EMBO/soda-nel huggingface dataset:
Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4 (2013)
Figure 4

Rab12 regulates mTORC1 activity and autophagy by controlling the PAT4 protein concentration. (A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated. (B) Quantification of the phospho‐S6K levels shown in (A). (C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown. (D) A schematic model of inhibition of autophagy followed by increased activation of mTORC1 in Rab12‐depleted cells. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; PAT4, proton‐coupled amino‐acid transporter 4; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.
Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment (2012)
Figure 1

PINK1 processing is regulated by MPP, PARL, m‐AAA and ClpXP. (A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP. (B) Immunoblots against indicated proteases in siRNA‐transfected cells. (C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.
Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment (2012)
Figure 3

The levels of mature 52‐kDa PINK1 are regulated by MPPβ, PARL, AFG3L2, ClpP and the proteasome. (A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour. (B) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA. (C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.
Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment (2012)
Figure 4

Knockdown of MPPβ leads to full‐length PINK1 accumulation at the outer mitochondrial membrane. (A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK). (B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.
Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment (2012)
Figure 5

MPPβ knockdown induces Parkin recruitment and mitophagy. (A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification). (B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA. (C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP. (D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.
FIP200 regulates targeting of Atg16L1 to the isolation membrane (2013)
Figure 1

Atg16L1 interacts with the ULK1–Atg13–FIP200–Atg101 complex. (A) Yeast two‐hybrid screening for Atg16L1‐interacting proteins. The Atg16L1‐interacting FIP200 clones are indicated. (B, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies. (D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP. (E) HEK293T cells were harvested and treated with DSP. (F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis. (G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.
FIP200 regulates targeting of Atg16L1 to the isolation membrane (2013)
Figure 4

Atg16L1Δ(230–300) mutant is impaired in isolation membrane targeting and partially restores the autophagic defects of Atg16L1 KO MEFs. (A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1–588), Atg16L1(1–230) or Atg16L1Δ(230–300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands. (B,C) Atg16L1 KO MEFs stably expressing GFP–ULK1 and either full‐length Atg16L1(1–588), Atg16L1(1–230) or Atg16L1Δ(230–300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1–230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset. (D, E) Cells stably expressing GFP–LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP–LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.
FIP200 regulates targeting of Atg16L1 to the isolation membrane (2013)
Figure 5

Atg16L1(1–230) localizes to aberrant membranes in a FIP200‐independent manner. (A) Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1–588), Atg16L1(1–230) or Atg16L1Δ(230–300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1–230), but not Atg16L1Δ(230–300), showed punctate structures under starvation conditions (inset). (B) FIP200 KO MEFs stably expressing either CFP–Atg16L1(1–588), CFP–Atg16L1(1–230) or CFP–Atg16L1Δ(230–300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1–230). (C,D) FIP200 KO MEFs stably expressing CFP–Atg16L1(1–230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset. (E) Hypothetical model of membrane targeting of Atg16L1. Interaction of Atg16L1 with FIP200 leads to proper targeting of this complex to the autophagosome formation site (middle). Without FIP200, puncta formation of Atg16L1 is impaired probably owing to a self‐inhibitory role of the C‐terminal WD‐repeat domain (left). If the WD‐repeat domain is deleted, the N‐terminal half of Atg16L1 localizes to aberrant membranes as well as the autophagosome formation site (right). KO, knockout; MEFs, mouse embryonic fibroblasts.
LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis (2021)
Figure 2

Figure 2 - Ctcflos stands out as strongest regulated lncRNA in brite adipogenesis in vitro and during iBAT activation in vivo.

(A) Venn diagram of lncRNAs positively regulated in brite adipogenesis in vitro (see Figure 1 E) and induced in cold-mediated activation of interscapular brown adipose tissue (iBAT) in vivo. Seven lncRNAs are upregulated in both conditions.

