EMBO/SourceData huggingface dataset:
Figure 1(B) RNA samples extracted from normal breast epithelial cell, MCF10A, and different subtypes of breast cancer cell lines as indicated were subjected to RT-qPCR analysis to examine the expression of circPVT1 (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).(C) RNA samples extracted from 28 pairs of ER-positive (ER+) breast tumor and adjacent normal tissues and 14 pairs of ER-negative (ER-) breast tumor and adjacent normal tissues were subjected to RT-qPCR analysis to examine the expression of circPVT1 (±s.e.m., *P < 0.05, **P < 0.01 by two-tailed Student's t-test).MCF7 cells were infected with control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPVT1 (sh-circPVT1#1 and sh-circPVT1#2) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of circPVT1 (H) (n = 3 biological replicates, ± s.e.m., ***P < 0. 001 by two-tailed Student's t-test).MCF7 cells were infected with control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPVT1 (sh-circPVT1#1 and sh-circPVT1#2) were subjected to colony formation assay (I) (n = 3 biological replicates, ± s.e.m., ***P < 0. 001 by two-tailed Student's t-test). (J) Quantification of the crystal violet dye as shown in (I) (n = 3 biological replicates, ± s.e.m., ***P < 0.001 by two-tailed Student's t-test).(O) Tamoxifen-resistant MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without tamoxifen (Tam, 5 μM, 72 h) followed by cell proliferation assay
Figure 2(B) Total RNAs extracted from MCF7 cells were incubated with or without RNase R (10 units/μg RNA) at 37℃ for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1 (C) MCF7 cells were treated with Actinomycin D (10 g/ml) for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1(F) MCF7 cells were subjected to cellular fractionation followed by RNA extraction and RT-qPCR analysis to quantify the amount of circRNA in both nucleus and cytosol of the cells. ACTIN and U6 snoRNA were served as purity control for cytosolic and nuclear fractions, respectively (n = 3 biological replicates, ± s.e.m.).(G) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were subjected to RNA-FISH analysis using probe specifically targeting circPVT1. Red: circPVT1; Blue: DAPI.
Figure 3(D, E) UCSC genome browser views of RNA-seq as described in (A) for TFF1 (D) and GREB1 (E) are shown.(F) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated (G) MCF7 cells infected with sh-CTL, sh-circPVT1#1, or sh-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated
Figure 4MCF7 cells transfected with control siRNA (si-CTL) or siRNAs specifically targeting circPVT1 (si-circPVT1#1 and si-circPVT1#2) were treated with or without estrogen (E2, 10-7 M, 6 h) followed by immunoblotting analysis (D) to examine the expression of ERα(E) Tumor samples as described in Fig 1K were subjected to RNA extraction and RT-qPCR analysis to examine the expression of ESR1 (n = 6 biological replicates, ±s.e.m., ***P < 0.001 by two-tailed Student's t-test).(H) Wild-type circPVT1-luc (circPVT1-luc (WT)) and its mutant form with the potential miR-181a-2-3p or miR-6715b-5p binding site mutated (circPVT1 (MT)-luc) were transfected into HEK293T cells with or without CTL, miR-181a-2-3p, or miR-6715b-5p mimic followed by luciferase activity measurement (n = 3 biological replicates, ±s.e.m., ns: non-significant, **P < 0.01 by two-tailed Student's t-test).MCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor were subjected to RT-qPCR analysis using antibodies as indicated (n = 3 biological replicates, ±s.e.m., **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).MCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor to examine the expression of genes as indicated and immunoblotting analysis (M) using antibodies as indicatedMCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor were subjected to RT-qPCR analysis using antibodies as indicated (n = 3 biological replicates, ±s.e.m., **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).(P) MCF7 cells were infected with control shRNA (sh-CTL) or shRNAs specifically targeting circPVT1 (sh-circPVT1) in the presence or absence of miR-181a-2-3p inhibitor were subjected to colony formation assay. (Q) Quantification of the crystal violet dye as shown in (P) (n = 3 biological replicates, ± s.e.m., **P < 0.01 by two-tailed Student's t-test).
Figure 5(F) Tumor samples as described in Fig 1K were subjected RT-qPCR analysis to examine the expression of genes as indicatedTumor samples as described in Fig 1M were subjected to RNA extraction and RT-qPCR analysis (G)Tumor samples as described in Fig 1M were subjected to examine the immunohistochemistry analysis (H) using anti-CD8 antibody. Dark brown staining indicates CD8-positive cell.
