Journal: bioRxiv

Article Title: The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD

doi: 10.1101/2023.08.08.552343

Figure Lengend Snippet: ( a ) A hypothetical heterodimer of human AKT2/SIRT5 suggests a potential protein-protein interaction between these proteins. The ribbon structures of AKT2 and SIRT5 are colored orange and cyan, respectively. Residues related to the AKT2 and SIRT5 active sites are shown in yellow. The model suggests that both proteins have active sites with potential exposure to substrates and ligands, indicating that the AKT2/SIRT5 complex is catalytically active. ( b ) Co-immunoprecipitation study showing that SIRT5 and AKT2 are binding partners in AKT2-pcDNA and SIRT5-HA-pcDNA overexpressing ARPE19 cells, compared to untransfected controls. n=3. ( c ) Pulldown of AKT2 with anti-AKT2 antibody and subsequent incubation of the pulldown complex with TFEB (recombinant; rec) in vitro to evaluate phosphorylation of TFEB using the phospho-tag gel staining, showed increased p-TFEB/total TFEB in AKT2 overexpressing ARPE19 cells, compared to control. This TFEB phosphorylation was reduced when cells were overexpressing both AKT2 and SIRT5, indicating SIRT5 can modulate AKT2 activity and subsequent TFEB phosphorylation. n=3. ( d ) Pulldown assay using anti-GFP magnetic beads from ARPE19 lysates overexpressing either GFP-AKT2 or GFP-AKT2 and SIRT5-HA, showed a decrease in lysine malonylation (K-Mal) of AKT2 pull down complex in presence of SIRT5 overexpression, compared to cells overexpressing only GFP-AKT2. n=3. Western blot analysis showing reduced expression of SIRT5 and PGC-1α in RPE cells from ( e ) Akt2 KI, ( f ) PGC-1α KO, and ( g ) Sirt5 KO mice, as well as ( h ) iPSC-derived RPE cells from CFH (H/H) donors, compared to controls. n=4 (mice) and n=3 (iPSC cells). All values are Mean ± S.D. **P<0.01, *P<0.05.

Article Snippet: The primary antibodies AKT2 (3063S), p-AKT2 (8599S), SIRT5 (8782S), CTSD (69854S), ULK1 (8054T), FKBP51 (8245S), p-TFEB (37681S), K-Malonyl (14942S) and GFP (2555S) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Binding Assay, Incubation, Recombinant, In Vitro, Staining, Activity Assay, Magnetic Beads, Over Expression, Western Blot, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Overexpressed Malat1 Drives Metastasis through Inflammatory Reprogramming of Lung Adenocarcinoma Microenvironment

doi: 10.1101/2023.03.20.533534

Figure Lengend Snippet: (A) Representative H&E and GFP IHC staining of liver and lymph node (LN) metastasis (Met) of the long-term experiment of KPC animals infected with mA1 dRNA; (B) Quantification of metastasis of the mice analyzed in (A) (n=10 mice per group, unpaired t test); (C) Survival curves of the animals shown in (A). Log-rank test was used for statistical analysis. Presence of ethical endpoint criteria were used as endpoint; (D) IHC staining for the metastasis markers Nkx2.1 and Hmga2 of KPC animals analyzed in (A); (E) Quantification of Nkx2.1 staining in images in (D) (n>20 tumors, unpaired t-test); (F) Contingency analysis showing the percentage of positive tumors for Hmga2 immunostaining (Chi-square 28.68, P<0.001, n=79 tumors); (G) IHC staining for Hmga2 marker in the KC the cohort; (H) Contingency analysis showing the percentage of positive tumors for Hmga2 immunostaining in KC cohort (Fisher exact test P=0.504, n=157 tumors).

Article Snippet: Afterwards, tissues were incubated with primary antibodies: Hmga2 (BioChek, 59170AP; 1:500), GFP (Cell Signaling Technology, 2555S; 1:2000), α-Sma (ThermoScientific, 14-9760-82; 1:000), Cd68 (Abcam, ab125212; 1:100), Ki67 (D3B5, Rabbit mAb Mouse Preferred IHC Formulated; 1:500), pHH3 (Cell Signaling Technology, 9701S; 1:500), and Cdh1 (Cell Signaling, 24E10, 1:1000) at 4°C overnight.

Techniques: Immunohistochemistry, Infection, Staining, Immunostaining, Marker

Journal: bioRxiv

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1101/2023.03.16.532226

Figure Lengend Snippet: ( A ) Western blot of Triton-soluble lysate (left lanes) and YFP-PKCβII immunoprecipitated from COS7 cells using GFP-Trap ® Agarose. Cells were pre-treated with 20 μM MG-132 for 3 hours followed by 30 min of 200 nM PDBu treatment prior to lysis. Blots were probed with indicated antibodies. ( B ) Quantification of PDBu-induced ubiquitination of immunoprecipitated PKCβII. Relative ubiquitination was determined (Ubiquitin / PKC) for immunoprecipitated samples and each condition was normalized to DMSO-treated control (1.0) to determine fold-increase in ubiquitination after PDBu stimulation. Data represent mean ± SEM from four independent experiments. ns = not significant, **** P < 0.0001 by two-way ANOVA and Šídák’s multiple comparisons test.

Article Snippet: Antibodies against GFP (catalog no. 2555S), Vinculin (catalog no. 4650S), Ubiquitin (catalog no. 3933S), and GAPDH (14C10, catalog no. 2118) were from Cell Signaling Technologies and used at 1:1000 dilution.

Techniques: Western Blot, Immunoprecipitation, Lysis

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Chlamydomonas expressing KAP-GFP. Indicated are the measured areas of KAP-GFP localization: the two basal bodies and cilia. Scale bar is 5 μm. (B) Super plot quantification of basal body fluorescence in (A) after a 2-h treatment with BCI at the indicated concentrations. Error bars are the mean with the 95% confidence interval for the averages from the trials. P = 0.5126, which was determined by an ordinary one-way ANOVA. (C) Ciliary length plotted against ciliary fluorescence for cells in DMSO (orange circles) or 30 μM BCI (purple) after a 2-h treatment (n = 40, N = 1). Simple linear regression was used to generate linear regression lines and comparisons. For DMSO, y = 0.1171x + 0.4305 and r 2 = 0.05676. For BCI, y = 0.1174x + 0.4051 and r 2 = 0.2636. Comparing slopes, F = 4.048e-006 (1, 76) and P = 0.9984. Comparing intercepts, F = 0.005429 (1, 77) and P = 0.9415. (D) Comparison of total ciliary fluorescence shown in (C). P -values were determined using a t test. (E) Comparisons of ciliary fluorescence in DMSO or BCI per micrometer of cilia in (C). P -values were determined using a t test. (F) Example kymographs collected from total internal reflection fluorescent microscopy of KAP-GFP movement in cilia in cells treated with DMSO (top) or 30 μM BCI (bottom) for 2 h. Vertical scale bars are 4 μm. Horizontal scale bars are 2 s. (G, H, I) KAP-GFP dynamics quantified from the kymographs (n = 20, N = 1). Error bars are the mean with the 95% confidence interval (n = 20, N = 1). P -values were calculated from a two-tailed unpaired t test for pairwise comparisons. (G) Frequency of KAP-GFP trains measured as the total amount of trains counted over the total amount of time the kymograph was collected. For DMSO, n = 40 (1 and 2 h). For BCI, n = 30 (1 h) and 34 (2 h). (H) Velocity of KAP-GFP trains measured as the distance traveled in μm over time in seconds. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). (I) Relative injection size of KAP-GFP trains measured as the relative total fluorescent intensity of each train relative to the maximum measurement. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). P -values were determined using a t test.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Two Tailed Test, Injection

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Immunofluorescent images of KAP-GFP cells during regeneration in either DMSO or 30 μM BCI. Scale bars are 5 μm. Red arrows point to basal bodies, which are the object of quantification in (D). (B) Western blot for KAP-GFP expression in regenerating cells in either DMSO (D) or 30 μM BCI (B). Total protein was measured with amido black. (C) Quantification of (B). Error bars are the mean with SD for three independent experiments. P -values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (D) Quantification of KAP-GFP fluorescence at the basal bodies in (A). Error bars are the mean with the 95% confidence interval for the averages from three independent trials (n = 30, N = 3). The P -value was calculated using an unpaired t test with Welch’s correction.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Western blot of KAP-GFP expression compared with total protein shown with amido black. Cells were treated for 2 h with either 0.5% DMSO, 20 μM BCI, 50 μM MG132, or both 50 μM MG132 and 20 μM BCI. (A, B) Quantification of (A). Error bars are SD of the mean (N = 3). The P -value was determined by an unpaired t test between BCI and BCI + MG132.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: BCI inhibits phosphate removal from MAPK. This ultimately alters or inhibits ciliogenesis, KAP-GFP dynamics in cilia, KAP-GFP protein synthesis, NPHP4 protein localization at the transition zone, membrane trafficking, and microtubule organization.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques:

Journal: Nature Communications

Article Title: Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A

doi: 10.1038/s41467-023-36693-9

Figure Lengend Snippet: A Sites of CIP2A-B56α cross-links (in red; PDB: 6NTS) mapped in relation to indicated LxxIxE-binding groove of B56α. Residues indicated in light purple constitutes the LxxIxE-binding region of B56α between amino acids 212-271. Residues in dark purple indicate amino acids involved in PP2A-A interaction as explained in Fig. . B CIP2A(1-560)V5 out-competes prototypical LxxIxE motif target BubR1(LxxIxE peptide 647-720) from its direct association with B56α. Source data are provided as a Source Data file. C Quantification of GST pull-down data from ( B ) shown as a mean + S.E.M from N = 3 biological repeats. Two-sided t-test. D B56α competition assay using GST-BubR1(647-720) alone, or in combination with CIP2A N-terminal head peptide (aa. 18-40). Source data are provided as a Source Data file. E Quantification for data from D shown as a mean + S.E.M from N = 4 biological repeats. Two-sided t-test. F In vitro binding assay using purified recombinant GST-tagged CIP2A(1-560) and B56α WT or B56 variants Y215Q and R222E, which contain mutations that prevent interaction with LxxIxE groove substrate proteins . Source data are provided as a Source Data file. G Quantification of data from ( F ) shown as mean + S.E.M from N = 3 biological repeats. H GFP-tagged B56α WT or indicated triple mutant were expressed in HEK-293-T cells. The amount of PP2A-A subunit bound to GFP-tagged B56α upon GFP trap pull-down was quantified by anti-PP2A-A immunoblotting. Shown are the mean values + S.E.M of the ratios of the quantified anti-PP2A-A signal versus the quantified anti-GFP signal, relative to the B56α WT (set at 100 %) from N = 3 biological replicates. A two-sided one-sample t-test. I Triple B56α mutant (K181A/K217A/K227A) exhibits loss of K227 (B56)-D117(PP2A-A) salt bridge. Overlay of PP2A-B56α (PDBID 6NTS: PP2A-A, yellow; B56α, beige) and PP2A-B56γ (PDBID 2IAE: PP2A-a, light cyan, B56γ, cyan), with PP2A-A D177 and B56α/B56γ K227/K202 residues shown as sticks and labelled. H-bond interactions are shown using dotted lines. Image was generated using Pymol.

Article Snippet: The following antibodies were used: anti-V5 (monoclonal mouse (mM) Ab (E10/V4RR), Thermo Fisher Scientific, MA5-15253; 1:5000), anti-V5 (mM, Thermo Fischer Scientific, R960-25; 1:2000), anti-CIP2A (mM Ab(2G10-3B5), sc-80659, 1:1000), anti-GST (polyclonal rabbit (pR Ab), Thermo Fisher Scientific CAB4169; 1:10,000), anti-GST (pR Ab, Cell Signalling 2622 S; 1:10,000), anti-GST (mM Ab (B-14), sc-138; 1:10,000), anti-PR65 (pR Ab (H-300), sc-15355; 1:1000 and 1:5000), anti-B56α (mM Ab (23), sc-136045; 1:500 and 1:5000), anti-β-Actin (mM Ab (C4), sc-47778; 1:5000 and 1:10,000), anti-B56γ (mM Ab (E6), 374380; 1:500), all from Santa Cruz Biotechnology), anti-c-Myc (mM Ab (9E10), sc-40, 1:1000), anti-PP2Ac (pR Ab, Cell Signalling 2038S; 1:1000 and 1:5000), anti-GAPDH (mM Ab (6C5), 5G4-6C5 Hytest, 1:10,000), anti-PR65 (clone C5.3D10, 1:1000) and anti-PP2Ac (clone F2.6A10, 1:500), both generously supplied by Dr. S. Dilworth at Middlesex University, London, UK), anti-GFP (pRb Ab, 2555 S, Cell Signalling; 1:1000), anti-cleaved PARP (mM Ab(E51), ab32064 abcam; 1:1000), anti-Phospho-MEK1/2 (Ser217/221) (pR Ab, 9121S Cell Signallings; 1:1000), anti-phospho Vimentin (Ser39) (pR Ab, 13614 Cell Signalling; 1:1000), anti-HA tag (mRb Ab (C29F4), 3724 Cell Signalling; 1:200 and 1:1000).