(B) Table of co-regulated lncRNAs. Three Ctcflos transcripts stand out as strongest regulated lncRNA candidates.

(C-E) Ctcflos meets the three selection criteria for positively regulated lncRNAs in brite adipogenesis. (C) Correlation of Ctcflos (mean of transcript 1; 2; 3) with Ucp1 transcript levels across differentiated brite adipocytes derived from the five mouse strains (transcript levels in RPKM). Mean values, n=3 (biological replicates), pearson correlation, *p<0.05. (D) Ctcflos transcript levels (mean of transcript 1; 2; 3) in differentiated compared to undifferentiated brite adipocytes of 129S6 mice (transcript levels in RPKM). Mean and individual values, n=3 (biological replicates), unpaired t test, ****p< 0.0001. (E) Ctcflos transcript levels (mean of transcript 1; 2; 3) in differentiated brite (+rosi) compared to differentiated white (-rosi) adipocytes of 129S6 mice (transcript levels in RPKM). Mean and individual values, n=3 (biological replicates), unpaired t test, *p< 0.05.

(F, G) Induction of Ctcflos along with Ucp1 in vivo in cold-activated iBAT Data ref: (Maurer S, 2018). Time course of (F) Ctcflos and (G) Ucp1 transcript levels in iBAT of 0, 6, 24 or 48 hours cold (4°C) exposed C57BL/6J mice (transcript levels in RPKM). Mean and individual values, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.
LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis (2021)
Figure 3

Figure 3 - Ctcflos is a polyadenylated, nuclear enriched and adipose tissue predominantly expressed lncRNA. (A) Ctcflos locus. Location and structure of annotated Ctcflos isoforms (Ensembl 99). Green, blue and brown marks display primer binding sites, red arrows mark target areas of LNA Gapmer antisense oligos.

(B) Gel picture of Ctcflos transcripts expressed in differentiated brite adipocytes amplified by polymerase chain reaction (PCR) using transcript specific primers.

(C) Comparison of Ctcflos, Ucp1 and 45SrRNA transcript abundance in total RNA and poly A RNA fraction of primary differentiated brite adipocytes derived from 129S6 mice, assessed by qPCR, n=1.

(D) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice, assessed by qPCR. Mean values ± SD, n=1 (biological replicate) for Ucp1n=3 (biological replicates) for Ctcflos .

(E) Ctcflos, 45SrRNA and 12SrRNA transcript abundance in nuclear, cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice, assessed by qPCR. Mean values ± SD, n=6 (biological replicates), two-way ANOVA (Tukey-test), ****p< 0.0001.
Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2 (2016)
Figure 1

Figure 1. Regulation of the Hippo pathway and YAP during liver regeneration.

A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point.

B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted.

C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group.

D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence. Images are representative of 3 independent animals per condition.

E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal.

F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B & and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.
Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2 (2016)
Figure 2

Figure 2. Hepatocyte proliferation is impaired in aged mice.

A-C. Representative photomicrograph of young (6-8 weeks) and aged (>12 months) livers 40 h post PH stained with Ki67 and visualized by IHC and H & E for histology. Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2.0 software. A proliferation index (p.i.) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as (+) regenerating, p.i.>10% or (-) non-regenerating, p.i.>10%.

D. Number of young (6-8 weeks) and aged (>12 months) mice with (-) non-regenerating and (+) regenerating or livers based on p.i., 40 h post PH. Animals deemed non-surviving for ethical reasons were euthanized 24 hours post PH. Representative results of a single experiment with n=9 aged and n=6 young animals per group.
Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2 (2016)
Figure 3

Figure 3. Hippo signalling and YAP activation are impaired in aged mice.

A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SD

B. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.
Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2 (2016)
Figure 4

Figure 4. Inhibition of MST provokes hepatocyte proliferation in vivo.

