Journal: Antioxidants

Article Title: Nrf2 Activation Does Not Protect from Aldosterone-Induced Kidney Damage in Mice

doi: 10.3390/antiox12030777

Figure Lengend Snippet: Localization of the activated transcription factor Nrf2. Representative pictures of the Western blots of the expression of total Nrf2 phosphorylated at Ser40 (pNrf2, 110 kDa) in ( a ) nuclear and ( b ) cytosolic extracts of kidneys of mice as well as the quantification of band densities of the above-mentioned protein measured via ImageJ and related to the housekeepers lamin B2 (68 kDa) and GAPDH (37 kDa). Percentage of pNrf2-positive stained nuclei in cortex ( c ) and medulla ( d ). For the quantification of positive Nrf2 nuclei, 10 visual fields of cortical and 3–5 visual fields of medullary kidney sections were analyzed per animal via ImageJ. ( e ) Representative images of double stained kidney sections used for the localization of pNrf2 in kidney cells. Double staining on paraffin-embedded kidney sections was carried out using antibodies against pNrf2 (brown staining) and against calbindin (purple staining), a marker for distal tubule and early collecting duct cells. Examples of pNrf2-positive stained nuclei are indicated by black arrows; white arrows indicate the corresponding section of the tubulus system. Proximal tubules were identified by the presence of the brush border, whereas glomeruli were identified by their capillary tuft (blue circles). Distal tubules and the early collecting duct were visualized by positive calbindin staining. The late collecting duct was identified by the absence of positive calbindin staining and brush border. ( f – i ) Quantification of pNrf2-positive nuclei in the four kidney structures related to the number of nuclei in regions positive for the specific kidney cell identifiers in 10 visual fields. For the quantification in the glomerulus, 50 glomeruli were evaluated. Aldo: aldosterone, C: control, Nrf2: nuclear factor erythroid 2-related factor 2, Sulf: sulforaphane, WT: wild type. Data are shown as mean + SEM, n = 7–8. * p ≤ 0.05 vs. WT-C, # p ≤ 0.05 vs. WT-Sulf, ° p ≤ 0.05 vs. WT-Aldo, ^ p ≤ 0.05 vs. Nrf2ꜛ-C.

Article Snippet: After visualization of antibody binding, the protocol was repeated with an antibody against calbindin (1:200, #2173, Cell Signaling, Herts, UK) to identify distal tubular cells and cells of the early collecting duct.

Techniques: Western Blot, Expressing, Staining, Double Staining, Marker, Control

Journal: International journal of molecular medicine

Article Title: Aqueous extract of pomegranate seed attenuates glucocorticoid-induced bone loss and hypercalciuria in mice: A comparative study with alendronate.

doi: 10.3892/ijmm.2016.2622

Figure Lengend Snippet: Figure 3. Expression of calcium transport proteins in the kidney. (A and B) The protein levels of calcium transporters [transient receptor potential vanilloid (TRPV)5, TRPV6, calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase1 (PMCA1)] were measured in the kidney. Values are expressed as the means ± SEM, n=8 in each group. *P<0.05 versus vehicle group; #P<0.05 versus dexamethasone (DXM) group. Vehicle, vehicle group (n=8); DXM group, mice were injected intramuscularly with 5 mg/kg body weight DXM three times/week for 12 weeks (n=8); ALN group, mice rece ived alendronate orally at a dose of 0.1 mg/kg/day in combination with DXM for 12 weeks (n=8); pomegranate seed (PS) group, mice received daily drinks of aqueous extract of pomegranate seed (AE-PS) in combination with DXM for 12 weeks (n=8).

Article Snippet: After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following antibodies: transient receptor potential vanilloid (TRPV)5 (sc-398345; 1:500), TRPV6 (sc-28763; 1:500), osteoprotegerin (OPG; sc-8468; 1:1,000) and receptor activator of nuclear factor-κB ligand (RANKL; sc-7628; 1:2,000) , CaSR (sc-32181; 1:500), calbindin-D9k (CaBP-9k; sc-367381; 1:500), plasma membrane Ca2+-ATPase1 (PMCA1; sc-16488; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), overnight at 4 ̊C.

