EMBO/soda-nel huggingface dataset:
GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes (2021)
Figure 5

Fig. 5 | Conserved expression of GPR15-C10ORF99 pairing in amniotes.

(a) CCR9 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata.

(b) CCR9 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis.

(c) CCR9 expression in the small intestine (SI) and colon of Rana catesbiana Shaw and Rana nigromaculata.

(d) GPR15 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata.

(e) GPR15 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis.

(f) C10ORF99 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata.

(g) C10ORF99 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis.

(h) GPR15 and C10ORF99 expression in the small intestine (SI) and colon of Rana catesbeiana Shaw and Rana nigromaculata.

(i) GPR15 expression in CD4+ T cell from chicken small intestine (SI) and colon.

(j) The migration of CD4+ cells from chicken small intestine (SI) and colon induced by ligand C10ORF99.

Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).
GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes (2021)
Figure 6

Fig. 6 | The correlation of GPR15 and C10ORF99 expression and diet in the mouse colon.
(a) The scheme of three batches of just-weaned mice (18 mice per batch) were fed with a chicken, corn, or mixture of corn and chicken diet for 5.5 months, representing a carnivore diet, an herbivore diet, and an omnivore diet, respectively

(b-e) The expression levels of GPR15, C10ORF99, FOXP3, and IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice.

(f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography.

(h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays.

(i) Luciferase activities of constructs with the mouse or (j) Japanese quail C10ORF99 promoter in HEK293 cells.

Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).
GPER is a mechano-regulator of pancreatic stellate cells and the tumor microenvironment. (2018)
Figure 1

Figure 1. Tamoxifen decreases pancreatic fibrosis and regulates macrophage infiltration and polarization. (A) Representation of mice treatment. (B-E) IHC images for collagen, aSMA, CD68, and CD204. The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining. For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg). Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side. Histogram bars represent mean ± s.e.m. (Anova and Tukey's tests **, p<0.01; ***, p<0.001). For all panels, three experimental replicates.
GPER is a mechano-regulator of pancreatic stellate cells and the tumor microenvironment. (2018)
Figure 4

Figure 4. Tamoxifen deactivates YAP in PSCs and in pancreatic tissues: (A) Immunofluorescence images of PSCs stained for YAP. (B) Quantification of the nuclear/cytoplasm YAP in PSCs (four experimental replicates). (C) qPCR mRNA levels for YAP target genes connective tissue grow factor (CTGF) and Ankyrin Repeat Domain 1 (ANRKD1), (three experimental replicates). (D) Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8). (F) IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μm. (G) Quantification of YAP positive nucleus. n=5 control mice, and ≥3 mice for 2mg and 5 mg. In all cases, bars indicate mean ± s.e.m. t- test for B, C, and E and Anova and Tukey's test for G, *, p<0.05; ***, p<0.001. Three experimental repeats.
GPER is a mechano-regulator of pancreatic stellate cells and the tumor microenvironment. (2018)
Figure 5

Figure 5. Tamoxifen decreases phosphorylation of mlc-2 in PSCs and mice pancreatic tissues and suppresses RhoA activation in PSCs. (A) IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μm. (B). Quantification of staining in A. Each data point represents a cell. (C) Western blot for total and phosphorylated mlc-2. (D) Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control condition, (E) IHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm. (F) Quantification of staining in E. In all cases, Histogram bars indicate mean ± s.e.m. t- test for B, C, and D and Anova and Tukey's test for F, *, p<0.05; ***, p<0.001. Three or more experimental repeats for all panels. (G) Illustration of the effect of tamoxifen on GPER activation at the cellular level (left panel) and tissue level (right panel). In PSCs, tamoxifen suppresses the activation of YAP and MLC-2 (by phosphorylation to pMLC-2) via the axis GPER/RhoA. This inhibits mechanosensing and the ability to apply endogenous forces in PSCs, which are required to maintain the myofibroblastic phenotype in PSCs. Consequently, PSCs adopt a mechanically inactive state (not myofibroblast like cell). Pancreatic tissues from KPC mice treated with tamoxifen have reduced tissue stiffness and desmoplastic reaction (decreased collagen deposition). The expression of the myofibroblast marker a-SMA is reduced consistent with the mechanical inactivation of PSCs. The recruitment of macrophages, their M2 polarization, and the invasion of cancer cells, are also reduced in these tissues.
Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus (2022)
Figure 1

Fig. 1. Mili is preferentially expressed in aNPCs and depleted in neurogenesis.