Techniques: Binding Assay, Competitive Binding Assay, In Vitro, Purification, Recombinant, Mutagenesis, Western Blot, Generated

Journal: Nature Communications

Article Title: Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice

doi: 10.1038/s41467-023-36780-x

Figure Lengend Snippet: a Schematic of experimental design for electrophysiology recordings in the whole-cell patch-clamp configuration. Horizontal brain slices containing BLA and NAc from CRH-ires-CRE mice that were injected with Cre-dependent ChR2-EYFP in BLA. b Representative traces of optically evoked IPSCs (oIPSCs). These were blocked in the presence of GABA A receptor antagonist picrotoxin. c A neurobiotin (brown) filled neuron from which oIPSCs were recorded. Note spines (arrows) suggesting the cell is a medium spiny neuron (MSN). Arrowheads denote ChR2-expressing boutons from BLA-origin CRH + axons (CRH; blue) on the soma. d oIPSCs amplitudes pre and post picrotoxin ( n = 7 neurons, 5 mice). e Time-course plot of normalized oIPSCs amplitudes throughout the recording and following application of picrotoxin. Black triangles in e denote trace recordings in b and timepoint analysis in d and e . f Representative trace of a NAc cell showing no response from optically evoked EPSC (oEPSC) at −70 mV, obtained after verifying oIPSCs at 0 mV in the presence of picrotoxin. g Representative trace showing spontaneous EPSCs (sEPSCs) were still present; gray box shows magnified view recording. In d and e , bars represent mean. In e circles represent mean ± SEM. Two-sided paired t -tests in d and e . d oIPSCs (current) pre vs. post PTX: P = 0.0338. e oIPSCs (normalized) pre vs. post PTX: P = 0.0004. 3 V = third ventricle, Hip Hippocampus, LV lateral ventricle. Source data are provided as a Source Data file.

Article Snippet: Sections were rinsed in PBS-T and then processed for CRH (1:20,000, PBL#rC68) or ChR2-EYFP (rabbit anti-GFP in 1:2000, Cat # 2555 S, Lot # 2, Cell Signaling, US) immunostaining.

Techniques: Patch Clamp, Injection, Expressing

Journal: Nature Communications

Article Title: Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice

doi: 10.1038/s41467-023-36780-x

Figure Lengend Snippet: a , b Coordinate locations of a , DREADD injection and CNO infusion and b opsin injection and fiber placement. c schematic of the reward tasks. d , e Chemogenetically stimulating the CRH/GABA BLA → NAc projection with microinfusion of CNO in the medial NAc shell suppressed d , palatable food consumption ( n = 10 male mice; 12 female mice) and e preference for a sex-cue ( n = 12 male mice; 12 female mice) in males, but not females. f , g Inhibiting the CRH/GABA BLA → NAc projection in TR male mice did not increase f , palatable food consumption ( n = 11 mice) or g , preference for a sex-cue ( n = 8 mice). h , i stimulating the CRH/GABA ChR2-expressing BLA → NAc projection decreased h palatable food consumption ( n = 9 mice) and i , approach time to a sex-cue ( n = 11 mice). In d – i bars represent mean. Two-way ANOVA with repeated measures followed by post hoc tests ( d , e ). d hM3Dq BLA → NAc: Sex x Treatment— F = 5.691, DFn = 1, DFd = 40, P = 0.0219; post hoc with Tukey’s multiple comparison (Veh vs. CNO: Male— P = 0.0016; Female— P = 0.9366). e hM3Dq BLA → NAc: Treatment— F = 6.448, DFn = 1, DFd = 44, P = 0.0147; post hoc with Sidak’s multiple comparison (Veh vs. CNO: Male— P = 0.0081; Female— P = 0.8241). Two-sided paired t -tests ( f – i ). f hM4Di BLA → NAc: P = 0.1036; g hM4Di BLA → NAc: P = 0.1139. h ChR2 BLA → NAc: P = 0.0064; i ChR2 BLA → NAc: P = 0.0034. PF palatable food. Gray = vehicle/light off, teal = CNO, blue = light on. Source data are provided as a Source Data file.

Article Snippet: Sections were rinsed in PBS-T and then processed for CRH (1:20,000, PBL#rC68) or ChR2-EYFP (rabbit anti-GFP in 1:2000, Cat # 2555 S, Lot # 2, Cell Signaling, US) immunostaining.

Techniques: Injection, Expressing