EMBO/soda-nel huggingface dataset:
Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 6

Figure 6. The mitochondrial Akt-MICU1 axis is critical for tumor growth Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activity. Lung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blotting. Western blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activity. Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting. Western blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activity. Glioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting. (G-I) Number of soft agar colonies (n=3 independent experiments) (G), xenograft tumor volumes (H), and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructs. (J and K) Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit. ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure. Speculative model of the mitochondrial Akt-MICU1 axis in the regulation of tumor growth. Data information: (G, I) Means ± SEMs. **p<0.01 (Student&quot;s t-test); (H) Means ± SEMs. *p<0.05; **p<0.01 (Multiple t-tests); (K) Means ± SEMs. ****p<0.0001 (one-way ANOVA).
Integrator is recruited to promoter-proximally paused RNA Pol II to generate C. elegans piRNA precursors (2020)
Figure 1

Figure 1. Integrator is required for motif-dependent piRNA precursor abundance and their short length.

A. Average mature piRNA abundance relative to EV in EV and ints-11 RNAi samples normalized using miRNA-derived size factors. Error bars represent the standard error of the mean (5 replicates).

B. Distributions of log2 fold change in piRNA abundance in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes in 5 replicates. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.

C. Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).

D. Distributions of log2 fold change in piRNA precursor abundance in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes in 5 replicates. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.

E-F. Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.
Integrator is recruited to promoter-proximally paused RNA Pol II to generate C. elegans piRNA precursors (2020)
Figure 2

Figure 2. Integrator localizes to sites of piRNA biogenesis in germ cells.

A. Live imaging of a rescue transgene expressing an eGFP-tagged version of Integrator subunit 6 (INTS-6) in the germline of C. elegans. M=mitotic region, TZ=transition zone.

B. Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).
Integrator is recruited to promoter-proximally paused RNA Pol II to generate C. elegans piRNA precursors (2020)
Figure 3

Figure 3. Chromatin fractionation identifies nascent piRNA precursors.

A. Experimental design. Germ nuclei isolated from empty vector or ints-11 RNAi-treated nematodes were fractionated into chromatin-bound and nucleoplasmic fractions. RNA corresponding to each fraction was purified and used as input for short capped RNA libraries.

B. Length distribution of chromatin-bound and nucleoplasmic piRNA precursors in wild type N2 nematodes, calculated from two merged replicates.

C. Length distribution of piRNA precursors generated from loci with weak and strong AT content enrichment downstream of the 21U-RNA in wild-type nematodes, calculated from two merged replicates.

D. Distributions of log2 fold change in piRNA precursor abundance in nucleoplasmic and chromatin fractions relative to N2 empty vector controls, for N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes in two replicates. Significance for each distribution to be different from no change was tested using a two-tailed paired Wilcoxon rank sum test, with all p-values<2.2e-16. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.

E. Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).

F-G. Distribution of the position of the ~28 nt (F) and ~48 nt (G) nascent RNA peaks in 2000 bootstrapped subsamples of 3000 precursors. Sampling probability was weighted by precursor abundance.
Integrator is recruited to promoter-proximally paused RNA Pol II to generate C. elegans piRNA precursors (2020)
Figure 5

Figure 5. Integrator knockdown results in transcriptional readthrough in a subset of motif-dependent piRNA loci.

A. Number of unique CAGE-supported TSSs mapping to a 10 nucleotide region upstream of motif-dependent 21U-RNAs. A clear enrichment of TSS 2 nt upstream of 21U-RNAs is observed.

B. Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.

C. Heatmap showing changes in CAGE signal upstream of motif-dependent 21U-RNAs upon ints-11 knockdown in an N2 and a tfiis mutant background. The total cpms corresponding to each nucleotide position upstream to 21U-RNAs are shown as a barplot (top part).

D. Length distributions of CAGE fragments initiating 2 nt upstream of motif-dependent 21U-RNAs. The total counts in each length bin are normalized to counts per million of mapped reads.

E. Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range

F. Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range
Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting (2021)
Figure 2

Figure 2: Genetic ablation of iNOS prevents LPS-driven deregulation of the TCA cycle and energy production.

Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized and the metabolome of tibialis anterior muscles was analyzed through LC-MS/MS.

Scores scatter plot of partial least square discriminant analysis (PLS-DA) model of metabolites from WT and iNOS KO mice treated with or without LPS. Metabolomic data were range-scaled and mean-centered.

Pathway Analysis using MetaboAnalyst 4.0 Software comparing significantly altered pathways from WT saline to LPS as well as iNOS KO saline to LPS. Pathways are ranked by their significance and filtered based on a Pathway Impact Score >0.1. Metabolomic data were range-scaled and mean-centered. P-values were obtained using GlobalTest and the -log(p-value) corresponding to a p-value of 0.05 is indicated by the red dashed line.

