Journal: bioRxiv

Article Title: Extra-ciliary role for polycystins in regulation of Ezrin and renal tubular morphology

doi: 10.1101/2025.11.05.684799

Figure Lengend Snippet: (A) Western blot for Ezrin abundance in immortalized mouse Pkd1 fl/fl Pax8rtTA, TetO-Cre cortical (C) renal epithelial cells, clone #302 ( Pkd1 +/− (C)) and clone #313 ( Pkd1 +/− (M)), cultured with vehicle (DMSO = Pkd1 +) or doxycycline (DOX = Pkd1 -). LC is loading control calculated from total lane protein. (B) Quantification of Ezrin abundance in A, n=4 for each clone and each treatment group; +/− SEM, p<0.05, ANOVA with secondary Dunnett’s for multiple comparisons. (C,D) Immunofluorescent labeling of Ezrin and ZO1 in Pkd1 (M) (clone #313) in 2D culture in control (C) or after treatment without doxycycline (D) with quantification. Ezrin localizes to apical surface and does not overlap with ZO1 in Pkd1 + control cells. Inducible knockout of Pkd1 (DOX = Pkd1 -) led to significant reduction in Ezrin signal. Exposure and Intensity matched, representative of n=4 experiments for each treatment group; n=45 cells for measured Ezrin immunofluorescence integrated intensity, +/− SEM, p<0.05, two tailed Student’s T-Test). (E-H) Tubuloids grown in 3D culture using Pkd1 (M) cells (clone #313) treated with DMSO (E, control Pkd1 + (M)) or doxycycline (G, DOX, Pkd1 - (M)) imaged in brightfield and with labeling for Ezrin (green) and DAPI (blue). (F) Close up of control (DMSO) tubuloids showing Ezrin localization on the luminal surface (white arrow). Pkd1 knockout ( Pkd1 -) results in large hollow spheres containing few cells with Ezrin (G). (H) Quantification of 3 separate experiments ( Pkd1+(M) (DMSO), n=34 tubuloids, Pkd1- (m) (DOX), n=18 tubuloids, +/− SEM, p<0.05, two tailed Student’s T-Test).

Article Snippet: Vectashield Mounting Media with DAPI (Vector Labs H1200) was then added with a cover slip.

Techniques: Western Blot, Cell Culture, Control, Labeling, Knock-Out, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Extra-ciliary role for polycystins in regulation of Ezrin and renal tubular morphology

doi: 10.1101/2025.11.05.684799

Figure Lengend Snippet: (A) Mouse immortalized Pkd2 fl/fl Pax8rtTA, TetO-Cre (medullary clone#125) cells grown in 2D culture, treated with vehicle DMSO ( Pkd2+ ) or doxycycline ( Pkd2- ) for 7 days and assessed for Ezrin and Pkd2 abundance by Western blot and (B) quantified (n= 7 for each treatment, +/−SEM, P<0.05 two tailed student’s t-test). (C,D) Same Pkd2 clone #125 cells grown in 2d culture treated with vehicle DMSO ( Pkd2+ ) or doxycycline ( Pkd2- ), fixed and labeled for Ezrin (green), ZO1 (purple), and DAPI (blue), (representative of 3 unique experiments for each treatment, 40x, scale bar 20um). White box represents the enlarged area in Ci and Di. (E) Tubule fragments (from Pkd2 fl/fl Pax8rtTA;TetOCre; mTmG mice) were used to generate 3D tubuloids treated with vehicle or doxycycline, lysed, and used for western blotting to quantify PC2 and Ezrin abundance in the 3D structures. (F) Quantification showed a significant decrease in both PC2 and Ezrin abundance when compared to controls (n=3, +/ SEM, p-value <0.05, two tailed student’s t-test). (G, H) Immunofluorescent images demonstrate the mTmG ( Pkd2 + red, Pkd2 -green) reporter following treatment with doxycycline alongside ezrin (purple) and Pkd2 -(green) with overlap (white). Magnification 20X, Scale bar 50μm. Representative of n=3 unique experiments for each treatment. (I,J) The colocalization quantification of ezrin and Pkd2 signal exhibits a statistically significant decrease in Pkd2 -cells compared to Pkd2 + (n=19 unique tubuloids, and 214 total cells, two-tailed p-value <0.0001). (K) Pkd2 fl/fl Pax8rtTA; TetOCre mice (P10) were treated with doxycycline for a short induction period to inactivate Pkd2 in the kidneys . Representative P15 kidneys (time point before significant cyst development) were stained from both control (L) and doxycycline (M) treated mice for ezrin (green). Magnification 20X, scale bar 100μm. (N, O) Higher magnification images of ezrin in control and doxycycline treated mice co-labeled with apical AQP2 (red) and junctional ZO1 (purple). Magnification 40X, scale bar 50μm.(C) There is a significant decrease in ezrin intensity (PTP/TP) in doxycycline treated mice (n=3 male mice, 5 field of views each) when compared to controls (n=3 male mice, 5 field of views each) (two tailed p-value<0.0001).

