EMBO/soda-nel huggingface dataset:
Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 5

Figure 5 | ATG5 regulates neuronal cAMP/PKA/pCREB signaling.

(A-B) Western Blot analysis of pCREB protein levels in Atg5flox:CAG-CreTmx WT/ KO cultured neurons at DIV14, treated with forskolin (WTDMSO set to 100%, WTForskolin: 214.17± 48.87%, KODMSO: 63.26± 15.85%, KOForskolin: 33.50± 10.84%, pWT DMSO/WT Forskolin=0.042, pWT Forskolin/KO Forskolin=0.002, two-way ANOVA with Tukey's multiple comparisons). n=4. The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB/CREB ratio.

(C-D) High-content screening microscopy analysis of pCREB fluorescence intensity in neurons isolated from 8-12-week-old Atg5flox:CamKIIα-Cre:Ai9 WT/KO mice, treated either with DMSO or with 2µM, 10µM Forskolin or 40mM KCl (WTDMSO set to 1, KODMSO: 0.76±0.07, WTForskolin 2µM: 1.31±0.34, KOForskolin 2µM: 0.68±0.04, WTForskolin 10µM: 1.78±0.31, KOForskolin 10µM: 0.87±0.15, WTKCl 40mM: 1.64±0.23, KOKCl 40mM: 0.66±0.13, pWT Forskolin 10µM/KO Forskolin 10µM= 0.047, pWT KCl 40mM/KO KCl 40mM= 0.034, two-way ANOVA with Tukey's multiple comparisons test). n=3.

(E) Next-generation RNA sequencing-based Kallisto transcriptome output of FACS-sorted neurons isolated from 3-week-old Atg5flox:Slc32a1-Cre:Ai9 WT/KO mice (cut-off p<0.02, FC<-2/>2) (nWT=4, nKO=5). See also Dataset EV2 and Appendix Figure S8D for differential gene expression analysis.

(F, G) EnrichR-based analysis of transcription factor-binding site enrichment using ChEA2016 databank. Analysis was applied to differentially downregulated (F) and upregulated (G) gene sets (cut off p<0.01, log2FC<-1/>1), obtained using Sleuth-based algorithm for gene expression analysis of RNA transcriptome shown in (E).

(H) Top downregulated CREB1 target genes in autophagy-deficient FACS-sorted Atg5flox:Slc32a1-Cre:Ai9 KO neurons.

(I, J) Representative fluorescent images and subsequent analysis of primary cortico-hippocampal neurons containing nuclear pCREB either under DMSO-treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89. Neurons were identified by their polarized morphology in DIC. White rectangular boxes indicate panel magnified to the right. In the inserts, LUT gradient was added to highlight the pCREB intensity in the nucleus. Scale bar: 50 µm. DMSO: 52.50±2.757, starvation: 70.00±2.697, starvation+H89: 53.92±3.147; pDMSO/starvation<0.0001, pstarvation/starvation&H89=0.0004, one-way ANOVA with Tukey's multiple comparisons test, n=30 images from n=3 independent experiments.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001. All data represent mean ± SEM. All n represent biological replicates.
Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift. (2010)
Figure 1.

Rb−/− myoblasts transiently differentiate to form short myotubes that do not twitch, express autophagy markers, and slowly degenerate. (A) Western blot analysis for pRb in DM-2 cultures. (B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments. (D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green). (E) LC3-RFP expression in control (ctrl) and Rb−/− myotubes. Insets show nuclear DAPI stain (blue). (F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest. (G) Representative Western blot for LC3 in E16.5 control and mgRb:Rb−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student’s t test. (I) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.
Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift. (2010)
Figure 2.

The Rb−/− muscle defect is characterized by perinuclear mitochondrial/autophagosome aggregates, reduced mitochondrial DNA content, and reduced ATP production. (A, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student’s t test. (C) MitoTracker (red) and LC3 immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/− myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/− myotubes (total) or Rb−/− myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student’s t test. (E) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrial DNA/nuclear DNA in the indicated cultures (n = 3). (G) Ratio of mitochondrial DNA/nuclear DNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F–H) Error bars represent SD. *, P < 0.001.
Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift. (2010)
Figure 3.

