EMBO/SourceData huggingface dataset:
Figure 6A) IFNγ cytokine measured in the amniotic fluid (AF) and fetal liver supernatant (FL) of IFNγKO (IFNγ-/-) mice one day following injection of cytokine on E14.5. ND = not detectable. Data represent mean of 3 fetuses/condition and error bars represent + SD.C-H) Total cellularity of C) HSPCs, D) LT-HSCs, E) ST-HSCs, F) MPP2, G) MPP3, and H) MPP4 in saline or IFNγ exposed littermates of each genotype (IFNγ +/- or -/-), as derived from the cross (IFNγR +/- dam). n = 19-23 fetuses from 3 litters/condition.J-O) Total cellularity of J) HSPCs, K) LT-HSCs, L) ST-HSCs, M) MPP2, N) MPP3, and O) MPP4 in saline or IFNγ exposed littermates of each genotype (IFNγ +/- or -/-), (IFNγR -/- dam). n = 19-21 fetuses from 3 litters/condition. For all analysis bars represent mean. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Figure 2B, single optical plane in the HPuHG of wildtype mice and Alk1EndoKO mice showing dramatic vascular defect in Alk1EndoKO mice. Severely tangled and tortuous vasculatures were outlined in white dotted rectangles.C, 8 μm skin section of Cre- control (CT) and Alk1EndoKO mice stained for Cdh5 (red), Ki67 (green), Erg (white), and Dapi (blue). Examples of Cdh5/Ki67 double positive cells were enlarged in the white dotted inset for visualization purposes.D-G, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 9-16 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of number of ECs in HPuHG area from C. E, Percentage of proliferative ECs (percentage of Ki67+ Cdh5+ cells in total Cdh5+ cells) in HPuHG from C. F, Percentage of proliferative ECs (percentage of Ki67+/Cdh5+ cells in total skin Cdh5+ cells) from C G, Percentage of proliferative ECs (percentage of BrdU+ Cdh5+ cells in total Cdh5+ cells) in total skin from PD17-24 BrdU-pulsed CT and Alk1EndoKO mice
Figure 3A-C, Immunofluorescence staining images of 12 μm skin section from PD17 induced PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice stained for Endomucin (EMCN, red), CD31 (green), and Dapi (blue). HPuHG region was outlined in yellow dotted lines. D-F, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 8-9 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of EMCN+/CD31+ area from images like those in A-C, shown as percentage of BV in the HPuHG. E, Quantification of absolute EMCN+ area (e.g. BV area) in HPuHG in images like those in A-C. F, Quantification of EMCN-/CD31+ area from images like those in A-C as LV area in the HPuHG.G-J, Maximum projection through optical Z-stacks of 60 μm skin sections from mice TM-induced at PD17 and sacrificed at PD20 telogen CT, PD25 early anagen CT, PD32 Anagen CT, and PD25 telogen Alk1EndoKO mice stained for VEGFR3 (red), LYVE1 (green), and Dapi (blue). Lymphatic capillaries were outlined in white dotted lines. K, Quantification of lymphatic capillary caliber from images like those in G-J in PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one lymphatic capillary. N = 3 mice per group, and n = 9-25 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.
Figure 4B, Immunofluorescence image of 12 μm skin section from mice treated as shown in A, stained for VEGFR3 (green) and Dapi (blue). C, Quantification of tdTomato+/VEGFR3+ area from images like those in B, shows high induction efficiency. Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice, and n = 7-8 images per mouse.F, Quantification of lymphatic capillary caliber from images like those in D in CT and Alk1LymphKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement of one lymphatic capillary. N = 3 mice per group, and n = 13-16 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.G, Immunofluorescence images of 12 μm skin section of PD17 induced PD25 CT and Alk1LymphKO mice stained for Ki67 (red), Itga6 (green) and Dapi (blue) showing representative hair follicle. Hair follicles were outlined with yellow dotted lines. Note Ki67+ cells in the hair germ (HG) area indicative of HFSC activation. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. Experiments were repeated at least 2 times with at least 3 skin sections/each mouse analyzed with N = 6/4 mice/group. See Table 1 for quantification detail.
Figure 5B, Immunofluorescence image of 12 μm skin section from CT and Alk1EndoKO mice used for RNA-seq stained for Ki67 (red) and Dapi (blue) show that HFSCs are in quiescence.
