EMBO/SourceData huggingface dataset:
Figure 4C African green monkey kidney epithelial (Vero) cells were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D MEFs were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL and western blotting analysis of MLKL and β-actin. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown. β-actin serves as an internal control (D).E MEFs were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for 24 h. Cell viability was determined by measuring ATP levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F HEK293T cells were transfected with NC or miR-324-5p, together with pmirGLO-mouse MLKL-3'UTR or the mutated form of mouse MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n = 5, biological replicates).
Figure 5A U937 cells were treated with 100 ng/ml IFN-α, 100 ng/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR analysis for the expression of MLKL. C U937 cells were treated with PBS, IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. qPCR analysis for the expression of miR-324-5p. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.B U937 cells were treated with IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. and western blotting analysis of MLKL and β-actin (left). Quantification of MLKL normalized to β-actin levels (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D, E U937 cells were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with IFN-β (D) or IFN-γ (E) for 24 h. Western blotting analysis of MLKL and β-actin.F, G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN-β (F) or IFN-γ (G) treatment. After 24 h, qPCR analysis for the expression of miR-324-5p. After 48 h, western blotting analysis of p-STAT1, STAT1, MLKL and β-actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.H, I U937 cells were transfected with NC, or STAT1 siRNA oliogs (siSTAT1). After 48 h, cells were treated with IFN-β (Η) or IFN-γ (Ι). qPCR analysis for the expression of miR-324-5p. Western blotting analysis of STAT1, MLKL and β-actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.J HT-29 cells were treated with IFN-γ for 24 h. The cell lysate was collected for ChIP analysis. STAT1 binding to miR-324-5p promoter DNA region was determined by Chip-qPCR. The amount of precipitated DNA was calculated as percent input. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Figure 6A U937 cells were transfected with NC, siMLKL-3'UTR, or miR-324-5p. After 48 h, cells were exposed to 100 ng/ml IFN-γ plus 20 μM z-VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.B qPCR analysis for the expression of MLKL (left) and miR-324-5p (middle) in PBMC-derived macrophages that were treated with PBS or IFN-γ for 24 h. The corresponding western blotting analysis of MLKL and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.C, D PBMC-derived macrophages (C) and HT-29 cells (D) were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were exposed to S+Z or IFN-γ+S+Z for 24 h. Cell viability was determined by measuring ATP levels (left). The corresponding western blotting analysis of MLKL and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.E qPCR analysis for the expression of miR-324-5p in miR-324-5p knockout HT-29 cells (miR-324-5p-/- cells) . Altered miR-324-5p DNA sequences were shown in miR-324-5p-/- clone 19# and 36#. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F MiR-324-5p+/+ HT-29, miR-324-5p-/- -19# , and miR-324-5p-/- -36# cells were treated with T+S+Z for the indicated times. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.G, H MiR-324-5p+/+ HT-29, and miR-324-5p-/- HT-29 cells were treated with IFN-β (G) or IFN-γ (H) plus 20 μM z-VAD for 24 h. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Figure 7A qPCR analysis for the expression of IFN-β (left) and IFN-γ (right) in PBMC-derived macrophages that were infected with IAV (H1N1 strain PR8) at an multiplicity of infection (MOI) of 0.2 for 24 h. B qPCR analysis for the expression of MLKL (left) and miR-324-5p (right) in PBMC-derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV (MOI=0.2) for 24 h. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.C PBMC-derived macrophages were transfected with NC, siMLKL, or miR-324-5p. After 48h, cells were treated with S+Z in the presence or absence of IAV (MOI=0.2) for 24 h. Cell viability was determined by measuring ATP levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D qPCR analysis for the expression of M gene of IAV in PBMC-derived macrophages that were transfected with NC, siMLKL or miR-324-5p for 48h, followed by the infection with IAV (MOI=0.2) for 24 h. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Figure 1A Freeze-fracture electron micrograph of wild-type yeast treated with DTT for 3 h. The left panel shows two spherical structures, one in a vacuole membrane invagination and one inside the vacuole. The middle panel shows the boxed area of the left panel at higher magnification. Arrows indicate membrane layers that are free of intramembrane particles. The right panel shows the protoplasmic fracture face (P face) of cortical ER with a high density of intramembrane particles. Cy, cytosol; N, nucleus; V, vacuole.B Fluorescence images of cells expressing GPD promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. The upper and lower panel show vacuole-associated and perinuclear GFP-Pho8 stretches and an intravacuolar GFP-Pho8 ring, respectively. Scale bars: 2 µm.C Correlative light and electron microscopy of cells expressing GPD promoter-driven GFP-Pho8 and Sec63-mCherry. The lower panel shows the boxed area in the electron micrograph at higher magnification. The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack.D Fluorescence images of cells expressing Hmg2-GFP and either additionally expressing Sec63-mCherry or stained with CMAC. The upper and lower panel show cytosolic and vacuolar Hmg2-GFP rings, respectively. Scale bars: 2 µm.
Figure 2A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm.B Electron micrograph of a cell expressing GPD promoter-driven GFP-Pho8. Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination. Black arrowheads point to vacuole membrane that encases the bent stack and the whorl.C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks. n = 43.The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.
Figure 3Fluorescence images of ∆atg7 and ∆atg8 cells (A) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.Fluorescence images of ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3.D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type.F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control. G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4.
Figure 4A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3.Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.
Figure 5A Fraction of wild-type (WT), ∆vps23, ∆snf7 and ∆vps4 cells with GFP-Pho8 structures. Mean + SEM, n ≥ 3.B Fraction of GFP-Pho8 structures classified as stacks, cytosolic whorls, uptake intermediates or vacuolar whorls. The number of GFP-Pho8 structures in WT, ∆vps23, ∆snf7 and ∆vps4 cells was 562, 345, 367 and 449, respectively. The table shows the results of Chi-Square tests for independence for all pairs of strains. ***, highly significant (P < 0.00001); ns, not significant (P > 0.05).C Fraction of stacks classified as peripheral, perinuclear or vacuole-associated. The number of stacks in WT and ∆snf7 cells was 252 and 351, respectively. The observed distributions in the two strains were analyzed with a Chi-Square test for independence. ***, highly significant (P < 0.00001).Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.
Figure 6A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test.C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.
Figure 1Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm.Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.
Figure 2pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.
Figure 3H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays.Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR.
Figure 4Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.
Figure 5De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.
Figure 6Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.
Figure 1A The mRNA expression of FAM189A2/ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines. In qRT-PCR analyses, the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16. Relative expression ratios shown as mean + SD from three biological replicates.B Relative expression of FAM189A2/ENTREP in the normal (white) and cancer (green) tissues of breast (Curtis Breast), lung (Okayama Lung), colorectal (TCGA Colorectal) and head and neck (Estilo Head-Neck). Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots. C Relative expression of FAM189A2/ENTREP in the primary (green) and the metastatic sites (blue) including lymph node, bone, liver, lung, soft tissues of prostatic cancer (Grasso Prostate) (left) and in the normal (white), primary (green) and metastatic sites (three cases of sentinel lymph node) (blue) of breast cancer (TCGA Breast) (right). Note that only three cases of the metastatic breast cancer were available in the dataset. Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter