Journal: Nature Communications

Article Title: Covalent modification of a glutamic acid inspired by HaloTag technology

doi: 10.1038/s41467-026-68999-9

Figure Lengend Snippet: a mCitrine lifetime (τ) measurement in HEK293T cells with mCitrine-Rheb only, or donor-acceptor system with mCitrine-Rheb bound to mCherry-PDEδ, or donor-acceptor system with DeltaTag treatment for 10 min. First column: fluorescence intensity distribution mCitrine-Rheb; second column: zoomed-in views for cellular mCitrine-Rheb distribution; third column: mCitrine-PDEδ; fourth column: global lifetime distribution; fifth column: lifetime decay of mCitrine and fitting with mono-exponential decay. Scale bar, 25 µm. Images represent biological replicates n = 3 (each with technical replicates N = 6). b Average lifetime of mCitrine. Data are presented as mean ± s.d., representative of biological replicates n = 3. Unpaired t -test, two-tailed p -value = 0.0036 (**). c Immunofluorescence staining of PA-TU-8902 cells with anti-Rheb antibody, overnight after treatment, representative of biological replicates n = 3. Intensity profiles (arbitrary unit, A.U.) along the lines are plotted against distance (µm). Scale bars, 20 µm. d Plot of kinase z -scores (two-tailed p -value ≤ 0.05) after kinase-substrate enrichment analysis (KSEA App with NetworKIN, substrate count cutoff = 2, NetworKIN score cutoff = 1) – of significant hits from phosphoproteome profiling upon treatment of 6a (see Supplementary Fig. for details). e Reactome pathway overrepresentation of downregulated kinases, with Voronoi visualisation zooming into mTOR signalling. The scale of colour intensity is an indication of the p -value of pathway overrepresentation. f Western blot analysis of S6P phosphorylation on Ser235 and Ser236 (S235/S236) and total S6P (tS6P) in PA-TU-8902 cells. (−/−) represents unstimulated DMSO-treated cells. Quantification of per cent pS6P/tS6P ± s.d. was normalised to EGF-stimulated DMSO control at each time-point, representative of biological replicates n = 3. Unpaired t -test, two-tailed p -values comparing each condition to EGF-stimulated DMSO control (−/+): 3 h: vs 1 , p = 0.78; vs 6a , p = 0.035 (*); 5 h: vs 1 , p = 0.138; vs 6a , p = 0.0009 (**); 8 h: vs 1 , p = 0.102; vs 6a , p < 0.0001 (***); p -value > 0.05 are labelled as non-significant (ns). j Schematic representation of inhibition along the PDEδ-Rheb-mTORC1 axis, created in BioRender. Zhang, R. (2026) https://BioRender.com/dxvyoxr . Source data are provided as a Source Data file.

Article Snippet: Cells were subsequently blocked by 2% BSA in PBS-T (0.1% Tween 20 in PBS) for 1 h at room temperature, incubated with the primary antibody Anti-Rheb (Santa Cruz Biotechnology Cat# sc-271509, RRID: AB_10659102, 1:500) overnight at 4 °C in blocking buffer, then washed with PBS-T three times and incubated with Alexa-555-conjugated secondary antibody (Invitrogen # A-31570, RRID:AB_2536180, 1: 1000) and DAPI (1 μg/ml) in blocking buffer for 1 h at room temperature with protection from light.

Techniques: Fluorescence, Two Tailed Test, Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Control, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC7A5 regulates B cell metabolism and plasma cell differentiation independent of leucine transport