Figure 6C, Images of 20 μm skin section of PD20 and PD25 BMP4-LacZ; Alk1f/f; Cdh5-CreERT2 negative, injected with tamoxifen TM at PD17 (labelled as CT), and PD20/PD25 Cdh5CreERT2; Alk1f/f; BMP4-LacZ mice (labelled as Alk1EndoKO) TM injected at PD17, all stained for CD31 (IHC method, brown) and X-gal (blue). Hair follicles (HF) were outlined with black dotted lines. Arrows point to X-Gal+ vascular structure in HPuHG area.G-J, Immunofluorescence images of 12 μm skin section of PD17 induced stained for (Top panel) Ki67 (red), Itga6 (green), and Dapi (blue) and (Bottom panel) CD31 (red) and Dapi (blue). HPuHG region was outlined with white dotted lines.K, Quantification of hair follicle percentage of PD17-induced PD25 CT, Alk1EndoKO, Alk1 BMP4dKO, and BMP4EndoKO at telogen, anagen I, and anagen II stages. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. P-value was generated from Chi-square test. See Table 1 for quantification detail.
Figure 1Validation of upregulated lncRNAs (ΔCT) in PBMC-derived M2-like macrophages (stimulated with IL-4 stimulated for 24 hours, M2). n = 6 biological replicates, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with M1-like macrophages (LPS/IFNɣ-stimulated for 24 hours, M1).Representative images of fluorescence in-situ hybridization of ADPGK-AS1 (magenta) and nuclei (blue) in IL-4 stimulated M2-like macrophages. β-actin served as the cytoplasmic control and MALAT1 as the nuclear control. Scale bar = 10 µm.RNA expression analysis of ADPGK-AS1, 47S ribosomal RNA (nuclear control), and β-Tubulin (cytoplasmic control) in IL-4 stimulated M2-like macrophages after subcellular fractionation. n = 3 independent experiments. Data represent the percentage of total detected RNA.Subcellular fractionation of IL-4-stimulated M2-like macrophages and isolation and analysis of ADPGK-AS1 abundance in the cytoplasmic and mitochondrial fractions The mitochondrial encoded RNA mtATP6 served as the mitochondrial control and β-tubulin as the cytoplasmic control. n = 3 independent experiments, Data represent the percentage of total detected RNA.
Figure 2(A) Validation of the ADPGK-AS1-interacting protein MRPL35 by RNA immunoprecipitation (RIP) with followed by qPCR for ADPGK-AS1. n = 4 independent experiments, ***p ≤ 0.001 compared with IgG control.(B, C) Co-staining for ADPGK-AS1 (magenta) and MRPL35 or MRPL15 (yellow) with respective fluorescence intensity across co-stained macrophages (see white line) for ADPGK-AS1 and MRPL35 or MRPL15. Scale bar = 10 µm (left), 5 µm (right).(E) Number of M1-like or M2-like macrophages based on TMA data for lung cancer patients with either high (above median) or low (below median) expression of MRPL35.(F, G) Kaplan-Meier curve estimate of disease-free survival (DFS) or overall survival (OS) of patients with high (above median) or low (below median) expression of MRPL35 in M2-like macrophages. n = 200 biological replicates.
Figure 3(C, Representative images and quantification of mitochondria by MitoTracker (red) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).D) Representative images and quantification of mitochondria by TOM20 (green) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).(E) Representative immunoblot for mitochondrial protein expression (TOM20, αb-crystallin, PCG-1, MRPL35, cytochrome C) in THP1 control and ADPGK-AS1 OE cells. n = 4 independent experiments.(G) Gene expression analysis (fold change) of mitochondrially encoded genes (ND1, ND2, ND4, ATP6, ATP8, CO1, CO2, CyB) after isolation of mtDNA from THP1 control and ADPGK-AS1 OE cells. n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with control.I) full oxygen consumption plot (n = 8) by the seahorse assay in THP1 control and ADPGK-AS1 OE cells. **p ≤ 0.01, ***p ≤ 0.001 compared with control.(K) Representative confocal fluorescence images and quantification of mitochondrial shape with the mitochondrial marker TOM20 (yellow) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). n = 35 cells, Scale bar=10µm (left), 5µm (magnification, right).(L) Transmission electron microscopy images of mitochondrial shapes in THP1 control and ADPGK-AS1 OE cells. n = 4 cells, Scale bar = 5 µm (left), 1 µm (right, magnification).
