Journal: PLoS Pathogens

Article Title: Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

doi: 10.1371/journal.ppat.1007475

Figure Lengend Snippet: (A) Fluorescence microscopy analysis. mNG THT1 and mNG THT2 localise to the cell surface in bloodstream and insect stage T . brucei , respectively, as shown by direct mNeonGreen (mNG) fluorescence. (B) Differential RNA-seq expression analysis of THT1 and THT2 genes in BSF and PCF showing developmental control. Total number of reads at the THT1/THT2 locus, applying an uniqueness filter of MapQ>1, are depicted using the Artemis genome browser ; raw transcriptomic data was derived from Hutchinson et al . (C) Western blot analyses indicating stage specific expression of in situ tagged mNG THT1 and mNG THT2 in BSF (left) and PCF and cells differentiated in DTM medium (right). WT, wild-type; EF1α serves as a loading control. (D) Differentiation in DTM medium of mNG THT expressing cells ( mNG THT2 cells shown) was validated by immunofluorescence microscopy with stage specific surface markers, VSG-2 for BSF and EP procyclin for PCF. In A and D, DNA was counter stained with DAPI; Scale bars 5 μm.

Article Snippet: A codon-optimised mNeonGreen (mNG) sequence was cloned using AvrII and PacI (NEB) restriction sites in the pNAT vector [ ].

Techniques: Fluorescence, Microscopy, RNA Sequencing Assay, Expressing, Derivative Assay, Western Blot, In Situ, Immunofluorescence, Staining

Journal: EMBO Reports

Article Title: Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion

doi: 10.1038/s44319-024-00190-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The initial plasmid backbone used was the pLenti-spCas9-T2A-Crimson-P2A-puro (kindly provided by Dr. Feng Zheng) and derived versions of this plasmid were also generated to express mNeonGreen (Addgene #122183), TagRFP-T (Addgene #122200) or iRFP670 (Addgene # 122182) fluorescent proteins.

Techniques: Isolation, Recombinant, Sequencing, Knock-Out, DNA Ligation, Transfection, Cell Viability Assay, Blocking Assay, Protease Inhibitor, Virus, Software, Electroporation, Imaging, Polymer, Microscopy, Flow Cytometry, Membrane

Journal: Cell

Article Title: The Golgi Outpost Protein TPPP Nucleates Microtubules and is Critical for Myelination

doi: 10.1016/j.cell.2019.08.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LGC Biosearch Technologies Stellaris RNA Fish, Custom Recombinant DNA Plasmid: tdTomato-MannII-N-10 Addgene Cat#58110 Plasmid: mCherry-EB3 Gift from Erika Holzbaur N/A Plasmid: EB3-EGFP Gift from Erika Holzbaur N/A cDNA: human EB3 (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"AY893969","term_id":"60832325","term_text":"AY893969"}} AY893969 ) Harvard PlasmID Database Plasmid ID: HsCD00331591 cDNA: human TPPP (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"BC131506","term_id":"124297936","term_text":"BC131506"}} BC131506 ) Harvard PlasmID Database Plasmid ID: HsCD00347208 cDNA: rat TPPP NCBI Reference Sequence {"type":"entrez-nucleotide","attrs":{"text":"NM_001108461","term_id":"157823714","term_text":"NM_001108461"}} NM_001108461 Plasmid: pHAT2 Addgene Plasmid ID: 112583 Plasmid: TPPP-mNeonGreen This paper N/A Plasmid: pEGFP-C1 Takara Clontech Replaced with: Cat#632468 Plasmid: TPPP-EGFP This paper N/A Software and Algorithms FIJI/ImageJ ( Schindelin et al., 2012 ) Version 2.0.0-rc-69/1.52n; http://fiji.sc Neurolucida 360 MBF Bioscience https://www.mbfbioscience.com/neurolucida360 Neurolucida Explorer MBF Bioscience https://www.mbfbioscience.com/neurolucida-explorer Photoshop CS6 Adobe Systems Version 13.0 ×64 Prism Graph Pad Version 8 SoftWoRx GE Healthcare Life Sciences DeltaVision Imaging System Other 12-well plate containing cell crown inserts of Mimetix aligned fibers (PLLA, 2-micron fiber diameter) Electrospinning UK via Amsbio Cat#TECL-006 Tissue-Plus O.C.T.

Techniques: Western Blot, Staining, Recombinant, Affinity Chromatography, Fluorescence, Immunoprecipitation, Sequencing, Modification, Knock-Out, Plasmid Preparation, Software, Imaging, Microscopy, Electron Microscopy, Transferring