Article Snippet: Vectashield Mounting Media with DAPI (Vector Labs H1200) was then added with a cover slip.

Techniques: Western Blot, Two Tailed Test, Labeling, Staining, Control

Journal: Skeletal Muscle

Article Title: AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

doi: 10.1186/s13395-024-00349-z

Figure Lengend Snippet: Low NRIP expression in the spinal cord and skeletal muscles of SOD1 G93A mice. A NRIP expression in the spinal cord at age of 56 days WT and SOD1 G93A mice. Mice were euthanized, and transcardial perfusion with PBS was performed to remove blood from tissues. Total proteins from L3-L5 spinal cord were subjected to Western blot (WB) analysis for NRIP expression, with GAPDH serving as the loading control. The asterisk indicates the representative band for NRIP expression. The right panel represents the quantification of NRIP expression conducted through densitometry analysis. B NRIP expression in the gastrocnemius (GAS) muscles. The asterisk indicates the representative band for NRIP expression. The right panel illustrates the quantification. C NRIP expression in the tibialis anterior (TA) muscles. The asterisk indicates the representative band for NRIP expression. The right panel shows the quantification. WT, N = 3 mice; SOD1 G93A, N = 5 mice. D NRIP expression in the lumbar segment of the spinal cord at the age of 56 days WT and SOD1 G93A mice. The 30 μm-thick sections were co-stained with anti-ChAT (green) and anti-NRIP (red) antibodies to detect NRIP expression in spinal cord motor neurons. DAPI was used as the nuclear counterstain. Arrows indicate high NRIP expression in ChAT-positive neurons, while arrowheads indicate low NRIP expression in ChAT-positive neurons. Scale bar: 100 μm. Right panel: Quantification of the immunofluorescence intensity of NRIP in ChAT-positive neurons from WT mice ( N = 3) and SOD1 G93A mice ( N = 3). Data are presented as mean ± SEM. Statistical analysis was conducted using the Student t-test. * P < 0.05 and *** P < 0.001

Article Snippet: Finally, sections were washed in PBS for 10 min three times in dark and mounted with DAPI Fluoromount-G (SouthernBiotech).

Techniques: Expressing, Muscles, Western Blot, Control, Staining, Immunofluorescence

Journal: Journal of Leukemia

Article Title: Beta-Hydroxyisovalerylshikonin Inhibits the Growth of U266 Multiple Myeloma Cells by Triggering the Mitochondrial Pathway

doi: 10.4172/2329-6917.1000155

Figure Lengend Snippet: Figure 3b: Immunofluorescence staining, observed via a fluorescence microscope at 72 h after beta-HIVS treatment, at x 400 magnification. Cleaved caspase-3 was marked by green fluorescence (FITC), and cytochrome c was marked by red fluorescence (Cy3). The nucleus was stained by DAPI.

Article Snippet: The main reagents are listed as follows: beta-HIVS (Dibo Chem, Shanghai, China); RPMI-1640, fetal bovine serum (FBS) (Gibco-BRL, Carlsbad, CA, USA); DMSO, methylcellulose (Sigma-Aldrich Corporation, St. Louis, MA, USA); Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan); SYBR-Green PCR Master Mix (Roche Diagnostics GmbH, Mannheim, Germany); RIPA protein lysis buffer, paraformaldehyde, DAPI dye, DNA Fragmentation kit, Cy3conjugated anti-mouse IgG, FITC-conjugated anti-rabbit IgG, mouse anti-human β-actin, anti-cytochrome c, rabbit anticleaved PARP and anti-cleaved caspase-3 (Asp175) (Beyotime Institute of Biotechnology, Shanghai, China); rabbit antihuman Bcl-2 and anti-Bax (Abcam, Cambridge, MA,USA); rabbit anti-caspase-9 and anti-caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA);