Figure 4(B) Expression analysis of ADPGK-AS1 and MRPL35 (fold change) in THP1 control and ADPGK-AS1 OE macrophages transfected with a siRNA against ADPGK-AS1, MRPL35 or negative control. n = 4 independent experiments. ***p ≤ 0.001 compared with ADPGK-AS1 OE siRNA control.(C) Representative images of JC-1 monomers (green) and aggregates (red) in THP1 control and ADPGK-AS1 overexpressing macrophages transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. Scale bar = 100 µm.(E) Representative images (left panel) and quantification (right panel) of ROS accumulation in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 7. ***p ≤ 0.001 compared to control.(F) Spare capacity (oxygen consumption rate) measured with the seahorse assay in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 3 independent experiments. ***p ≤ 0.001 compared with control, §p ≤ 0.05, §§§p ≤ 0.001 compared with ADPGK-AS1 OE siRNA negative control.
Figure 5(B) mRNA expression analysis (ΔCT) of macrophage markers (TNFα, IL-8, CD206, CSF-1R) in co-cultured PBMC-derived macrophages with tumor cells or macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.(C) Expression of ADPGK-AS1 lncRNA in macrophages co-cultured with tumor cells (A549) or macrophage controls (M0). n = 4 biological replicates. *p ≤ 0.05, compared with M0.(D) Mass spectrometry analysis of TCA cycle metabolites (fumarate, malate, citrate, succinate) in macrophages co-cultured with tumor cells (A549) and macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.(G) Expression of IDH mRNA in A549 cells transfected with siRNA specific for IDH (IDH KD) or negative control. n = 5 independent experiments. ***p ≤ 0.001 compared with A549 control.(H) RNA expression analysis of ADPGK-AS1 in THP1 macrophages stimulated to have an M1-like or M2-like phenotype, untreated (M0) or treated with conditioned medium (CM) of A549 cells transfected with a siRNA specific for IDH (IDH KD) or negative control. n = 5 biological replicates. ***p ≤ 0.001, compared with tumor cell control CM.
Figure 6(A, B) mRNA expression analysis (ΔCT) of M1 (TNFα, IL-8, CXCL10) and M2 (CSF-1R, CD206, IL-10, ALOX15, TGFβ, IL-1Ra) macrophage marker genes in THP1 control and ADPGK-AS1 OE cells. n = 4 independent experiments. *p ≤ 0.05, ***p ≤ 0.001 compared with control.(E) Migration (right panel) of A549 cells treated with CM from THP1 control or ADPGK-AS1 OE macrophages. Representative membrane images are shown in the left panel. n = 5 independent experiments. ***p ≤ 0.001 compared with control. Scale bar = 400 µm.(G-H) mRNA expression analysis (ΔCT) of M1 (TNFα, IL-8, CD80, CCR7) and M2 (CSF-1R, CD206, IL-10, TGFβ) macrophage marker genes in primary M1-like (M1) and M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control. n = 5 biological replicates, *p ≤ 0.05, **p ≤ 0.01, compared to M2 control.(K) Migration (right panel) of tumor cells (A549) treated with CM from PBMC-derived M1-like (M1) or M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control. Representative membrane images are shown in the left panel. n = 5 biological replicates. *p ≤ 0.05, compared with M2 control. Scale bar = 400 µm.
Figure 7(C) Representative brightfield images of tumors (scale bar = 2mm) and fluorescence images (Ki67, cleaved caspase 3, scale bar = 100 µm; cleaved caspase 3 (green) and cytokeratin 18 (magenta), scale bar = 50 µm) and LDH enzyme activity (scale bar = 25 µm) after co-injection with M1-like (M1) or M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control (ctrl) together with A549 tumor cells or A549 cells alone as a control.(D) Quantification of Ki67+ cells and cleaved caspase 3+ of co-injected tumors. n = 5 biological replicates. **p ≤ 0.01, ***p ≤ 0.001 compared with M2 control.(G) Representative immunoblot of the mitochondrial-related proteins MRPL35, Opa1, Mfn1, pDRP1, and DRP1 about β-actin in isolated tumor tissue. n = 3 biological replicates.
Figure 8(C, D) Representative fluorescence images of PCLS with proliferative (EdU+) and apoptotic (TUNEL+) cells (red) and nuclear dye (DAPI+, blue) in the tumor area (cytokeratin+, green). Scale bar = 250 µm (c, upper panel), 150 µm (d, upper panel), and 50 µm (c and d lower panel, magnification).(F) RNA expression analysis of ADPGK-AS1 and MRPL35 in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK-AS1 or a negative control or seeded with THP1 control or ADPGK-AS1 OE cells. n = 5 independent experiments. *p ≤ 0.05, ***p ≤ 0.001 compared with control GapmeR, §p ≤ 0.05, §§§p ≤ 0.001 compared with EV control.(G) Multispectral flow cytometry analysis of M1-like and M2-like macrophage populations in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK-AS1 or a negative control or seeded with THP1 control or ADPGK-AS1 OE cells. n = 6 biological replicates. *p ≤ 0.05 compared with Gapmer negative control, §p ≤ 0.05 compared with EV control.