Rescue of Rb-deficient myotube degeneration by Bcl-2 and autophagy inhibitors. (A) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad.GFP or Ad.Bcl-2. Each point represents the mean ± SD of six fields from six independent experiments. Bcl-2–rescued, twitching Rb−/− myotubes are shown in Video 2. (inset) Western blot for Bcl-2 in Ad.Bcl2-transduced versus uninfected primary myoblasts. (B) Immunostaining for MCK (green) in Ad.Bcl-2–transduced control (ctrl) and Rb−/− myotubes at DM-6. (C) Western blot for MCK, myogenin, MHC, and troponin T in indicated cultures at DM-6 after transduction with Ad.Bcl-2. Tubulin was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4). (E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA–rescued Rb−/− myotubes at DM-8. 3-MA–rescued, twitching Rb−/− myotubes are shown in Video 4. (F) Immunostaining for BrdU (green) and MHC (red) of 3-MA–treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10. (G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.
Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift. (2010)
Figure 4.

Hypoxia rescues muscle degeneration after chronic or acute inactivation of Rb by inducing glycolysis via HIF-1α. (A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine. (B) Representative immunoblot (left) and mean expression of myogenin and MCK normalized for MHC expression (right; n = 3). (C) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunoblotted for pRb. Tubulin was used as a loading control. (D) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunostained for pRb (red). (insets) DAPI staining for nuclei (blue). (E) Immunostaining for MHC in Rbf/f myoblasts transduced with Ad.Cre and induced to differentiate in normoxia (top and middle) or hypoxia (bottom). (F, top) Brightfield images of Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. (bottom) Larger view of the boxed field. Arrowheads point to rescued Rb−/− myotubes. This field is shown in Video 6. (G) Quantification of myotube formation in Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. Counts are mean of six representative fields (n = 3). *, P = 0.004. (H) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions. Myotube counts are the mean of six fields (n = 4). (I) MHC staining (red) of the indicated myoblasts transduced with Ad.NF-κB/HIF-1α and induced to differentiate in normoxia. (J) Quantification of myotube formation in the indicated myoblasts transduced with Ad.EV or Ad.NF-κB/HIF-1α and induced to differentiate in normoxia. Myotube counts are the mean of six fields (n = 3). Error bars represent SD.
An integrated workflow for crosslinking mass spectrometry (2019)
Figure 1

Figure 1.

Sequential digestion workflow compared to repeated analysis and parallel digestion.

A. Sequential digestion workflow. Proteins or protein complexes are crosslinked and digested with trypsin. After splitting the sample into four aliquots, one remains single digested with trypsin (T) while the others are sequentially digested with either AspN (A), chymotrypsin (C) or GluC (G). Samples are enriched by SEC and the three high-MW fractions are analysed by LC-MS, submitted to xiSEARCH and xiFDR analysis.

B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs.

C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B.

D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.
An integrated workflow for crosslinking mass spectrometry (2019)
Figure 2

Figure 2.

Sequential digestion of the affinity-purified complexes OCCM (S. cer.) (Residue pairs observed in tryptic (green) and non-tryptic (orange) peptides)

A. Unique residue pairs mapped to the OCCM complex (PDB|5udb) and the key link Mcm2 - Orc5 (Mcm2-850-Orc5-369).

B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).

C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria (2019)
Figure 1

Figure 1: Transcriptional regulation of micV

A) Alignment of micV sequences, including the promoter regions, from various Vibrio species. The -35 box, -10 box, the TSS, the highly conserved potential seed region, and the rho-independent terminator are indicated. Lower part: Consensus motif of E. coli σE-dependent promoters.

B) V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.

C) V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.

D and E) V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 (D) or PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.

F) V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.

Data information: In (C-E), data are presented as mean ± SD, n = 3. In (F), the box plots indicate the median, 75th and 25th percentiles (boxes) and 90th and 10th percentiles (whiskers), n = 8. Statistical significance was determined using one-way ANOVA and post-hoc Holm-Sidak test.
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria (2019)
Figure 3

Figure 3: Patterns of target regulation by VrrA and MicV
A, B and C) Predicted base-pairing of MicV with the 5'UTR of ompT (A), with the 5'UTR of ushA (B) or VrrA with the 5'UTR of lpp (C). Mutations introduced in (D, E, F) are indicated.

D, E and F) V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.

G, H and I) V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.
Data information: In (D-I), data are presented as mean ± SD, n = 3.
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria (2019)
Figure 4

Figure 4: A conserved sRNA seed sequence inhibit OMP production
A) Alignment of the seed pairing sequences of VrrA, MicV and RybB.

B) V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.

C) E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.

