Journal: NeuroImage

Article Title: A face-off of MRI research sequences by their need for de-facing

doi: 10.1016/j.neuroimage.2023.120199

Figure Lengend Snippet: Image Types Tested. All sequences used Siemens 3T Prisma scanners unless otherwise noted.

Article Snippet: 3D T2-FLAIR , Siemens sagittal 3D T2-FLAIR sequence (TR/TE/TI = 4800/441/1650 ms, variable flip angle, resolution 1.0 × 1.0 × 1.2 mm, 3x GRAPPA acceleration, fat suppression OFF).

Techniques: Sequencing, Diffusion-based Assay, Imaging, Functional Assay, Extraction, Derivative Assay, Synthesized

Journal: bioRxiv

Article Title: A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

doi: 10.1101/2025.11.24.690135

Figure Lengend Snippet: a) Western blot analysis showing a reduction in phospho-PERK, phospho-eIF2α and ATF4 expression following PERK inhibition with GSK2606414 (0.5 μM for 24 hours) in iPSC RPE CFH Y402H cells, even in the context of AKT2 inhibition, highlighting the role of PERK in regulating ATF4. ER stress inducer thapsigargin (1μM) was used as a positive control. b) Subcellular fraction immunoblots demonstrating increased shuttling of ATF4 to the nucleus under AKT2 inhibition, which is diminished upon PERK inhibition, even in the presence of AKT2 inhibition. c) Quantification of nuclear ATF4 expression shows an increase after AKT2 inhibition, whereas treatment with the PERK inhibitor leads to a reduction in the nuclear localization of ATF4. d) Immunofluorescence analysis reveals that a reduction in ATF4 targets CHOP expression and an increase in ATF4 targets phospho-NRF2 localization in the nucleus following AKT2 inhibition under serum-starved conditions, with these effects magnified in thapsigargin-treated conditions. Increase in NRF2 indicates that cells are adapting to the stress for survival. e) Co-IP of ATF4 demonstrated critical interactions with several autophagy-related genes (ATG), including ATG3, ATG4B, ATG5, and ATG7, indicating the induction of autophagy. Treatment with a PERK inhibitor (PERKi) resulted in a reduced interaction between ATF4 and these ATG genes. f) Untargeted proteomics of ATF4 pull down from DMSO and AKT2i treated conditions in iPSC RPE identified reduced expression of ribosome biogenesis proteins RPSA, RPL5, and RPL26, indicating stalled protein synthesis upon AKT2 inhibition. The expression of molecular chaperones, including HSPB1, HSP90B1, and HSP90AA1, as well as the endo-lysosomal trafficking protein Rab7a, which are crucial for protein folding and endoplasmic reticulum (ER) function, was elevated following AKT2 inhibition. RNA sequencing analysis of iPSC RPE CFH Y402H cells treated with either DMSO or AKT2i revealed upregulation of ERphagy receptor genes. g) The heatmap of hierarchical clustering with euclidean distance and ward’s method shows increased expression of CCPG1, TEX264, and the ER lumen protein SEC61G upon AKT2 inhibition. h) ERphagy flux was assessed using a tandem GFP-RFP-KDEL construct. The GFP-RFP-KDEL construct was transfected into human fetal RPE (hFRPE) cells using Lipofectamine 3000 reagent. After 48 hours of transfection, the cells were treated with either DMSO or AKT2 inhibitor to evaluate ERphagy dynamics. The analysis involved quantifying the intensity of GFP and RFP signals as well as the ratio of RFP to GFP to assess ERphagy flux quantitatively. An increase in the RFP puncta ratio upon AKT2 inhibition indicates a shift towards autolysosome formation and enhanced ERphagy flux. i) Quantification of yellow puncta (indicative of autophagosome formation) and red puncta (representing autolysosomes) was performed in a total of 50 cells per condition to assess ERphagy dynamics. The AKT2 inhibitor treatment leads to an increased frequency of red puncta, indicating enhanced ERphagy, while a reduction in yellow puncta suggests defective ERphagy is observed under DMSO treated conditions. Values represent mean ± s.d. (n = 4). j) CCPG1-LAMP1 staining (pending), k) Increased ER-lysosome tethering sites were observed following AKT2i treatment. Co-staining of the ER-lysosome tethering protein PDZD8-Flag tag with LAMP1 was performed to visualize these tethering sites. l) Quantification shows the number of tethering sites per cell, with n = 10 cells per condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The PERK inhibitor GSK2606414 (S7307, Selleckchem) was used at 0.5 μM for 24 hours to determine the role of the PERK–eIF2α–ATF4 pathway in ERphagy induction.

Techniques: Western Blot, Expressing, Inhibition, Positive Control, Immunofluorescence, Co-Immunoprecipitation Assay, RNA Sequencing, Construct, Transfection, Staining, FLAG-tag

Journal: bioRxiv

Article Title: High-throughput discovery and deep characterization of cyclin-CDK docking motifs

doi: 10.1101/2024.12.03.625240

Figure Lengend Snippet: ( A ) Numbers of exclusive and shared docking motifs for the major cell cycle cyclins (D1, E1, A2, B1) among wild-type peptides tested by SIMBA. ( B ) Exclusive (left) and shared (right) cyclin interactors identified by SIMBA. The width of the cyclin-substrate connecting line shows the SIMBA score. Proteins are colored by UniProt keywords. ( C ) Comparison of wild-type peptide SIMBA scores for sequential cyclins illustrates fluctuating docking specificity during the cell cycle. ( D ) Schematic illustration of cyclin docking diversification. ( E ) CDK activity reporter based on the C-terminal IDR of PKMYT1 fused to FLAG tag, GFP and nuclear localization signal (NLS). The heatmap shows SIMBA scores of the introduced docking motifs. ( F ) PKMYT1-based reporter proteins with different cyclin docking motifs were captured from human cell lysates and probed for interacting cyclins. ( G ) Cells released from palbociclib-induced G1 arrest were collected at different times and monitored for DNA content by flow cytometry. ( H ) Western blot images showing cyclin expression in synchronized cell cultures after release from palbociclib-induced G1 arrest. ( I ) The impact of docking motif specificity on phosphorylation of the PKMYT1-based reporter in palbociclib-synchronized cell cultures was monitored using Phos-tag SDS-PAGE and α-FLAG Western blot.

Article Snippet: Then, the medium was replaced with DMEM containing 150 nM palbociclib (Selleck Chem) to arrest cells in G1 phase and 1 µg/ml doxycycline to induce the expression of CDK reporters.

Techniques: Comparison, Activity Assay, FLAG-tag, Flow Cytometry, Western Blot, Expressing, SDS Page