EMBO/SourceData huggingface dataset:
Figure 4Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).
figf1(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.(b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.(e) Flow cytometric analysis of the activation and proliferation of OVA peptide-stimulated OT-II and Irgm1-/- OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells.(f) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c-f) independent experiments with similar results (mean and s.d. of triplicate cultures, a-d,f).
figf2(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control.(b) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected.(c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells.(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.(e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later.(f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide.(g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).
figf3(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer.(b) Survival of M. avium-infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype).(c-e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c-e represents an individual mouse. Data are representative of two independent experiments.
figf4(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.(b) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml).(c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations.(d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).
figf5(a-f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b-f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a-c,e,f), × 1,000. Data are representative of two experiments.(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).
Figure 1Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.
Figure 2Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.Testis weight of 3-month-old males.Sperm count of 3-month-old males.Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.
Figure 3Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.
Figure 4The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.
Figure 6A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.B-D Body weight (B), testisweight (C) in Tex11 KI(V749A)/KO males.B-D Significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).E Mating test of 3- to 5-month-old males.F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.
Figure 1Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.Expression of CALIC in primary skin tumors (P) and their metastases (M). Expression of CALIC in primary skin tumors of patients without (w/o) or with (w) metastases.qRT-PCR analysis of CALIC expression in early (I/II) and late (III/IV) stage colon cancers. [Normal (N) n = 8, stage I/II n = 14, stage III/IV n = 26]Kaplan-Meier curves for disease-free survival of colon cancer patients whose primary tumors expressed low (blue, n = 113) or high (red, n = 250) levels of CALIC.
Figure 2Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.(Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls.Immunoblotting analysis of proteins precipitated by RIP assays using anti-hnRNP-L antibody are shown (D).ChIRP-immunoblotting analysis of hnRNP-L association with endogeneous CALIC in HCT116 cells.Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)
Figure 3RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated.Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk.Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score.Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.
Figure 4qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICimmunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICqRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mutHCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibodyChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells.ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full.ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ.Migratory activity of AXL knockdown HCT116 cells.Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control.Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.
Figure 1B. RT-PCR analysis for forebrain (FOXG1), optic cup (VSX2), dorsal forebrain (PAX6 and EMX2) and ventral forebrain (NKX2-1) identity in organoids. All expression values (2-ΔCt) calculated relative to reference gene TBP. Analysis was performed on 5-6 organoids from multiple independent differentiations - control (7), dorsal (7), extended ventral (9) and short ventral (5). Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's comparison of means. The central band depicts the median, the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values.F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling. G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.
Figure 2C. UMAPs depicting the expression of GABAergic markers DLX2 and DLX5, progenitor marker TOP2A, MGE marker SOX6, CGE marker NR2F2, and striatal marker ZFHX3.
Figure 3C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis. D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis.
Figure 4B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification.
Figure 5C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed.
Figure 6C. Mean square displacement (MSD) in μm2 over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the MSD is shown for a time delay corresponding to 24 hours of imaging.F. Bar plot depicting the fold-change in composition of cluster groups - directed, exploratory or confined - of treatment groups relative to the control distribution. Bars are color-coded to denote the corresponding cluster group. The dotted line represents the control baseline for visualization of the fold changes between the groups.
Figure 1Growth curves of the MCF7 (left) and T47D cells (right) upon gene silencing with siRNAs. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.Relative protein levels of HNRNPC upon siRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.Relative protein levels of HNRNPC upon CRISPR-mediated gene knock-down, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Anchorage independent cell growth assays of the HNRNPC-deficient MCF7 (top) and T47D (bottom) cells.Relative protein levels of HNRNPC upon shRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus-mediated stable gene knock-down. Each group has 6 mice. The error bars represent the ± SEM.Images, weights, and growth records of the MCF7-derived xenograft tumors, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.
Figure 2Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using siRNAqPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using CRISPR-Cas911 TFs involved in the interferon response and signaling, which were identified by MARINa as master TFs upon HNRNPC knock-down. In each row, all genes were sorted (from left to right) by their differential expressions in siHNRNPC vs. control cells. The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars. All the TFs were sorted by the P-values (FDR-corrected) from the MARINa analysis. Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right.
Figure 3Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.