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Phylogenetic/guide trees obtained following multiple amino acid sequence alignment of the CDK7, cyclin H, and Mat1 homologs from C. neoformans ( Cn ), Homo sapiens ( Hs ), S. cerevisiae ( Sc ), and S. pombe ( Sp ). The trees show that Cn CDK7 and Cn Mat1 proteins cluster with the human homologs. Cn CDK7 (CNAG_06445 and XP_012053336.1 ), Hs CDK7 ( NP_001790.1 ), Sc CDK7/ Sc Kin28 ( NP_010175.1 ), Sp CDK7/ Sp Mcs6 ( NP_596349.1 ), Cn Cyclin H (CNAG_04405 and XP_012051791.1 ), Hs CycH ( NP_001230.1 ), Sc CycH/ Sc Ccl1 ( NP_015350.1 ), Sp CycH/ Sp Mcs2 ( NP_595776.1 ), Cn Mat1 (CNAG_05877 and XP_012050601.1 ), Hs Mat1 ( NP_002422.1 ), Sc Mat1/ Sc Tfb3 ( NP_010748.3 ), and Sp Mat1/ Sp Pmh1 ( NP_596334.1 ). Numbers represent sequence distances.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Sequencing

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 forms a CAK complex with Mat1 and cyclin H that localizes to the nucleus. ( A ) Schematic depicting the addition of the mNeonGreen (mNG), V5 epitope (V5), and 6× His (His) tags on CDK7, CycH, and Mat1, respectively, in the CDK7 triple-tagged strain as described in the Supplemental Method. ( B ) Immunoprecipitation (IP) and Western blotting with antibodies to each tag demonstrates formation of a CAK complex: in the left panel, Cn CDK7 was immunoprecipitated from the CDK7 triple-tagged strain with anti-mNG trap and subjected to SDS-PAGE and Western blotting with anti-mNG, anti-6× His, and anti-V5, detecting CDK7, CycH, and Mat1, respectively (lane 2). In the right panel, CycH was immunoprecipitated from the CDK7 triple-tagged strain with V5-Trap, followed by Western blotting with the various antitag antibodies (lane 2). In both panels, the non-tagged parent KN99 WT strain (WT) was taken through the same procedure as a negative control (lane 1). The total lysates used for each IP were probed with anti-PSTAIR to demonstrate the presence of Cdc2 in all IP samples. Epifluorescence microscopy validated the fluorescence of Cn CDK7 in the CDK7 triple-tagged strain ( C ) and the nuclear localization of the Cn CAK complex ( D ). DIC, Differential interference contrast.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Immunoprecipitation, Western Blot, SDS Page, Negative Control, Epifluorescence Microscopy, Fluorescence

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Chemical structures of the human CDK7 inhibitors tested in this study. Inhibitors are arranged based on their molecular scaffold (in blue): pyrimidine (THZ1, SY-1365, and CDK7-IN-3), pyrrolidinopyrazole from p21-activated kinase (PAK4) (CDK7-IN-1, YKL-5-124, and IV-361) and pyrazolopyrimidine (samuraciclib). Red regions indicate the covalent warhead. THZ1 served as a molecular starting point for the creation of all compounds (indicated by the arrows), except Samuraciclib.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques:

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 is inhibited by human CDK7 inhibitors. Kinase assays were performed over a 300 min time course in the absence (DMSO) and presence of the indicated concentrations of each CDK7 inhibitor, using pulled-down CAK, CDK7 peptide substrate, and Kinase-Glo reagent. The latter allowed ATP consumption (due to the phosphorylation of the peptide substrate by CDK7) to be measured as a relative luminescence unit. The full dose–response time curves are shown in . Only CDK7 enzyme activity at 300 min is plotted and is expressed as “relative activity (%)” after normalization as described in Materials and Methods. The CDK1/CDK2/CDK4/CDK5 inhibitor, Purvalanol A, is included as a negative control. The results represent the mean relative activity ± SEM ( n = 2–3 independent experiments). Statistical analysis was performed using ordinary one-way analysis of variance with Dunnett’s multiple comparison test, comparing each concentration to the DMSO control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Activity Assay, Negative Control, Comparison, Concentration Assay, Control

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Phosphoproteomic analysis of SY-1356-treated Cn strain H99. ( A ) K-means clustering using the STRING database was performed on the 122 proteins that had reduced phosphorylation following CDK7 inhibition with SY-1365 (see the Supplemental Method and for details). This resulted in four functional clusters (PPI enrichment P value <5 × 10⁻⁴). Proteins in pink are discussed in the main text. ( B ) Representative Western blot of n = 3 independent experiments (see scatter dot plot), validating reduced phosphorylation of the MAPK, Hog1 (Hog1p) following treatment with SY-1365, as compared to total Hog1 (Hog1t). In the scatter dot plot, the reduction in Hog1p levels after 1 h treatment with SY-1365 is statistically significant ( *P = 0.013, unpaired t -test; bars represent mean intensity ± SD).