A. Eight weeks old mice were injected with liposomes coupled to scrambled siRNA (siScr) or sequences targeting MST1 & MST2 (siMST). Each dot is representing an independent animal. The % of Mst1 & Mst2 mRNA remaining in the liver was tested by RT-PCR, 1, 3 and 6 days post injection and calculated compared to control non-transfected livers. Unpaired, two-tailed Student t-test was used to calculate the significance of mRNA remaining in comparison to non-transfected control livers at each time point.

B. Western blot detection of MST1, p-LATS1 and β-actin 1, 3 and 6 days post siRNA injection. A representative animal from each time point is shown.

C. Western blot detection of YAP1, p-YAP1, H3 and p-H3, 3 days post siRNA injection. Two representative animals from each group are shown. All samples were run on the same blot. The blot is split due to non-consecutive loading of the samples.

D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, 1, 3 and 6 days post injection. Log2 fold change was calculated using non-transfected mouse liver as control. Unpaired two tailed Student t-test was used to calculate the significance of fold change of in comparison to a panel non-transfected control livers.

E. HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes. Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts/biliary cells with an arrow in tissue 3 days post siRNA delivery. The percentages of positive hepatocytes at day 3 and day 6-post injection are graphed. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes in comparison to non-transfected control livers.

Data information: Throughout the figure representative results of a two independent experiment with n = 3 animals for group are depicted. Error bars represent mean ± SD.
Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2 (2016)
Figure 5

Figure 5. Inhibition of MST improves liver regeneration in aged mice.

A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. “n” indicates the number of animals per group.

B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image.

C. Representative photomicrograph of an aged liver treated with siMST and stained with H&E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion.

D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH.

E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls.

F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group.

G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.

Data information: Error bars represent mean ± SD
Contribution of intertwined loop to membrane association revealed by Zika virus full-length NS1 structure (2016)
Figure 1

Figure 1. Overall structure of ZIKV NS1 dimer and extended surface for membrane association.

A. NS1 dimer with one subunit in grey and the other colored by domain (green, β-roll; orange, wing with magenta connector subdomain; blue, central β-ladder). N-linked glycosylation sites are shown in sticks, and the glycans are shown in spheres.

B. Topology diagram for the NS1 monomer, colored as the pattern in (A). Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles.

C. Side views of the NS1 dimer from the wing (left) and the end of the β-ladder (right). The β-roll (green), connector subdomain (orange) and the intertwined loop (yellow) of the wing domain form a discontinuous protrusion on one face of the β-ladder with the spaghetti loop (cyan) on the other face. The wing domain is omitted from the left image for clarity.
Contribution of intertwined loop to membrane association revealed by Zika virus full-length NS1 structure (2016)
Figure 2

Figure 2. Model of the ZIKV NS1 hexamer.

The ZIKV NS1 hexamer is modeled by using the full-length DENV2 NS1 structure (PDB code: 4O6B) (Akey et al, 2014). Each monomer is colored by domains (green, β-roll; orange, wing; magenta, connector subdomain; blue, central β-ladder). The intertwined loop “spike” of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines.
Contribution of intertwined loop to membrane association revealed by Zika virus full-length NS1 structure (2016)
Figure 3

Figure 3. Variation in electrostatic surface potential of flavivirus NS1 proteins.

ABC. Electrostatic surface views of NS1 from DENV2 (PDB: 4O6B, A) and WNV (PDB: 4O6C, B) (Akey et al, 2014), as well as ZIKV©, showing diverse characteristics on certain regions. Surfaces are colored by electrostatic potential at neutral pH from -2 kT (red) to +2 kT (blue) using PyMOL (http://pymol.org/).
Contribution of intertwined loop to membrane association revealed by Zika virus full-length NS1 structure (2016)
Figure 4

Figure 4. NS1 sequence conservation mapped onto the protein surface and models of membrane association for ZIKV, WNV and DENV2.

A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.

B. Models of intertwined loop “spike” interacting with the cellular membrane. The disordered “spike” regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.