(A) Levels of Piwil1 (Miwi), Piwil2 (Mili) and Piwil4 (Miwi2) transcripts in RNA seq. data from undifferentiated aNPCs (Days of differentiation -DIF- 0) and differentiating neuroblasts (DIF 4-14); insets in left and right panels show the same data with smaller scales in the ordinate axes.

(B) Western blot (inset) and quantification (bar graph) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral-induced neurogenesis (DIF4-14).

(C, D) Western blot (insets) and quantification (bar graphs) of Miwi and Mili protein abundance in lysates from postnatal mouse testis, hippocampus and undifferentiated aNPCs cultures.

(E) Western blot (inset) and quantification (bar graph) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs.

(F) Representative immunofluorescence micrograph of Mili (white) and Td+ NSCs (red) in hippocampal subgranular zone (SGZ); arrows indicate Td+ Mili + double-positive cells.

(G) Scheme of the experiment (top) and Mili mRNA expression in sorted Td+ and Td- cells after in vivo transduction with split-Cre viruses in the hippocampus (bottom).

Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test (in B-D, G) and two-tailed Student's t-test (in E). In F: GCL, granular cell layer; H, Hilus. The scale bars represent 10 µm.
Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus (2022)
Figure 2

Fig.2. Identification and validation of piRNAs in aNPCs.

(A-C) (A) Size distribution of the piRNA reads showing (B) uridine bias at piRNA 5' ends and (C) probability of distances from the 5'ends of putative secondary piRNAs to the 5'ends of putative primary piRNAs. Note in panel C that 5' termini of putative primary and secondary piRNAs derived from the same cluster tend to concatenate around the '0' mark, as reported in other animals. Distance probability was assayed for unique piRNAs (length between 15-35 nucleotides), without taking into account abundance, by locally weighted smoothing linear regression (LOWESS).

(D) Relative expression of transcripts bearing piRNA-cluster consensus sequences (piCS) of the top abundant piRNAs, and two control snoRNAs (202 and 135) in aNPC, upon treatment with sodium periodate (NaIO4) or water and alkaline ß-elimination. Synthetic RNA oligos were used as negative (Unmethylated, UnMet) and positive (2'-O-methylated, 2'OMet) controls, respectively. Note that presence of 3'-end 2'-O-methylation in positive control and piRNAs confers them resistance to periodate oxidation and alkaline ß-elimination, in contrast to the depletion in UnMet negative control and snoRNAs.

(E) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA (Control) or shRNA targeting Mili (Mili KD).

(F) Expression of four of the most abundant piCS in control and Mili KD aNPCs.

(G-I) Western blot (G), analysis by capillary electrophoresis (H), or quantification by qPCR (I) of the endogenous piCS after co-immunoprecipitation (IP) with endogenous Mili (IP Mili), or control IgG (IP IgG) in lysates of DIF4 aNPCs. In the qPCR abundance of the indicated piCS in the IP Mili was normalized to its respective level in the control co-immunoprecipitation (IgG); error bars in I represent standard deviation.

Data information: data are expressed as mean ± SEM unless differently indicated, n = 2 biological replicates (A-C) and n = 3 biological replicates (D-F, I). ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.
Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus (2022)
Figure 4

Fig.4 Depletion of Mili and piRNAs impairs neurogenesis and increases astrogliosis.

(A) Representation of the targeting regions of GapmeR1 and 3 on the Mili transcript.

(B) Scheme of the in vivo experiment.

(C) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 against Mili (Mili KD).

(D) Representative light-microscopy (left) and confocal (right) micrographs of postnatal hippocampal sections, immunostained for GFAP at 30 dpi of scrambled (Control, left hemisphere) and GapmeR1 against Mili (Mili KD, right hemisphere).

(E) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled (Control) and GapmeR1 against Mili (Mili KD); Right panels: Fold change in GFAP fluorescence intensity level (upper graph) in a hippocampal region of interest (ROI) of 500µm2 in brain slices upon Mili KD compared to Control; Gfap mRNA levels (lower graph) in the DG from mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 (Mili KD).

(F) (Left) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP (green), BrdU (red), NeuN (white) and nuclear DNA (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili (Mili KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells.

(G) Relative mRNAs expression of reactive astrocyte markers in the hippocampus 48h upon injection of scrambled (control, n=6 mice) or GapmeRs against Mili (Mili KD, n=3 mice GapmeR1 and n=3 GapmeR3).