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Inhibition, Functional Assay, Western Blot

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 phosphorylates Ser5 and Ser2 in the CTD of the Rpb1 subunit of RNAPII in vivo ( A ) Representative Western blot of three independent experiments (see adjacent scatter dot plot) showing that SY-1365 inhibits phosphorylation of Rpb1 on Ser5 (Rpb1-Ser5p). In the adjacent scatter dot plot, the reduction in Rpb1-Ser5p after 1 h treatment with SY-1365 when normalized to DMSO treatment is statistically significant (* P = 0.013, unpaired t -test; bars indicate mean intensity ± SD). ( B ) Representative Western blot of three independent experiments (see adjacent scatter dot plot) showing that SY-1365 inhibits phosphorylation of Rpb1 on Ser2 (Rpb1-Ser2p). In the adjacent scatter dot plot, the reduction in Rpb1-Ser2p after 1 h treatment with SY-1365 when normalized to DMSO treatment is statistically significant (* P = 0.0145, unpaired t -test; bars indicate mean intensity ± SD). In panels A and B , the levels of Rpb1-Ser5p and Rpb1-Ser2p in the SY-1365- and DMSO-treated samples were normalized to histone H3, which was used as a loading control.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: In Vivo, Western Blot, Phospho-proteomics, Control

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: RNA-seq data analysis of SY-1365-treated Cn identifies CDK7 functions including splicing regulation. RNA-seq was performed on untreated and SY-1365-treated Cn ( n = 3) as described in Materials and Methods. ( A ) A principal component analysis plot shows that replicates correlate well, with each treatment group having a distinct cluster profile. Gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package to identify Gene Ontology categories ( B ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( C ) that were significantly enriched following SY-1365 treatment. Diamond plots display the enriched pathways, where size represents the percentage of enriched genes and color indicates the statistical significance of enrichment. ( D ) Table presenting alternative splicing events (ASEs) determined upon SY-1365 treatment. Inhibition of Cn CDK7 leads to more intron retention events over intron exclusion. Normalized coverage plots of representative ASE with ( E ) intron retention and ( F ) intron exclusion upon SY-1365 treatment.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: RNA Sequencing, Alternative Splicing, Inhibition

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Model depicting the role of Cn CDK7 in regulating the cell cycle, transcriptional processes, and translation. Cell cycle regulation: in the nucleus, Cn CDK7 in the CAK complex activates Cdk1 to promote cell cycle progression through G 2 /M. Activated Cdk1 also phosphorylates Cdc24 to promote actin polymerization. Cn CDK7 either directly phosphorylates components of the Hog1 signaling pathway or acts through an intermediary protein affected by Cn CDK7-dependent phosphorylation. Transcription: also in the nucleus, TFIIH-associated CAK phosphorylates Ser5 on the CTD of the Rpb1 subunit of RNAPII (see ), allowing RNAPII release from the promoter to initiate transcription. Ser5 phosphorylated-RNAPII pauses ~30 bp post-initiation and interacts with the capping enzymes, Ceg1p (RNA guanylyltransferase) and Cet1p (RNA triphosphatase), to form a capping complex. This complex allows the co-transcriptional formation of the 7-methylguanosine (7 mG) cap (red circle) on the 5′ end of newly synthesized (nascent) mRNA, ensuring mRNA stability, nuclear export, and translation. Cbc1 (mammalian Cbp80 homolog) and Cbc2 (mammalian Cbp20 homolog) then form a heterodimeric cap binding complex (CBC) that binds to the 7mG cap to stimulate formation of the pre-initiation complex (PIC) via the transcription regulator, Mot1. Post-release of the RNAPII transcriptional pause, Cn CDK7 is essential for Ser2 phosphorylation of Rpb1 to allow transcription elongation (see ). This occurs via Cn CDK7-mediated activation of CDK9. The CBC also links capping to splicing by promoting the recruitment of U1 snRNP to the 5′ splice site to initiate spliceosome assembly. The spliceosome, comprising U1–U6 snRNPs, Sf3b1, Msl5 (binds to the branch point sequences of the intron), and Cwf19 (facilitates spliceosome disassembly and mRNA release), mediates intron removal. As in humans, Cn CDK7 may also enhance spliceosome maturation by activating Cn CDK11 to phosphorylate Sf3b1 . Translation: in the cytoplasm, Pab1 binds the 3′ poly(A) tail of mature mRNAs, protecting them from degradation. Two major deadenylation complexes then act sequentially to regulate mRNA stability and turnover: the Pan2/Pan3 complex trims the poly(A) tail, while the Ccr4-NOT complex trims it further, leading to mRNA decay. The CBC at the 5′ end is replaced by eIF4E, which, along with eIF4A and eIF4G, initiates recruitment of additional eIFs to assemble ribosomal subunits on the mRNA, ultimately leading to the formation of a functional ribosome and the initiation of translation. Proteins with solid lines were identified in the CDK7 phosphoproteome (see ), while proteins, interactions, and functions delineated by broken lines are based on studies in higher eukaryotes and the presence of the homologous protein in Cn .

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Synthesized, Binding Assay, Activation Assay, Functional Assay

Journal: Cell reports

Article Title: A PINK1 input threshold arises from positive feedback in the PINK1/Parkin mitophagy decision circuit

doi: 10.1016/j.celrep.2023.113260

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The lentiviral doxycycline-inducible PINK1 expression vector, pLv-TetOn-FKBP-PINK1(110–581)-mNeonGreen ( Neo ), was created using the backbone from pLv-TetOn-MCS1-P2A-MCS2 ( Neo ) (Addgene, #89180).

Techniques: Recombinant, Bradford Assay, Mutagenesis, Plasmid Preparation, Microscopy, Cell Culture, Software, Sequencing