Techniques: Expressing, Clinical Proteomics, Membrane, Injection

Journal: International journal of molecular medicine

Article Title: Aqueous extract of pomegranate seed attenuates glucocorticoid-induced bone loss and hypercalciuria in mice: A comparative study with alendronate.

doi: 10.3892/ijmm.2016.2622

Figure Lengend Snippet: Figure 4. Expression of calcium transport proteins in the duodenum. (A and B) The protein expression of calcium transporters [transient receptor potential vanilloid (TRPV)5, TRPV6, calbindin-D9k (CaBP-9k)] was mea sured in the duodenum. Values are expressed as the means ± SEM, n=8 in each group. *P<0.05 versus vehicle group; #P<0.05 versus dexametha sone (DXM) group. Vehicle, vehicle group (n=8); DXM group, mice were injected intramuscularly with 5 mg/kg body weight DXM three times/week for 12 weeks (n=8); ALN group, mice received alendronate orally at a dose of 0.1 mg/kg/day in combination with DXM for 12 weeks (n=8); pomegranate seed (PS) group, mice received daily drinks of aqueous extract of pomegra nate seed (AE-PS) in combination with DXM for 12 weeks (n=8).

Article Snippet: After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following antibodies: transient receptor potential vanilloid (TRPV)5 (sc-398345; 1:500), TRPV6 (sc-28763; 1:500), osteoprotegerin (OPG; sc-8468; 1:1,000) and receptor activator of nuclear factor-κB ligand (RANKL; sc-7628; 1:2,000) , CaSR (sc-32181; 1:500), calbindin-D9k (CaBP-9k; sc-367381; 1:500), plasma membrane Ca2+-ATPase1 (PMCA1; sc-16488; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), overnight at 4 ̊C.

Techniques: Expressing, Injection

Journal: Cell reports

Article Title: RNA-programmable cell-type monitoring and manipulation in the human cortex with CellREADR