Figure 6(A) MCF7 cells infected with control shRNA (sh-CTL) or shRNAs specifically targeting MAVS (sh-MAVS) or STING (sh-MAVS) were transfected with control siRNA (si-CTL) or siRNA specifically targeting circPVT1 (si-circPVT1) followed by RT-qPCR analysis to examine the expression of genes as indicated (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).MCF7 cells infected with sh-CTL, sh-MAVS, sh-MDA5, or sh-RIGI were transfected with si-CTL or si-circPVT1 followed by using antibodies as indicated and RT-qPCR analysis (C) to examine the expression of genes as indicated (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).HEK293T cells transfected with HA-tagged MAVS-FL, MAVS-NTD, or MAVS-CTD were subjected to RIP using anti-HA antibody followed by immunoblotting analysis(K) using antibodies as indicated(L) HEK293T cells transfected with HA-tagged MAVS and Flag-tagged RIGI in the presence or absence of circPVT1 were subjected to immunoprecipitation (IP) with anti-Flag M2 agarose followed by immunoblotting analysis using antibodies as indicated.
Figure 7MCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected to RT-qPCR analysis to examine the expression of genes as indicatedMCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected immunoblotting analysis E) using antibodies as indicatedMCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected to RT-qPCR analysis to examine the expression of genes as indicated(I, J) MCF7 cells were treated with ASO-CTL, ASO-circPVT1 (50 nM), or fulvestrant (ICI, 1 μM) for duration as indicated followed by cell proliferation assay (I) and colony formation (J) (n = 3 biological replicates, ± s.e.m., **p < 0.01, ***p < 0.001, day 4 by two-tailed Student's t-test).Tumor samples as described in (K) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of genes as indicatedTumor samples as described in (K) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated
Figure 1(B) Example images of brightfield and fluorescently labelled HPG (Alexa Fluor 647) of wild type cells (PN1) assayed for global cellular translation. Scale bars represent 5 µm. (C) Change in HPG labelling signal with different durations of HPG incubation (in PN1), measured by flow cytometry. Population medians of at least 200,000 cells are shown. The red line is the ordinary least square (OLS) linear regression fitted on the medians between 0 and 5 minutes. ((E) Example images of brightfield and fluorescently labelled EU (Alexa Fluor 488) of hENT1 and hsvTK cells (PN10567) assayed for global cellular transcription. Scale bars represent 5 µm. (F) Change in EU labelling signal with different lengths of EU incubations, measured by flow cytometry (in PN10597). The mean and standard deviation (SD) of the population medians of at least 200,000 cells in experimental triplicates are shown in black. The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes.
Figure 2(H) Medians of global translation (solid black line) and IQR (shaded area) of cdc25-22 (PN143) cells grown at 30 °C and grouped in length bins of 2 µm. Bins containing more than 30 cells are shown. The solid red lines represent a locally estimated scatterplot smoothing (LOESS) function fitted on the single-cell data.(I) Global cellular translation of cells shown in (H) divided by their cell length. The solid black line represents the LOESS function fitted in (H).
Figure 3(D) For each of the 5 length bins boxed in (C), both 1C and 2C medians are normalised to their respective 1C global cellular translation values. The normalised values are represented as dots, each dot corresponding to one of the 5 length bins, the mean and SD of the normalised values are shown for each DNA content. For each DNA content, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.(F) Example images of brightfield and EGFP-PCNA fluorescence in CCP∆ EGFP-pcn1 cells (PN6001). The dashed lines in the EGFP-PCNA channel delimit the cell masks generated from the brightfield image. Cells with visible foci in the EGFP-PCNA channel are highlighted in yellow and marked with arrows. Scale bars represent 5 µm.(K) For each of the 4 length bins boxed in (J), G1, S, and G2/M medians are normalised to their respective G1 global cellular translation value. The normalised values are represented as dots, each dot corresponding to one of the 4 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each cell cycle stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (J). The p-value is calculated using a Welch's unequal variances t-test.