(H) (left) Schematic representation of the experiment; Mili mRNA (left graph) and piCS-1 (right graph) expression in sorted GFP+ NSCs from Nestin-GFP mice treated with Saline (Control) or Kainic Acid (KA).

Data information: data are expressed as mean ± SEM, n = 3 (C, D, E mRNA, H) and 5 (E, F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. UTR, untranslated region; CDS, coding sequence; GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 1 mm (D, left), 10 µm (D, right) and 100 µm (E, F).
Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus (2022)
Figure 5

Fig. 5. Depletion of Mili and piRNAs in aNPCs results in senescence-associated phenotypes.

(A) Heatmap illustrating the expression of genes encoding proteins involved in immune-modulatory and senescence-associated phenotype in differentiating neuroblasts (DIF4) upon Mili KD, compared to control cells. Expression heatmap correlation plots were computed by the k-means clustering method. Scale bar indicates Z-scores.

(B) Representative bright-field microscopy images (left) and quantification (right) of ß-galactosidase+ aNPCs as percent of total cells upon Mili KD, or control 48 h after induction of spontaneous differentiation.

(C) Representative fluorescence microscopy images (left) and quantification (right) of control or Mili KD neuroblasts 48h after spontaneous differentiation, immunostained with anti-BrdU (white) and Ki67 (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells.

(D) Representative cell cycle analysis of propidium iodide staining by flow cytometry in neuroblasts 48h after spontaneous differentiation; Percentage of cells in G0/G1 and S is reported in the text.

(E) Relative mRNA expression of genes encoding proteins involved in the regulation of cell cycle in control and Mili KD neuroblasts 48 hours after spontaneous differentiation.

(F) Transcript abundance expressed in transcripts per million (TPM) of genes encoding proteins involved in the regulation of cell cycle and differentiation (left) or cell cycle (right) in DIF4 neuroblasts upon Mili KD, compared to control cells.

(G) Representative light microscopy images of the ß-galactosidase staining of postnatal hippocampal sections, 30 dpi of scrambled (Control, left hemisphere) and GapmeR1 against Mili (Mili KD, right hemisphere). See Fig. EV4 E for a larger image of this section.

(H) (left) Scheme of the in vivo experiment; (right) Mili mRNA expression in Nestin-GFP+ sorted cells from young (6 weeks) and old (56 weeks) mice.

Data information: data are expressed as mean ± SEM, n = 3 (A, D-F, H) and 5 (B, C) biological replicates (in A each biological replicate was sequenced with two separate flow cells). ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm (B, C), 500 µm (G).
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 1

Figure 1. Regulation of circRNA-FNTA expression and function in response to androgen receptor (AR) in bladder cancer (BCa) cells.

(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).

(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.

(C) Schematic illustration showing the 4 circRNAs identified that could be regulated by AR.

(D) Schematic illustration showing the position of the targeting shRNA to knock down the circRNAs (sh-circ) by targeting the specific 5'-3' splice junction.

(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.

(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.

(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.

(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.

(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.

(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.

Data information: (B, E, F, G, H, I and J) Data shown represent the means ± SD (n = 3 biological replicates). Student's t-test was used for the statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001)
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 2

Figure 2. circFNTA expression is higher in BCa and promotes invasion and cisplatin chemo-resistence in BCa cells.

(A) The schematic diagram shows the genomic location and splicing patterns of circFNTA (hsa_circ_0084171).

(B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.

(C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.

(D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.

(E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.

(F) The structure of PWPI-oecircFNTA, PWPI-control-vector and PWPI-oelinearFNTA overexpression constructs.

(G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).

(H-I) Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) and oecircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.

(J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).

(K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.

(L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).

Data information: (B, C, E, G, H, I and J) Data shown represent the means ± SD (n = 3 biological replicates). Student's t-test was used for the statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, NS=Not significant). (K-L) Student's t-test was used for the statistical analysis (*P < 0.05, ***P < 0.001).
Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion (2020)
Figure 3

Figure 3. AR regulates circFNTA via the RNA editing gene ADAR2.

(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.

(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.

(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.

(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.

(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.

(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.

(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.

(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.

(I) The sketch shows the position of the ARE binding sites in the ADAR2 promoter region.

(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).

(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.

(L) Cloning of the 1 kb ADAR2 promoter into the pGL3 luciferase reporter. Site-directed mutagenesis was used to generate a mutant ARE.

(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.

Data information: (B, C, E, F, G, H, K and M) Data shown represent the means ± SD (n = 3 biological replicates). Student's t-test was used for the statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, NS=Not significant).