doi: 10.1016/j.celrep.2025.116037

Figure Lengend Snippet: (A) Human organotypic slice model. The human neocortex (middle and inferior temporal gyri; left) was sectioned at 350 μm and cultured on semi-permeable membranes for up to 16 days in vitro (DIV; right). CellREADR AAV vectors were applied to slices on DIV0. Orientation: A, anterior; D, dorsal; P, posterior; V, ventral. (B) Schematic of CellREADR design. In the single-virus design (left), binding of the sensor to the target initiates ADAR-mediated editing of a stop codon to express the downstream effector (mNeon). The binary system (right) uses a CellREADR virus (top right) to drive expression of tTA2 (Tet-advanced transactivator). A reporter virus, conditionally expressing a selected effector molecule (e.g., mNeon, ChIEF, or GCamp7f) under the control of the Tet-responsive element (TRE), is co-applied. (C) CellREADR targeting of diverse neuronal populations. Binary CellREADRs (mNeonGreen) designed against somatostatin ( SST ), Unc-5 netrin receptor B ( UNC5B ), parvalbumin ( PVALB ), forkhead box protein P2 ( FOXP2 ), and calretinin ( CALB2 ) were applied to the neocortex of at least 3 donors. Slices were immunostained for mNeon at DIV7. The CellREADR binary system drove robust expression of mNeon in differing lamina and in cells of various morphologies. Magnified images are indicated by dashed red boxes; throughout, pia and white matter (WM) are illustrated with dotted lines. Scale bars: 200 μm (large image) and 50 μm (magnified image). (D) Histological analysis of binary FOXP2 and CALB2 CellREADR targeting. CellREADR mNeon distribution (green, left) and protein expression of target (purple, middle) are depicted at DIV7. Fluorescence intensity profiles at far left reveal the depth distribution of cells targeted by CellREADR. Dashed boxes indicate areas depicted in (F). Scale bars: 200 μm. (E) Localization of CellREADR-targeted cells. CellREADR mNeon fluorescence intensity profiles measured across the depth of the cortex (0% = pial surface, 100% = WM boundary) averaged from 6 slices (tissues from 3 donors). The solid line shows the mean; the shaded area shows the SEM. FOXP2 -mNeon was relatively uniform throughout cortical layers, while CALB2 -mNeon was more restricted to outer layers. (F) Immunohistochemical characterization of CellREADR specificity. Representative images showing colocalization of CellREADR-mNeon with the corresponding target. Scale bars: 50 μm. (G) Quantification of CellREADR specificities as measured by immunostaining. Each point denotes the specificity of labeling measured from an individual donor’s tissue ( FOXP2 , n = 5 donors; binary CALB2 , n = 7; singular CALB2 , n = 3). Horizontal bars indicate mean specificity values for each CellREADR. Throughout the figures, data are presented as mean ± SEM. (H) Benchmarking CellREADR efficiency with a human interneuron enhancer virus DLX2.0-YFP. CALB2 CellREADR and DLX2.0-YFP viruses (rAAV2-retro) were applied to slices cut sequentially from the same neocortical tissue specimen. Tissue was immunostained at DIV7 against mNeon or YFP. Slice boundaries are indicated by the solid white line, and the WM is demarcated by a dashed white line; boxes mark the inset images on the bottom. Scale bars: 1 mm (top images) and 200 μm (bottom images). (I) Localization of DLX2.0-YFP expression. Representative image illustrating the efficiency and distribution of labeling achieved with a DLX2.0-YFP virus (left), with fluorescence intensity profile (right; averages from 3 donors) illustrating the distribution of labeled cells. Compared to CALB2 CellREADR labeling (D and H), DLX2.0-YFP + cells were less concentrated in the outer cortex. Scale bar: 250 μm. (J) DLX2.0-YFP targeting of the CALB2 population. Quantification of colocalization of DLX2.0-YFP expression and CALB2 measured by immunostaining (images not shown); note the inter-subject variability in the targeting of CALB2 cells ( n = 7 donors). (K) Efficiency of binary and singular CALB2 CellREADRs compared to DLX2.0-YFP enhancer virus. Quantification of cells labeled at DIV7 per unit area (binary CALB2 , n = 6 donors; singular CALB2 , n = 3; DLX2.0-YFP, n = 3). Note that although the CellREADRs were designed to target only CALB2 interneurons rather than multiple interneuron subclasses, they labeled more cells than the DLX2.0 virus (one-way ANOVA with Tukey’s post hoc).

Article Snippet: The following antibodies were used for immunohistochemical labeling of mNeon, mCherry, FoxP2, Calretinin and NeuN: mouse anti-mNeon Ab 1:500, Proteintech 32f6; chicken anti-mCherry Ab 1:500, Origene TA150127; rabbit anti-FoxP2 Ab 1:500, Abcam ab16046; rabbit anti-Calretinin Ab 1:500, Swant CR7697; mouse anti-NeuN Ab 1:500, Cell Signaling #94403S.

Techniques: Cell Culture, In Vitro, Virus, Binding Assay, Expressing, Control, Fluorescence, Immunohistochemical staining, Immunostaining, Labeling

Journal: eLife

Article Title: LKB1 coordinates neurite remodeling to drive synapse layer emergence in the outer retina

doi: 10.7554/eLife.56931

Figure Lengend Snippet:

Article Snippet: Calbindin D-28k , Full-length recombinant human Calbindin D-28K , Horizontal cells; amacrine cells; retinal ganglion cells , Novus biologicals; chicken polyclonal; NBP2-50028; no RRID , 1:2000.

Techniques: RNAscope, Multiplex Assay, Software