Figure 4(C) Cells shown in (A) and (B) are grouped in length bins of 1 µm. Medians of global cellular transcription (solid lines) and IQR (shaded areas) are shown for G1 (blue), S (yellow), and G2/M (red) subpopulations. The dashed line box marks the length bins which have G1, S, and G2/M median global transcription values. Bins containing more than 75 cells are shown.(D) For each of the 3 length bins boxed in (C), G1, S, and G2/M medians are normalised to their respective G1 global transcription value. The normalised values are represented as dots, each dot corresponding to one of the 3 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each cell cycle stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.
Figure 5(E) For each of the 3 length bins boxed in (D), the medians of each subpopulation are normalised to their respective "uninucleate, low nuclear synCut3-mCherry" global cellular translation value. The normalised values are represented as dots, each dot corresponding to one of the 3 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each mitotic stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (D). The p-value is calculated using a Welch's unequal variances t-test. For visual clarity, synCut3-mCherry is shortened to synCut3 in the figure.(G) Cells shown in (F) are grouped in length bins of 1 µm. Medians of global cellular transcription (solid lines) and IQR (shaded areas) are shown for uninucleates (blue) and binucleates (red), and with low (light) and high (dark) nuclear synCut3-mCherry signal subpopulations. Bins containing more than 25 cells are shown.
Figure 1(H, I) Representative H&E (H) and dystrophin immunofluorescence of images of TA muscle cross-sections in WT and Prmt5MKO mice. Scale bar: 100 μm.(J, Distribution of myofiber size (J) of WT (n=4) and Prmt5MKO TA muscles (n=4).(L) The total number of myofibers in EDL and Soleus from WT (n=5) and Prmt5MKO mice (n=6).
Figure 2(B-D) Exercise performance of maximum speed (B), running time (C), running distance (D) of WT (n=11) and Prmt5MKO mice (n=9) measured by treadmill.(E, F) Absolute (E) and specific force (F) (on the left panel) and maximal absolute and specific force (on right panel) on EDL muscle from WT (n=4) and Prmt5MKO mice (n=3). (G, H) Absolute (G) and specific force (H) (on the left panel) and maximal absolute and specific force (on right panel) on Soleus muscle from WT (n=4) and Prmt5MKO mice(n=3).
Figure 3(A) Representative immunostaining of Type I, IIA, and IIB myofibers in in EDL and Soleus muscles of 2-month-old WT and Prmt5MKO mice. (B, C) Quantification of abundance of various fiber types in EDL (B) and Soleus (C) muscles of WT and Prmt5MKO mice; (n=5).(D) Representative histochemical staining image of succinate dehydrogenase (SDH) enzymatic activity in EDL and Soleus muscles. (E, F) Quantification of abundance of SDH-low, -medium and -high myofibers in EDL (E) and Soleus (F) muscles of WT (n=5) and Prmt5MKO mice (n=4).(G, H) Metabolic rate of O2 consumption (G) and CO2 production (H) normalized to lean mass for a 48-hour cycle of 4-6-month-old WT (n=8) and Prmt5MKO (n=7) mice measured by an indirect calorimetry.
Figure 4(A, B) Representative images of ORO staining (A) and immunofluorescence (B) in TA muscle from WT and Prmt5MKO mice. Scale bar: 100 μm.(C) Western blotting analysis for protein markers of lipolysis, lipogenesis, and electron transport chain (ETC) complexes in skeletal muscles of WT (n=3) and Prmt5MKO mice; (n=3).(F) OCR was measured at basal state and after sequential addition of Oligomycin, FCCP, and Rotenone/Antimycin A to determine basal respiration (BS), proton leak (PL), ATP respiration (ATP R), maximal respiration (Max R), spare capacity (SC), and non-mitochondrial respiration (NR); (n=4, technical replicates).
Figure 5(A) C2C12 cells overexpressing PRMT5-GFP alone or PRMT5-GFP + SREBP1a-Flag were immunoprecipitated with Flag antibody and blotted with SYM10, GFP, Flag, PRMT5, and Tubulin antibodies. (B) C2C12 cells overexpressing SREBP1a-Flag alone or SREBP1a-Flag + PRMT5-GFP were immunoprecipitated with GFP antibody and blotted with Flag, GFP, PRMT5, and Tubulin antibodies.(E) HEK293 cells were transfected with PRMT5-GFP alone or PRMT5-GFP + SREBP1a-Flag for 24 hours, followed by cycloheximide (30 μg/ml) and protein analysis at 0, 4, 8 hours. Lysates were immunoblotted with Flag, GFP, and tubulin antibodies.(G) Relative expression of lipogenesis genes (Dgat1, Dgat2, Fabp4) in C2C12 transfected with PRMT5 and SREBP1a; (n=4, technical replicates).
