, (****) p<0.0001.
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 3

Figure 3: Hh activity is modulated by Lsd-2

A-B'') Representative images showing that Lsd2YFP is localised to the surface of LDs (yellow arrows).

C-D'') Lsd2YFP co-localises with Hh antibody staining in the cortex glia (yellow arrows) but not surface glial cells (white arrow).

E-G) Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed effectively reduces LD number in CB (outlined in yellow dashed lines).

H-K) Representative images showing that NB EdU index is rescued upon Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed, quantified in (K) (n=11, 14; 16, 14; 6, 14 brain lobes). The NP2222-GAL4>w1118 vs hhOE columns depict the same data as Fig 2K. EdU+ NBs are circled with yellow, dashed lines.

Data information: (B-B'', D-D'') are zoomed in images of (A and C). Scale bar=50 μm in (A, C, E-J). Scale bar=10 μm in (B-B'' and D-D''). Error bar represents SEM. In (K): unpaired t test, (***) p=0.0001; unpaired t test, (**) p=0.0053; unpaired t test, (*) p=0.0349.
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 4

Figure 4: Activation of htlACT but not InRwt and EgfrACT induces cortex glial overgrowth

A-D') Representative images showing that pan-glial overexpression of htlACT, but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs. Glial cells are marked with repo-GAL4 >GFP, and NBs are marked with Mira. (A', B', C' and D') are zoomed in images of (A, B, C and D), respectively.

E-K) Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell (Repo+) numbers and cortex glial membrane size, quantified in (K) (n=10, 10, 10; 7, 12 brain lobes) and (H) (n=10, 10 brain lobes), respectively. NP2222-GAL4>mGFP is used to mark cortex glial membrane in (E-G) and wrapper-GAL4> is used in (I, J).

L) Glial (repo-GAL4>) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs (n= 10, 10, 8, 11 brain lobes).

M) Glial (repo-GAL4>) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB NBs (marked with Mira and pH3 in (A-D')) (n= 10, 10, 8, 11 brain lobes).

Data information: Scale bar=50 μm. Error bar represents SEM. In (H): Welch's t test, (****) p<0.0001. In (K): Welch's t test, (****) p<0.0001; Welch's t test, (ns) p=0.7905; unpaired t test, (ns) p=0.0941. In (L): unpaired t test, (ns) p=0.9140; Welch's t test, (*) p=0.0170; unpaired t test, (ns) p=0.9825. In (M): unpaired t test, (**) p=0.0038; unpaired t test, (ns) p=0.4172; unpaired t test, (ns) p=0.5523.
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 5

Figure 5 Cortex glial overgrowth mediated by htlACT overexpression triggers NB cell cycle delay

A-E) Representative images showing that both pan-glial (repo-GAL4>) and cortex glial (NP2222-GAL4>) htlACT overexpression significantly reduce NB EdU index, quantified in (E) (n=15 ,14; 10, 16 brain lobes). (A, B, C, D) are single sections with Mira and EdU staining, and (A', B' C', D') are Z-projection of the EdU staining.

F-H) Representative still images from ex vivo CNS live imaging at 72ALH showing that pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, quantified in (F) (n= 25, 6 NBs imaged from 3 brains per genotype). The cell cycle length is measured as the length between consecutive divisions. NBs (Dpn::GFP, red; Histone RFP, grey) are circled with blue dashed lines.

I) Schematic depicting concurrent glial FGF activation (repo-LexA >LexAop-htlACT), and NB overexpression of fly-FUCCI (dnab-GAL4>UAS-GFP::E2F1, UAS-RFP::CycB). The fly-FUCCI system utilizes the fusion protein GFP::E2F1 (a marker for cells in G2, M and G1phase) and RFP::CycB ( a marker for cells in S, G2, M phase) to monitor cell cycle progression. Cells in G1 phase are GFP+ RFP- (green), cells in G2/ M phase are GFP+RFP+ (yellow), and cells in S phase are GFP-RFP+ (red), whereas, cells in G1-S transition are weakly labelled by both GFP and RFP (grey).

J-L) Representative images showing that the percentage of NBs in G1-S transition and G2/M phase are both significantly increased with significantly less cells remaining in G1 phase, quantified in (L) (n= 9, 10 brain lobes). NBs in G1 phase (Mira+, GFP+) are circled by green dashed lines; NBs in G1-S transition (Mira+, GFP-RFP-) are circled by grey dashed lines; NBs in S phase (Mira+, RFP+) are circled by red dashed lines; and NBs in G2/M phase (Mira+, GFP+RFP+) are circled by yellow dashed lines.