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Cell Death Discovery

Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances

doi: 10.1038/s41420-026-03027-9

Figure Lengend Snippet: A CellTrace™ Proliferation Assay was performed in ADA2 WT/WT and ADA2 -/- U-937 cells over 4 days. Cells were prepared for flow cytometry at baseline and after 2 and 4 days of incubation. B Cell death of ADA2 WT/WT and four different clones of ADA2 -/- U-937 cells was measured by flow cytometry during the incubation of the CellTrace™ Proliferation Assay. Dead cells were identified as Annexin V / Zombie® double-positive cells. C Immunofluorescence microscopy of ADA2 WT/WT and ADA2 -/- U-937 cells stained for ADA2. DAPI was used for nuclear staining. The scale bar represents 50 µm.

Article Snippet: Cells were harvested, fixed in 4% (v/v) paraformaldehyde (Electron Microscopy Sciences) for 10 min, and permeabilized with PBS containing 0.1% Tween®20 (Qbiogene Inc.) for 10 min. To prevent nonspecific antibody binding, cells were incubated in blocking buffer (1x PBS supplemented with 5% FCS and 5 mg/mL human IgG; IgG1 66.6%, IgG2 28.5%, IgG3 2.7%, IgG4 2.2%; Grifols) for 30 min. Nuclear staining was performed using DAPI (1 μg/mL in PBS / 0.1% Tween®20) for 15 min. For imaging, stained cells were transferred onto glass slides using a Cytospin 4 centrifuge (Thermo Fisher Scientific) at 800 rpm for 3 min with medium acceleration.

Techniques: Proliferation Assay, Flow Cytometry, Incubation, Clone Assay, Immunofluorescence, Microscopy, Staining

Journal: Advanced Science

Article Title: Beyond Catalytic Therapy: Copper‐Paeonol Nanozymes Disrupt Fascin‐Mediated Actin Bundling to Suppress Tumor Growth and Metastasis

doi: 10.1002/advs.202512186

Figure Lengend Snippet: CuPaeNs bound to FSCN1. A) Bubble chart of GO enrichment analysis top 10 of cellular component after CuPaeNs treatment. B) Immunofluorescence staining of B16 cells in different groups. DAPI (blue), FSCN1 (green), and F‐actin (red). C) Molecular simulation of CuPaeNs. D) Computational model of active sites related to the potential key target of FSCN1 with paeonol, a single paeonol‐Cu 2+ unit, and CuPaeNs. The residues of the ligand‐binding domain proteins, involved in hydrogen bonds, were illustrated and marked with yellow dotted lines. E) Molecular simulation illustrating the binding affinity and detailed intermolecular binding interactions of FSCN1 with paeonol, a single Cu 2+ ‐paeonol unit, and CuPaeNs, focusing on main hydrogen bonds and hydrophobic interactions. F) Isothermal titration calorimetry result demonstrating the direct binding of CuPaeNs to FSCN1. G) CLSM images showing intracellular FSCN1 binding with CuPaeNs in B16 cells (scale bar = 20 µm) and H) responding pixel intensity plot. I) Assessment of CuPaeNs's impact on FSCN1's actin‐bundling activity using the Co‐IP assay. (J) Protein expression of FSCN1, F‐actin, and β‐actin with B16 cells in different groups, along with quantitative analysis ( n = 3). Statistically significant differences are indicated with their respective p ‐values ( *** p < 0.001).

Article Snippet: Actin‐Tracker Red‐Rhodamine, DAPI staining solution, trizol, dimethylsulfoxide (DMSO, 99.9%), apoptosis and necrosis assay kit, ATP assay kit, mitochondrial membrane potential assay kit with 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide (JC‐1), β‐actin mouse monoclonal antibody (1:1000), PFKFB3 rabbit polyclonal antibody (1:1000), GAPDH mouse monoclonal antibody (1:1000), were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Immunofluorescence, Staining, Ligand Binding Assay, Binding Assay, Isothermal Titration Calorimetry, Activity Assay, Co-Immunoprecipitation Assay, Expressing

Journal: Brain, behavior, and immunity

Article Title: Irisin reprograms microglia through activation of STAT6 and prevents cognitive dysfunction after surgery in mice.