Figure 6(A, B) Immunoblots showing symmetric demethylation of H4R3 in skeletal muscles of WT (n=4) and Prmt5MKO mice (n=4) (A), and quantification of H4R3Me2s normalized to total H4 (B).(E) ChIP-sequencing results showing the Prmt5 binding peak on the Pnpla2 promoter of 3T3-L1 cells.(H, I) Enrichment of PRMT5 (H) and H4R3Me2s (I) on the proximal promoter region of the Pnpla2 gene in skeletal muscles of WT and Prmt5MKO mice (n=4 for H, and I, biological replicates).
Figure 7(D) Immunoblotting analysis showing PRMT5, ATGL and ETCs in muscle tissues (upper panel) and quantification of its relative protein levels in muscles of WT, Prmt5MKO, and Prmt5/Pnpla2MKO mice (bottom panel) (n=2, biological replicates).(E, F) Exercise performance in running distance (E) and griping strength test (F) in WT (n=11 for E, n=8 for F), Prmt5MKO (n=10 for E, n=9 for F) and Prmt5/Pnpla2MKO mice (n=9 for E, n=8 for F).(I) Representative images of ORO staining in TA muscles under brightfield (upper panel) and fluorescent imaging (bottom panel) from WT, Prmt5MKO, and Prmt5/Pnpla2MKO mice. Scale bar: 50 μm.
Figure 1(B) Flat-mounted retinas from OIR S1pr1f/stop/f and S1pr1 ECTG pups at P17. Blood vessels are stained with isolectin (left) and pathological neovascular tuft area are highlighted in yellow. Avascular area, total and average neovascular tuft areas are quantified (right)(C) Flat-mounted retinas from OIR S1pr1f/f and S1pr1 ECKO pups, at P17. Blood vessels are stained with isolectin and neovascular tufts are highlighted in yellow. Avascular area, total and average neovascular tuft areas are quantified.(D) Cross sections from OIR S1pr1f/stop/f, S1pr1 ECTG and S1pr1 ECKO P17 pups. Blood vessels are stained with isolectin (red) and nuclei with Hoechst (blue).
Figure 2(A) Retinal flat mounts from OIR S1pr1f/stop/f or S1pr1 ECTG or ECKO P17 pups. High-magnification view of neovascular tufts stained for VE-cadherin (green) and Claudin-5 (red). Junctional levels of VE-cadherin and Claudin-5 were quantified as described, for a minimum of 5 animals per genotype.(B) Retinal flat mounts from OIR S1pr1f/stop/f or S1pr1 ECTG or ECKO P17 pups. Sites of vascular leakage were assessed by staining for fibrinogen (red), blood vessels are delineated by isolectin (green). Extravascular fibrinogen was quantified as described for a minimum of 5 animals per genotype.(C) Pericytes stained by NG2 on the surface of isolectin-positive neovascular tufts at P17 post-OIR (left). EC-associated pericytes in neovascular tufts were quantified (right). 70 tufts from 3 different animals were assessed.(D) Flat-mounted retinas from OIR S1pr1f/f and S1pr1 ECKO pups at P21 stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. A minimum of 7 pups were analyzed per group.
Figure 3(A) Flat-mounted retinas from OIR Apom+/- and Apom-/- pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 10 pups /group)(B) Flat-mounted retinas from OIR WT and ApomTG pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 7 pups /group)(C) Flat-mounted retinas from SAR247799-treated, OIR WT pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 10 pups /group)(D) Flat-mounted retinas from ApoM-Fc-S1P-treated, OIR S1pr1f/stop/f or S1pr1 ECTG pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 6 pups /group)
Figure 1C. LITATS1 expression upon SMAD4 knockdown (as detected by RT‒qPCR) in MDA-MB-231 cells. Cells were serum starved for 16 h and TGF-β was added for 4 h. Representative results from a minimum of three independent experiments are shown. D. LITATS1 expression (as detected by RT‒qPCR) in HEK293T cells. Cells were transfected without (Co.vec) or with the constitutively active TGF-β type I receptor (caTβRI) ectopic expression construct. Representative results from a minimum of three independent experiments are shown. EJ. RNA fluorescence in situ hybridization was performed to evaluate LITATS1 expression and subcellular localization in A549 cells. Cells were treated with or without TGF-β for 2 h. Representative images are shown in the left panel, and signal quantification data are shown in the right panel. Scale bar=10 μm. Representative results from two independent experiments are shown.