D) V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.
Data information: In (D), the box plots indicate the median, 75th and 25th percentiles (boxes) and 90th and 10th percentiles (whiskers), n = 8. Statistical significance was determined using one-way ANOVA and post-hoc Holm-Sidak test.
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria (2019)
Figure 5

Figure 5: A conserved sRNA motif is enriched in laboratory selection experiments

A) Experimental strategy of the laboratory selection experiments: an sRNA library was generated using the rybB scaffold with nine randomized nucleotides at the 5' end, cloned into a broad-range plasmid backbone, and transferred into V. cholerae ΔrpoE cells. These colonies were pooled, grown to OD600 of 0.2 and treated with ethanol (3.5% final conc.) for 6 hours. Surviving cells were recovered on agar plates, pooled and subjected to another round of selection (3 selections total). After each selection, the plasmids of surviving cells were analyzed using high-throughput sequencing.

B) V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.

C) Plasmid contents of the strains carrying the sRNA libraries before selection (input) and after consecutive ethanol treatments (Sel1, Sel2, Sel3) were analyzed using high-throughput sequencing. Relative library complexity (left y-axis) was determined by counting sequence variants present in the normalized samples. To test for the enrichment of possible sequence motifs, the sequence variants present in each sample were counted and normalized for sequencing depth. The resulting data were analyzed for the enrichment of the conserved CRCUGCUUUU motif (right y-axis).
Data information: In (C), data are presented as mean ± SD, n = 2.
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria (2019)
Figure 6

Figure 6: Enriched sRNA variants mediate ethanol resistance by OmpA repression
A) V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.

B) V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).

C) V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.

D) V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.
Data information: In (C), data are presented as mean ± SD, n = 3. In (D), the box plots indicate the median, 75th and 25th percentiles (boxes) and 90th and 10th percentiles (whiskers), n = 8. Statistical significance was determined using one-way ANOVA and post-hoc Holm-Sidak test.
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 1

Figure 1. Induction of MAP4K3/GLK in epithelial cells of BALFs from COVID-19 patients.

A, B Distribution and classification of MAP4K3/GLK-positive cells in 20 ml BALFs of all 12 individuals (A), 3 healthy controls (HC, #1 - #3), 3 mild COVID-19 patients (#1 - #3), and 6 severe COVID-19 patients (#1 - #6) (B) from Cohort #1. Data were shown in UMAP. Single-cell gene expression of MAP4K3 (GLK) in individual cells was shown in color scale. Φ denotes macrophages; N denotes neutrophils; T denotes T cells; E denotes epithelial cells.

C, D The percentages of MAP4K3/GLK-positive KRT18+ epithelial (C) or KRT18+ TPPP3+ ciliated epithelial cells (D) in BALFs from Cohort #1.

E, F GLK mRNA levels in KRT18+ epithelial (E) or KRT18+ TPPP3+ ciliated epithelial cells (F) of Cohort #1. n (cell number) = 3, 14, 33 for HC; n = 65, 43, 74 for mild COVID-19 patients; n = 164, 62, 10, 4, 74, 66 for severe COVID-19 patients (E). n = 25, 43, 36 for HC; n = 209, 244, 141 for mild COVID-19 patients; n = 189, 223, 330, 792, 80, 243 for severe COVID-19 patients (F).

Data information: In (C, D), *, P value < 0.05 (ANOVA test); §, P value < 0.05 (Dunnett's test). In (E, F), *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001 (Kruskal-Wallis test).
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 2

Figure 2. The upregulated genes in GLK-positive epithelial cells belong to the pathways of intracellular/extracellular vesicle and viral process.

A-D Endogenous GLK levels were increased by the SARS-CoV-2 spike protein. Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells either infected with SARS-CoV-2 pseudovirus (A) or treated with the SARS-CoV-2 spike protein (C). Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells either infected with SARS-CoV-2 pseudovirus (B) or treated with the SARS-CoV-2 spike protein (D). The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group.

E-H KEGG (Kyoto Encyclopedia of Genes and Genomes) enriched pathways of the upregulated genes in GLK-positive KRT18+ epithelial cells or KRT18+ TPPP3+ ciliated epithelial cells from Cohort #1 (E and F) and Cohort #2 (G and H). Pathways belonging to different classifications are listed on the left side of the plot. Varied numbers of genes enriched in individual pathways are presented by different diameter sizes and numbers for individual dots. Adjusted P value is ranging from 0~1; less P value means greater intensiveness.

Data information: In (B, D), data are presented as means. Data shown are representative results of three (A, B, C, D) independent experiments.

Source data are available online for immunoblotting data of this figure.