Figure 1A-B Time dependent effects of ingesting 0, 0.1, 1, 5, 10, 30, and 50 mM vitamin C without any further nutrition on the survival of wild-type control flies (w1118) (A) males (B) females, n = 6. Kaplan-Meier survival analyses were performed to compare the survival rates of agar only and fed conditions at different concentrations of vitamin CF Electrophysiology mapping of 31 sensilla with control to 50 mM vitamin C, n = 10-18.G Tip recordings from the L4 and L6 sensilla of specific GRN-ablated flies with 50 mM vitamin C. +/- indicates the presence or absence of the transgene, respectively, n = 10-13.
Figure 2C Genetically recovered flies of Ir25a2 and Ir76b1 driven by their own GAL4 or Gr64f-GAL4 with the wild-type cDNA transgene. +/- indicate the presence or absence of the transgene, respectively, n = 10-15.D Genetically recovered flies of Gr5a, Gr61a, Gr64b, Gr64c, and Gr64e mutants driven by their own GAL4 or Gr64f-GAL4. +/- indicate the presence or absence of the transgene, respectively, n = 10-21.
Figure 3D Behavioral rescue of Gr5a∆5, Gr61a1, Gr64bLEXA, Gr64cLEXA, and Gr64eLEXA deficits in the vitamin C attraction via the expression of the respective UAS transgenes under the control of their specific GAL4s or Gr64f-GAL4, n = 6-8.
Figure 1B, Boxplots showing the cell size (y-axis) over time (x-axis) of all the samples as they progressed in the cell cycle. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.
Figure 2Each plot shows the relative mass isotopomer distribution (MID) values for metabolite species detected from cell extracts in each of the five independent experiments shown in Figure 1. Each value was divided by the average value of the entire time series (x-axis) for that species from the same experiment, and Log2-transformed (y-axis). Loess curves and the std errors at a 0.95 level are shown. The Log2(relative abundance) values of pyruvate(M1) and leucine(M6) showed the most significant changes
Figure 3B, The indicated strains (all in the prototrophic CEN.PK background; were spotted at 10-fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Exogenous amino acids were added at 1mM final concentration, as indicated in each case. The plates were incubated at 30 °C and photographed after 3-days.C, DNA content profiles of BAT1 and bat1Δ cells from asynchronous cultures, in SMM medium. Where indicated, exogenous amino acids were added at 1mM final concentration. On the x-axis of the histograms is fluorescence per cell, while on the y-axis is the cell number. Peaks corresponding to cells in G1 with unreplicated (1N) and cells in G2 and M phases with fully replicated (2N) DNA are indicated. The percentage of cells with G1 DNA content (%G1) from 3 independent measurements is shown in each case (mean and sd).
Figure 4A, The indicated strains were spotted at 10-fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Rapamycin was added at 30 ng/mL, as shown in each case. The plates were incubated at 30 °C and photographed after 5 and 7 days, as indicated.B, Immunoblots of total cell extracts from asynchronous BAT1 and bat1Δ cells, from four independent experiments. The signal from Pgk1 (α-Pgk1) is shown on the blot at the bottom, and from phosphorylated Rps6 (α-Rps6-P) is on the blot above, indicated for two exposures (2 min, top; 20 sec, middle).D, Exogenous addition of valine leads to sustained activation of TORC1 and phosphorylation of Rps6 in cells lacking Bat1. Wild type (BAT1+) and bat1∆ (two independent isolates, #4, and #5) strains were grown overnight in minimal (SMM) medium, diluted to 1E+06 cells/mL in fresh SMM media containing the indicated amino acid (at 1mM), and harvested when they reached 5E+06 cells/mL. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in immunoblots from total cell extracts in each case. The relative levels of Rps6-P/Pgk1 are shown in each case at the bottom. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.
Figure 5A, Immunoblots of total cell extracts from synchronous, elutriated wild-type (CEN.PK) cells in minimal (SMM) medium with exogenous Leu or Val added at 1mM immediately after elutriation. At the top, the percent of budded cells (%B), cell size (in fL), and time (in min) are indicated. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in each case.B, Quantification of the levels of phosphorylated Rps6 and Pgk1 from independent experiments done as in A. The relative levels of each protein across the cell cycle is shown on the y-axis, as a function of cell size (x-axis). Loess curves and the std errors at a 0.95 level are shown.