doi: 10.1016/j.bbi.2024.12.019

Figure Lengend Snippet: Fig. 6. Inhibiting STAT6 phosphorylation weakens the effects of irisin. (A) Schematic of the experiment to intervene the effects of irisin by prophylactic admin- istration of AS (i.p.) in adult C57BL/6 mice. Mice with normal cognition measured by OPR were divided into groups. Immunofluorescence and ELISA analysis of dorsal hippocampus were performed on postoperative day 1. (B, C) Effect of intraperitoneal (i.p.) administered irisin or AS + irisin on OPR memory in LPS induced neuroinflammation mice. (mean ± s.e.m.; paired two-sided t-test, *P < 0.05, **P < 0.01, Test vs. Sample; Two-sided one-way ANOVA with Bonferroni post-hoc test, #P < 0.05; B, Before drug administration: **P = 0.0018, *P = 0.0441, *P = 0.0449, *P = 0.0336 for Naïve, LPS, Irisin + LPS and Irisin + AS + LPS, respectively. P > 0.9999, LPS vs. Naïve; P = 0.7748, Irisin + LPS vs. LPS; P > 0.9999, AS + Irisin + LPS vs. Irisin + LPS. n = 10 per group. C, 1d post-operation: *P = 0.0008, P = 0.7383, *P = 0.0150, P = 0.1215 for Naïve, LPS, Irisin + LPS and Irisin + AS + LPS, respectively. #P = 0.0142, Irisin + AS + LPS vs. Irisin + LPS; n = 10 per group). (D) ELISA analysis for the level of irisin in dHPC is shown. (mean ± s.e.m.; Two-sided one-way ANOVA with Bonferroni post-hoc test; P > 0.9999, LPS vs. Naïve; *P = 0.0274, Irisin + LPS vs. LPS; P = 0.0747, AS + Irisin + LPS vs. LPS; P > 0.9999, Irisin + AS + LPS vs. Irisin + LPS. n = 7 per group). (E) Representative confocal images of the dHPC stained with anti-Iba1 (red), pSTAT6 and pPDH (green), and NeuN (magenta) and DAPI (blue) in Naïve, LPS, Irisin + LPS and Irisin + AS + LPS. Scale bars, 50 and 10 µm. (F, G) ELISA analysis for the effect of each treatment on the pro-inflammatory factor IL-1β and the anti-inflammatory cytokine Arg-1 in dHPC is shown. (mean ± s.e.m.; Two-sided one-way ANOVA with Bonferroni post-hoc test; *P < 0.05, ****P < 0.0001; IL-1β: ****P < 0.0001, ****P < 0.0001, *P = 0.0457; Arg1: P > 0.9999, *P = 0.0183, *P = 0.0431 for LPS vs. Naïve, Irisin + LPS vs. LPS, and Irisin + AS + LPS vs. Irisin + LPS. n = 7 per group). See also Figures. S8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Next, sections were washed 3 times with PBS for 5 min and incubated with secondary antibody solution containing DAPI (1:2000, 4083S, CST) dilution buffer for 1 h at room temperature in a dark place.

Techniques: Phospho-proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: Hydroxychloroquine Destabilizes Phospho-S6 in Human Renal Carcinoma Cells

doi: 10.1371/journal.pone.0131464

Figure Lengend Snippet: (A) In vitro kinase reaction of purified recombinant P70S6K was performed as described in Methods. Levels of phospho and total S6 determined by western blot. Number on top shows amount of HCQ added. Lane labeled RAD is reaction in which 10 μM RAD001 with ATP. (B) In vitro kinase activity was performed on immunoprecipitated P70S6K. Extracts were made from either control (C), RAD001 (R), or HCQ (H) treated ACHN cells. Lanes on left show reactions without added ATP. Lane labeled r70 contains recombinant epitope tagged P70S6K, which runs slightly slower than endogenous protein. Reaction mixture was tested for phospho-S6, total S6, phospho-P70S6K, and total P70S6K (C) Immunofluorescence of 769-P cells either treated with nothing, RAD001 or HCQ and stained with indicated antibody. Nuclear DNA was visualized by DAPI. (D) Cells of the indicated type were untreated or treated with RAD001 or HCQ. Extracts were then examined for phospho-S6, total S6, phospho-PP1 or total PP1.

Article Snippet: Nuclear DNA was visualized using a DAPI mounting solution (Vector Laboratories).

Techniques: In Vitro, Purification, Recombinant, Western Blot, Labeling, Activity Assay, Immunoprecipitation, Immunofluorescence, Staining