(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle. (right) Quantification of the pAMPK to AMPK ratio. Ratios are expressed relative to the saline treated controls.

Schematic of Glycolysis and the TCA cycle. White boxes denote detected metabolites. Grey boxes denote undetected metabolites. Dashed arrows indicate presence of multiple reactions between metabolites.

Relative concentrations of metabolites involved in glycolysis and TCA cycle.

Relative concentration of arginine.

Schematic of acylcarnitine metabolism. Grey circles denote key enzymes involved in metabolic processes highlighted.

Relative estimated activity of CPT1, CPT2, LCADH, and β-oxidation.

Data information: Individual data points represent values from individual mice, with a total of six mice per cohort (n = 6). (B) P-values were obtained using GlobalTest. (C-H) Error bars represent the standard deviation (SD) of the mean. Statistical comparisons were made between saline treated controls and LPS treated mice of same genotype. Plotted concentration data was relativized to saline controls of corresponding genotype. p-values were calculated with a Student's T-test (*P < 0.05; **P < 0.01). (C) Δ indicates the difference of mean values.
Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting (2021)
Figure 4

Figure 4: Pharmacological inhibition of iNOS reduces C26-induced derangement of amino acids and impairment of energy production.

Male BALB/C mice were injected subcutaneously with C26 cells (1.25x106 cells) or an equivalent volume of saline. After 5 days and everyday thereafter, the C26 tumor-bearing mice were injected with either saline or GW 5mg kg-1. After 16 days, mice were euthanized and the metabolome of tibialis anterior muscles was analyzed through LC-MS/MS.

Scores scatter plot of partial least square discriminant analysis (PLS-DA) model of metabolites from saline, C26, and C26 + GW treated mice. Metabolomic data were range-scaled and mean-centered.

Pathway Analysis using MetaboAnalyst 4.0 Software comparing significantly altered pathways from saline to C26 as well as C26 to C26 + GW. Pathways are ranked by their significance and filtered based on a Pathway Impact Score >0.1. Metabolomic data were range-scaled and mean-centered. P-values were obtained using GlobalTest and the -log(p-value) corresponding to a p-value of 0.05 is indicated by the red dashed line.

(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle (n = 3). (right) Quantification of the pAMPK to AMPK ratio relative to the saline treated control (n = 3).

Relative concentrations of metabolites involved in glycolysis.

Relative concentrations of amino acids.

Relative concentrations of odd-chain acylcarnitines.

Relative estimated activity of CPT1, CPT2, LCADH, and β-oxidation.

Data information: Individual data points represent values from individual mice, with a total of six mice per cohort (n = 6) unless otherwise stated. (B) P-values were obtained using GlobalTest. (C-G) Error bars represent the standard deviation (SD) of the mean. p-values were calculated with an ANOVA followed by Fisher's LSD test (*P < 0.05; **P < 0.01; ***P < 0.001). (C) Δ indicates the difference of mean values.
Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting (2021)
Figure 5

Figure 5: Cytokine treatment of C2C12 myotubes alters the levels of TCA cycle intermediates and activates AMPK in an iNOS-dependent manner.

C2C12 myotubes were treated with or without IFNγ (100U/mL) and TNFα (20ng/mL) and the indicated doses of GW. Protein content and metabolites were extracted from cells 24h after treatment and analyzed for AMPK phosphorylation, ACC phosphorylation, and total iNOS levels as well as GC-MS, respectively. Myotube integrity and widths as well as phosphorylation of S6 and S6K were assessed 48h after treatment.

Representative immunofluorescence imaging for myoglobin and myosin heavy chain (MyHC) in not treated (NT) controls and IFNγ/TNFα (IT) samples treated with or without GW. Nuclei were visualized with DAPI staining (n=4).

Quantification of mean fiber widths (n=4).

(top) Western blot analysis for iNOS and tubulin (n=3). (bottom) Media nitrite levels (n=3).

Heatmap visualizing mean concentration corresponding to metabolites of IFNγ/TNFα (IT) treated samples relative to not treated (NT) controls (n=3). Red and green indicate an increase or decrease in metabolite levels, respectively.

Western blot analysis for pThr172-AMPK (pAMPK), total AMPK (AMPK), pSer79-ACC (pACC), and total ACC (ACC).

Quantification of the pAMPK to AMPK ratio (left) and the pACC to ACC ratio (right) relative to the untreated control (n=3).

Cellular ATP content quantified as a percentage of the untreated control (n=3).

Western blot analysis for pThr389-S6K (pS6K), total S6K (S6K), pSer235/236-S6 (pS6), and total S6 (S6) (n=5).

Quantification of the (left) pS6K to S6K ratio and the (right) pS6 to S6 ratio relative to the untreated control (n=5).