M-O) Representative images showing that the number of EdU+ neurons generated per NB is significantly reduced upon pan-glial overexpression of FGF (repo-LexA > LexAop-htlACT; yellow arrows), quantified in (O) (Box plot, the boxes extend from the 25th to 75th percentiles; the median is marked by a central band inside the box; and the whiskers go down to the minimum value and up to the maximum value. n= 94, 127 NB lineages imaged from 5 and 7 brain lobes, respectively). NB lineages are marked with dnab-gal4 > GFP.

Data information: Scale bar=50 μm in (A-D', J-K'); Scale bar=10 μm in (G, H); Scale bar=20 μm in (M, N). Error bar represents SEM unless otherwise stated. In (E): unpaired t test, (****) p<0.0001; unpaired t test, (****) p<0.0001. In (F): Mann-Whitney test, (****) p<0.0001. In (L): G1: Mann-Whitney test, (****) p<0.0001; G1-S: unpaired t test, (**) p=0.0064; S: unpaired t test, (ns) p=0.1757; G2/M: unpaired t test, (*) p=0.0171. In (O): Mann-Whitney test, (****) p<0.0001.
Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 7

Figure 7 Fasn1 affects glial Hh palmitoylation to regulate its signalling to NBs

A) Schematic depicting Hh auto-processing, which starts with the cleavage of the protein into a C-terminal part (Hh.C, yellow) and a N-terminal part (Hh.N, blue), with simultaneous covalent addition of cholesterol. Palmitate, from either diet or de novo lipogenesis (via ACC and Fasn), is added onto Hh-N, in a reaction catalysed by an acyltransferase, encoded by rasp.

B) Inhibition of palmitoylation (via two independent rasp RNAis) rescues NB EdU incorporation defects induced by cortex glial (NP2222-GAL4>) htlACT overexpression, while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index (n=10, 15; 10, 16; 18, 21; 26, 29 brain lobes). The NP2222-GAL4> w1118 vs htlACT columns depict the same data as those in Fig 5E.

C-C') Representative images showing that Hh.N.EGFP (which cannot undergo cholesterol modification) are found as puncta on the surface of NBs (yellow arrows) when overexpressed in neighbouring cortex glial cells (NP2222-GAL4>).

D-G) Representative images showing that knockdown of Fasn1 in glial cells (repo-GAL4>), where Hh.N.EGFP is overexpressed, significantly reduces the number of Hh.N.EGFP puncta on the surface of NBs (yellow arrows), phenocopying the effect of Rasp knockdown, quantified in (G) (n=38, 38, 57 NBs from 4, 4, 8 brain lobes, respectively).

H) Cortex glial (NP2222-GAL4>) overexpression of Hh.N.EGFP significantly reduces NB EdU incorporation (n= 13,12 brain lobes).

I) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects, caused by glial Hh.N.EGFP overexpression, quantified in (I) (n=15, 13 brain lobes).

J) Schematic depicting our working model. During development, Hh tethered to LDs are localised to cortex glial cells, to activate gliogenesis. Non-autonomously, excessive glial Hh inhibits NB cell cycle progression. Upon cortex glial specific FGF activation, increased Hh modified by Fasn1 and Lsd-2 together with increased ROS prevent NB proliferation.

Data information: (C', D', E' and F') are zoomed in images of (C, D, E and F), respectively; NBs are marked with Mira. Scale bar=50 μm in (C, D, E, F). Scale bar=20 μm in (C', D', E' and F'). Error bar represents SEM. In (B): unpaired t test, (ns) p=0.9403; unpaired t test, (****) p<0.0001; Mann-Whitney test, (***) p=0.0004; Mann-Whitney test, (**) p=0.0047. In (G): Welch's t test, (****) p<0.0001; Welch's t test, (****) p<0.0001. In (H): unpaired t test, (****) p<0.0001. In (I): unpaired t test, (****) p<0.0001.

Expanded View Figure legends
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 1

Figure 1.

MiR-10b inhibition reduces viability and enhances apoptosis of GSC.

GSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled “miR-10b-i”) or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Statistical significance of the difference was determined by Student's t test, with p-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.

A. Cell viability was monitored at day 5 after transfection as described in Materials and Methods.

B. The number and size of GSC colonies were monitored at day 5 after transfection.

C. MiR-10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta-actin.

D. Flow Cytometry analysis of Annexin V and 7-AAD staining of GSC GBM8 at day 5 after miR-10b inhibition.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 2

Figure 2.

miR-10b regulates cell cycle and splicing related genes in GSC.

Three types of GSC (GBM4, GBM6, and GBM8) were transfected with miR-10b ASO, and gene expression was analyzed 24 hours later by the Affymetrix microarrays. The heatmaps colors intensity demonstrate altered expression of the genes (up- or down-regulated relative to the mock treated samples) with the fold change > 1.2 and p < 0.05 in at least two out of the three GSC cultures.

A. The genes associated with “cell cycle” bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR-10b inhibitor is indicated as “miR-10b-i”.

B. The genes associated with “RNA splicing” bioterm have been selected using Gene Ontology (GO). The treatment with the miR-10b inhibitor is indicated as “miR-10b-i”. Arrows depict the genes selected as candidate direct targets for further study.

C. miR-10b binding motifs are enriched in 5’UTRs of the genes up-regulated by miR-10b ASO. The graph shows the probability that enrichment of the miR-10b motifs in mRNAs up-regulated vs. unchanged (p<0.05) by anti-miR-10b does not occur by chance.

D. The miR-10b octamer motifs’ composition of the 5’UTRs was compared between transcripts up- and down-regulated on the microarrays. The relative frequencies of various miR-10b binding motifs are shown, indicating that mostly miR-10b 3’-end binding motifs are enriched in the up-regulated mRNAs.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 3

Figure 3.

Expression analysis of splicing factor mRNAs in various GBM datasets.

A. The genes encoding splicing factors down-regulated by miR-10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues.

B. In contrast, many splicing factors up-regulated by miR-10b are overexpressed in the GBM datasets.

Six high-content GBM microarray datasets from the Oncomine resource (https://www.oncomine.org/resource/main.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34) and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 4

Figure 4.

miR-10b regulates splicing factors through the non-canonical binding within 5’UTRs.

A. Putative miR-10b binding sites within 5’UTRs of candidate splicing factors mRNAs.

B. qRT-PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1 and other splicing factors are de-repressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.

C. Regulation of representative splicing-related proteins by miR-10b mimic in GSC, as demonstrated by Western blot analysis. The signals were quantified using ImageJ and normalized to beta-actin. The ratios between miR-10b mimic expressing and control samples are indicated.

D. miR-10b mimic regulates 5’UTR Luciferase reporter containing a single miR-10b complementary site.

E. miR-10b mimic regulates 5’UTR Luciferase reporters of some splicing factors genes bearing wild type (WT), but not mutated (Mut) miR-10b binding sites. Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks, p < 0.01 by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 5

Figure 5.

Intratumoral injections of miR-10b inhibitor reduce the growth of established intracranial GBM8 xenografts.

A. A schematic overview of in vivo experiments on orthotopic GBM8. The tumor growth was monitored by luciferase imaging (WBI), and expressed in photon flux per second. Mice assigned to the treatment and control groups were treated with miR-10b inhibitors or corresponding control oligonucleotides in different formulations.

B. 2’-O-MOE-PO miR-10b inhibitor (miR-10b-i) or non-targeting control (1 μg of each) formulated with InVivo-jet PEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR-10b inhibition was assessed by qRT-PCR analysis of the resected tumors, with miR-10b expression levels normalized to miR-125b.

C. qRT-PCR analysis demonstrates that miR-10b inhibition in orthotopic GBM8 leads do de-repression of its mRNA targets. mRNA expression levels were normalized to GAPDH.

D. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.

E. Inhibition of miR-10b markedly reduces tumor burden. The left panels illustrate tumor imaging in representative animals at day 29. The bars represent average signal ratios for each group at day 29 (after treatment) to day 20 (at the beginning of treatment). N = 7 animals per group at treatment initiation.

F. Growth curves of individual tumors, based on the ratios of bioluminescence signals to the baseline signals at day 5.

G. Each mouse was sacrificed when the tumor-generated signal reached 1.5x107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.

Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks, p < 0.01 by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 6

Figure 6.

Systemic treatment with miR-10b inhibitor reduces growth of the established intracranial GBM8 tumors.

A. Intravenously injected Cy5-labeled 2’-O-MOE-PS oligonucleotide is distributed to intracranial GBM8 tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. “T” – tumor, “B” – brain tissue. Each image is representative of three mice analyzed.

B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2’-O-MOE-PS miR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.

B. The left panels illustrate tumor images of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6.

C. Each mouse was sacrificed when the tumor-generated signal reached 5X107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.

D. Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6.

E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b.

F. qRT-PCR analysis demonstrates that miR-10b targets were de-repressed in orthotopic GBM8 upon systemic administration of the miR-10b inhibitor. Seven tumors per condition, and two specimens per tumor have been analyzed. mRNA expression levels were normalized to GAPDH.

G. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.

Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks, p < 0.01 by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 7

Figure 7.

Toxicity assessment of the systemic treatment with miR-10b inhibitor.

A. Uptake of the uncomplexed Cy5-labeled 2’-O-MOE-PS oligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy 24 h after injections.

B-D. Systemic treatment of intracranial GBM8 tumors with uncomplexed 2’-O-MOE-PS miR-10b inhibitor (miR-10b–i) or non-targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects.

B. No significant difference in average mice weight was observed between the anti-miR-10b and control treatment groups.

C. No significant difference in average organs’ weight was observed between the anti-miR-10b and control treatment groups.

D. No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti-miR-10b and control treatment groups.