Figure 2A. LITATS1 expression in different breast cells as measured by RT‒qPCR. Results from epithelial-like and mesenchymal-like cells are labeled in blue and green, respectively. Representative results from two independent experiments are shown.C. Quantification of LITATS1 expression levels by in situ hybridization in lung adenocarcinoma tissue microarrays. Representative images (bar=100 μm) and zoomed images (bar=20 μm) of in situ hybridization results in lung adenocarcinoma and matched adjacent normal tissues. The comparison of the LITATS1 staining index between the paired tissues is shown in the right panel. Tissue pairs with higher LITATS1 expression in the normal tissue (normal) than in the lung adenocarcinoma tissue (tumor) are highlighted in red, whereas tissue pairs with lower LITATS1 expression in the normal tissue than in the tumor tissue are highlighted in green.D. Kaplan‒Meier survival curves of relapse-free survival in 175 breast cancer patients stratified by LITATS1 expression. LITATS1 expression was measured by in situ hybridization in breast cancer tissue microarrays.Kaplan‒Meier survival curves of overall survival (E) in breast cancer patients stratified by LITATS1 expression. The data were generated via Kaplan‒Meier PlotterKaplan‒Meier survival curves of distant metastasis-free survival (F) and relapse-free survival (G) in breast cancer patients and overall survival (H) in non-small cell lung cancer patients stratified by LITATS1 expression. The data were generated via Kaplan‒Meier Plotter
Figure 3Effect of LITATS1 on TGF-β-induced EMT marker expression in MCF10A-M2 upon CRISPRa-mediated LITATS1 overexpression (A) GAPDH loading control. Representative results from a minimum of three independent experiments are shown.Effect of LITATS1 on TGF-β-induced EMT marker expression in MCF10A-M2 upon shRNA-mediated knockdown (B). α/β-Tubulin, loading control. The results of LITATS1 overexpression Representative results from a minimum of three independent experiments are shown.D, E. An IncuCyte chemotactic migration assay was performed to evaluate the effect of LITATS1 ectopic expression (D) or knockdown (E) on TGF-β-induced MDA-MB-231 cell migration. The results of LITATS1 overexpression and E. Representative results from two independent experiments are shown.F, G. In vivo zebrafish extravasation experiments with MDA-MB-231 cells upon ectopic LITATS1 expression (F) or LITATS1 knockdown (G). Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel. Whole zebrafish image, bar=309.4 μm; zoomed image, bar=154.7 μm. Representative results from two independent experiments are shown.
Figure 4C. Expression of TGF-β target genes (as measured by RT‒qPCR) in MDA-MB-231 cells without (Co.sh) or with (sh#1 and sh#2) LITATS1 depletion. Cells were serum starved for 16 h and treated with or without TGF-β for 4 h. Representative results from a minimum of three independent experiments are shown.D. Effect of LITATS1 knockdown on TGF-β-induced SMAD2 phosphorylation in MDA-MB-231 cells. Cells were serum starved for 16 h and stimulated with TGF-β for the indicated durations. The p-SMAD2 and total SMAD2 (t-SMAD2) levels were analyzed by western blotting. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown. E. Effect of LITATS1 knockdown on E-cadherin, N-cadherin and SNAIL expression in A549 cells. Cells were stimulated with vehicle control (-), SB431542 (SB; 10 μM) or TGF-β (Τβ) for 24 h, and protein expression was analyzed by western blotting. α/β-Tubulin, loading control. Representative results from a minimum of three independent experiments are shown. FH. In vivo zebrafish extravasation experiments with MDA-MB-231 cells upon LITATS1 knockdown and blockage of TGF-β signaling. Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel. Whole zebrafish image, bar=618.8 μm; zoomed image, scale bar=154.7 μm. Representative results from two independent experiments are shown.