Data information: Individual data points represent independent experimental replicates. Error bars represent the standard deviation (SD) of the mean. For statistical comparisons, Δ indicates the difference of mean values and p-values were calculated with an ANOVA followed by Fisher's LSD test.
Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis (2018)
Figure 2

Figure 2. tFT-Bcd reporter reflects average protein age.

A Schematic of tFT-Bcd reporter with mCherry and sfGFP fluorophores.

B Cartoon of the fluorescent protein maturation states in the tFT-Bcd reporter. mg and mr represent the sfGFP and mCherry maturation rates respectively (note, mr is an effective rate as mCherry has a two-step maturation process).

C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (see Methods for more details). (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).
Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis (2018)
Figure 4

Figure 4. Bcd is degraded by the proteasomes in vivo.

A,B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.

D The age of Bcd and its gradient are unaltered in fsd mutant. Comparison of a representative y/w embryo (left) and loss-of-function mutation fsdKG02393 (right) are shown. Both embryos have the tFT-Bcd construct in the same locus, are in early n.c. 14, were imaged side-by-side under identical conditions, and are displayed with the same settings. Embryo "shells" as in Figure 2 are displayed.

E Quantification of embryos as in f. Fluorescence intensity from sfGFP (green) and mCherry (magenta) for wt (N=8, filled circles) and fsd (N=6, open squares) at early cycle 14. Inset: tFT-Bcd ratio for wt (black) and FSD (gray) embryos.
Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis (2018)
Figure 5

Figure 5. The average age of Bcd proteins increases continuously during the cleavage divisions and the gastrulation.

Time-course of a representative y/w embryo with the tFT-Bcd reporter from the division cycle 10 to the early gastrulation. A movie of the same embryo with 6 min time resolution is available as Movie EV1.

A SPIM images of the embryo early development. Embryo "shells" as in Figure 2 are displayed. Left: Green fluorescence, center: Red fluorescence, right: Intensity-weighted ratiometric image (see Methods) of the mCherry/sfGFP ratio, reporting average protein age. The scale corresponds to 50 μm.

B Quantification of the same embryo as is A. Each line represents the fluorescence profile along the anterior-posterior axis of the embryo for a time frame (separated by 6 min). In the lower panel, where the mCherry/sfGFP ratio is shown, the posterior half of the embryo is not shown because the signal drops to the background levels (due to softer imaging conditions for the long term time-course). C The same data is shown as a function of time for different embryo segments.
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 1

Figure 1. PML deficiency inhibits necroptosis and apoptosis induction in BMDMs and MEFs.

(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates.

(B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates.

(C) Normal expression of RIPK1, RIPK3 and MLKL in Pml-/- BMDMs. Lysates from WT and Pml-/- BMDMs were analysed for their levels of RIPK1, RIPK3 and MLKL.

(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates.

(E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).

Data information: *p < 0.05 for multiple t-tests according to the Holm-Sidak method (A, B, D, E).
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 2

Figure 2. Attenuated phosphorylation of RIPK1[S166], RIPK3 and MLKL in PML-deficient cells following necroptosis induction.

(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.

(C) Pml+/+ and Pml-/- BMDMs were treated with AT-406 at the concentration indicated for 24 h, and cell viability was then determined by ATP assay. Data show mean ± SD of three biological replicates.

(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates.

(E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.

Data information: *p < 0.05 for multiple t-tests according to the Holm-Sidak method (A-E).
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 3

Figure 3. Diminished association of RIPK1 with FADD and caspase-8 in PML-deficient cells.

(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed.

(B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.

Data information: Data (A, B) are representative of three biological replicates.
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 4

Figure 4. Resistance to TNF-induced septic shock in Pml-/- mice.

(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).

(C, D) WT and Pml-/- mice were injected with TNF and serum samples from live mice were collected 30 h later. Levels of LDH (C) and GPT/ALT (D) were quantitated. Mean ± S.E.M. is shown. P = 0.0095 (C, D) by unpaired t-test with Welch's correction.

(E, F, G) Serum levels of TNF (E), IL-6 (F) and IL-1α (G) from WT and Pml-/- mice 12 h after TNF injection. Mean ± S.E.M. is shown. Indicated P values were calculated using unpaired t-test with Welch's correction.
Promyelocytic leukemia protein targets MK2 to promote cytotoxicity (2021)
Figure 5

Figure 5. PML deficiency enhances phosphorylation of p38 MAPK, MK2 and RIPK1[S321], and a MK2 inhibitor restores necroptosis.

(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting.

(B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined.

(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E). P-values were determined by two-way ANOVA for multiple comparisons (C, E) or by Log-rank (Mantel-Cox) test (D, F).