doi: 10.1093/jimmun/vkaf328

Figure Lengend Snippet: (A) Levels of phospho-mTOR (p-mTOR) in g Slc7a5 and gCtrl-transduced B cells after co-culture with 40LB cells for 24 hours. Representative flow cytometry plots (left) and mean fluorescence intensity (MFI) of p-mTOR normalized to the average of the gCtrl B cells in each experimental batch (right). B cells were stimulated with αRP105 for 2 days, transduced and restimulated with CpG for 24h. Cells were gated on FSC hi SSC lo NGFR + B cells as shown in Fig. S5A , where NGFR is a reporter for transduced cells. (B) S6 phosphorylation in g Slc7a5 or gCtrl-transduced B cells after co-culture with 40LB cells for 24h. Representative flow cytometry plots (left) and quantification of relative frequencies of phospho-S6 + B cells (right). Frequencies were normalized to the average of the gCtrl samples (indicated by the dotted line) in each experimental batch. Cells were gated on FSC hi SSC lo NGFR + B cells. (C) Schematic illustration of lysosome- and PI3K/AKT-dependent regulation of mTORC1 activity. (D-H) Extracellular flux (XF) analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) in g Slc7a5 and gCtrl-transduced B cells. B cells were stimulated with anti-RP105 for 3 days and re-stimulated with CpG for 1 day. (D) Representative time-series plot of ECAR at baseline and after challenge with 1 μM oligomycin (dotted line). (E) Quantification of baseline ECAR (left) and maximum ECAR (right). (F) Representative time-series plot of OCR at baseline and after sequential addition of 1 μM oligomycin, 1.5 μM FCCP, and 0.5 μM Rotenone/Antimycin A (each indicated by dotted lines). (G) Quantification of baseline OCR (left) and maximum OCR following addition of FCCP (right). (H) Plot of baseline OCR vs. ECAR. (I) PC differentiation of gCtrl transduced B cells and g Slc7a5 -transduced B cells expressing empty vector (−), myrAKT or RHEB Q64L . Representative flow cytometry plots (left) and quantification of CD138 + TACI + PC frequencies (right). Gating strategies for myrAKT and RHEB Q64L conditions are shown in Fig. S5C . (J) Analysis of phospho-S6 + gCtrl and g Slc7a5 -transduced B cells with or without ectopic expression of myrAKT or RHEB Q64L after 1 day co-culture with 40LB feeder cells. Representative flow cytometry plots (left) and quantification of p-S6 + cell frequencies (right). Data in A, B, D–J are from 6 mice and at least 2 independent experiments. Each dot represents a biological replicate. Shown are the mean ± SEM. Statistical significance was tested by two-tailed Student’s t test (A, I, J), paired t-test (E, G) with the following significance levels: * P < 0.05, ** P < 0.01, *** P < 0.001. For data in I and J, all experimental comparisons were prespecified; ANOVA was performed to confirm global significance, followed by hypothesis-driven individual comparisons tested using two-tailed Student’s t test.

Article Snippet: Expression constructs used in this study include: myrAKT (Addgene, 10841) ( 64 ) and RHEB Q64L (Addgene, 21050) ( 65 ).

Techniques: Activity Assay, Co-Culture Assay, Flow Cytometry, Fluorescence, Phospho-proteomics, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie

Article Title: Differences in the characteristics of subjects achieving complete, partial, or no resolution of macular edema in the READ-3 study

doi: 10.1007/s00417-021-05148-6

Figure Lengend Snippet: Clinical features of the study groups

Article Snippet: Persistent N = 82 Rebound N = 20 Resolved N = 21 p value Mean age (year ± SD) 65.0 ± 9.0 65.5 ± 8.5 60.9 ± 12.0 0.18 Females [% ( n )] 40.2 (33) 65.0 (13) 47.6 (10) 0.13 Mean BMI (kg/m 2 ± SD) 31.3 (8.4) 31.4 (6.1) 30.3 (5.8) 0.85 Race [% ( n )] Caucasian 66.7 (46) 81.3 (13) 57.1 (10) 0.52 African American 18.8 (13) 12.5 (2) 7.1 (1) Asian 7.3 (5) 6.3 (1) 14.3 (2) Native Hawaiian/Pacific Islander 4.4 (3) 0 (0) 14.3 (2) Other 2.9 (2) 0 (0) 7.1 (1) Smoking status [% ( n )] 29.3 (24) 40.0 (8) 38.1 (8) 0.55 Prior anti-VEGF therapy [% ( n )] 9.1 (6) 6.3 (1) 16.7 (3) 0.55 VMA at baseline 19.1 (13) 25.0 (4) 23.8 (5) 0.82 Mean baseline BCVA (ETDRS letters) 28.1 ± 10.0 26.7 ± 14.1 28.4 ± 12.1 0.85 Mean baseline CST (μm) 536.7 ± 127.1 424.6 ± 82.8 456.5 ± 108.7 0.0002* Mean baseline HbA1c (%) 7.8 ± 1.5 7.9 ± 2.0 7.3 ± 1.6 0.47 Dose of ranibizumab [%(n)] 2.0 mg 43.9 (36) 55.0 (11) 71.4 (15) 0.07 0.5 mg 56.1 (46) 45.0 (9) 28.6 (6) Mean number of intravitreal injections (number ± SD) 11.1 ± 1.34 8.8 ± 1.4 8.5 ± 1.9 < 0.0001* Median Interleukin-6 (pg/mL) 13.1 6.15 23.5 0.38 Open in a separate window BCVA best-corrected visual acuity, CST central subfield thickness, ETDRS Early Treatment Diabetic Retinopathy Study, SD standard deviation.

Techniques:

Journal: Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie

Article Title: Differences in the characteristics of subjects achieving complete, partial, or no resolution of macular edema in the READ-3 study

doi: 10.1007/s00417-021-05148-6

Figure Lengend Snippet: Mean change from baseline in best-corrected visual acuity (ETDRS letters) among the subjects in study groups

Article Snippet: Persistent N = 82 Rebound N = 20 Resolved N = 21 p value Mean age (year ± SD) 65.0 ± 9.0 65.5 ± 8.5 60.9 ± 12.0 0.18 Females [% ( n )] 40.2 (33) 65.0 (13) 47.6 (10) 0.13 Mean BMI (kg/m 2 ± SD) 31.3 (8.4) 31.4 (6.1) 30.3 (5.8) 0.85 Race [% ( n )] Caucasian 66.7 (46) 81.3 (13) 57.1 (10) 0.52 African American 18.8 (13) 12.5 (2) 7.1 (1) Asian 7.3 (5) 6.3 (1) 14.3 (2) Native Hawaiian/Pacific Islander 4.4 (3) 0 (0) 14.3 (2) Other 2.9 (2) 0 (0) 7.1 (1) Smoking status [% ( n )] 29.3 (24) 40.0 (8) 38.1 (8) 0.55 Prior anti-VEGF therapy [% ( n )] 9.1 (6) 6.3 (1) 16.7 (3) 0.55 VMA at baseline 19.1 (13) 25.0 (4) 23.8 (5) 0.82 Mean baseline BCVA (ETDRS letters) 28.1 ± 10.0 26.7 ± 14.1 28.4 ± 12.1 0.85 Mean baseline CST (μm) 536.7 ± 127.1 424.6 ± 82.8 456.5 ± 108.7 0.0002* Mean baseline HbA1c (%) 7.8 ± 1.5 7.9 ± 2.0 7.3 ± 1.6 0.47 Dose of ranibizumab [%(n)] 2.0 mg 43.9 (36) 55.0 (11) 71.4 (15) 0.07 0.5 mg 56.1 (46) 45.0 (9) 28.6 (6) Mean number of intravitreal injections (number ± SD) 11.1 ± 1.34 8.8 ± 1.4 8.5 ± 1.9 < 0.0001* Median Interleukin-6 (pg/mL) 13.1 6.15 23.5 0.38 Open in a separate window BCVA best-corrected visual acuity, CST central subfield thickness, ETDRS Early Treatment Diabetic Retinopathy Study, SD standard deviation.

Techniques:

Journal: bioRxiv

Article Title: Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation

doi: 10.1101/825828

Figure Lengend Snippet: A) C2C12 myoblast cells transfected with Myc-Nix or an empty vector control. Cells were treated with Rapamycin (500nM, 1h) or DMSO as vehicle control. Proteins were immunoblotted as indicated. B) C2C12 myoblast cells transfected with shNix, or a scramble control shRNA, and treated overnight with palmitate (200μM) conjugated to 2% albumin in low glucose media. Proteins were immunoblotted as indicated. C) C2C12 myoblast cells were transfected as in (a), and were treated with clenbuterol (500nM), cilomilast (10μM) or vehicle for 2 h. Proteins were immunoblotted as indicated. D) C2C12 myoblast cells were transfected as in (a). Cells were treated with mdivi-1 (20 μM) or vehicle for 1 h. Proteins were immunoblotted as indicated. E) C2C12 myoblast cells were transfected as in (a) and treated with 1-Butanol (1%) for 30 minutes. Proteins were immunoblotted as indicated. F) C2C12 myoblast cells were transfected as in (a), followed by phosphatidic acid assay and quantification. G) C2C12 myoblast cells were transfected as in (a), along with the diacylglycerol biosensor DAGR. Cells were treated overnight with palmitate and analyzed by FRET imaging. H) C2C12 myoblast cells were transfected with Myc-Nix, siRheb, siPLD6. Proteins were immunoblotted as indicated. I) C2C12 myoblast cells were transfected with Myc-Nix, Rheb, Myc-Nix-S212D, Myc-Nix-ΔTM, along with mito-pHRed. Cells were imaged by standard fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVA.

Article Snippet: The Rheb siRNA (19744) and the Pld6 (194908) were purchased from Dharmacon, and the scrambled siRNA control and control lentivirus were purchased from Santa Cruz (sc-37007, sc-108080).

Techniques: Transfection, Plasmid Preparation, Control, shRNA, Phosphatidic Acid Assay, Imaging, Fluorescence