Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Lens expression of p120-catenin subfamily proteins. (A–O) Immunofluorescent labeling of equatorially cryosectioned juvenile mouse lenses (P7) using antibodies specific for p120-catenin (A–E) δ-catenin (F–J) , and Arvcf (K–O) . (P) Diagram of a portion of the lens indicating the plane of section and approximate location within the lens for (A–O) . Expression within the lens epithelium (B,G,L) , the outermost elongating lens fiber cells near the fulcrum (C,H,M) , the outermost cortical lens fiber cells within 50 microns of the surface (D,I,N) , and lens fiber cells 100 microns from the surface (E,J,O) is depicted. Note that p120-catenin and Arvcf but not δ-catenin are localized to the junctions of lens epithelial cells and that δ-catenin and Arvcf are much stronger than p120-catenin in lens fiber cells. Arvcf is also localized strongly to bicellular lens fiber cell junctions while δ-catenin appears restricted to lens fiber cell tricellular junctions. (Q–S) Immunofluorescent labeling of transversely cryosectioned mouse embryos at the indicated ages immunofluorescently labeled for Arvcf (red). Blue signal for all panels represents Hoechst labeling of nuclei. A Plan-Apochromat ×40/1.3 Oil objective with wide-field fluorescent microscope was utilized. Scalebars = 25 microns.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Expressing, Labeling, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Loss of Arvcf protein leads to disrupted lens fiber cells and an increase in p120-catenin localization. (A–F) WGA immunolabeled (white) equatorial cryosections of 30 day old mouse lenses from animals with the indicated genotype. Magnified images of the boxed region are found to the right within each panel. (C,D) are images taken ∼300 microns from the lens surface and (E,F) are from near the lens nucleus. The yellow arrowheads point to examples of fiber cells that are often observed separated from each other in Arvcf −/− lenses and the asterisks depict regions where either the membrane is not stained or possibly missing. (G) RNA isolated from the eye and brain of animals with the indicated genotype was subjected to RT-PCR using primers specific to Gapdh and exons 3 and 4 of Arvcf . Exon 4 is predicted to be absent from the transcript due to the insertion of a polyadenylation signal site within intron 4–5. (H) An Arvcf-specific antibody was utilized on western blots from control, heterozygous, and homozygous lens and brain lysates. Left: A band near the predicted size of the longest isoform of Arvcf (105 kDa) was detected in the lens and the brain of lysates from control animals. Right: Western blots with the Arvcf antibody of lens lysates from each genotype above an image of the same gel with its total protein stained. (I–L) Images of equatorial cryosections of juvenile mouse lenses (P7) from Arvcf +/+ (I,K), Arvcf +/- or Arvcf −/− (L) animals immunofluorescently co-labeled for Arvcf (red) and nuclei (blue) (I) or Arvcf (red) and N-cadherin (green). (N) Fluorescent signal intensity was measured along the junctions labeled radial or circumferential as depicted in the diagram. The graph represents the normalized mean intensity of Arvcf signal along each junction orientation. Asterisks and # symbols represent a significant difference from the control group ( p < 0.05). A Plan-Apochromat ×40/1.3 Oil objective with wide-field fluorescent microscope was utilized. The scalebar is 25 μm.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Immunolabeling, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Labeling, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Arvcf is required for adherens junction protein membrane localization. (A–D) Equatorial cryosections of juvenile mouse lenses (P7) from Arvcf +/+ (A,C) or Arvcf −/− (B,D) animals immunofluorescently labeled for δ-catenin (A–B) or p120-catenin (C–D) . Magnified views of the lens fiber cells within ∼50 μm of the lens surface from (A–D) are displayed in the panels immediately to the right. (E–F) The graphs represent the mean and standard error of tricellular fluorescent intensity of δ-catenin (E) or the bicellular junctional intensity of p120-catenin (F) . The asterisks/# symbols represent statistically significant differences between the control and experimental gropus ( p < 0.05). (G–O) Equatorial cryosections of juvenile (P30) day old lenses from Arvcf +/+ (G,J,M) or Arvcf −/− (H,K,N) mice were immunofluorescently labeled with antibodies specific for N-cadherin (G–H) , β-catenin (J–K) , and αN-catenin (M–N) . Magnified views of the lens fiber cells within ∼50 μm of the lens surface are depicted to the right. The graphs in (I,L,O) represent the mean and standard error of junctional measurements taken as shown for . The asterisks/# symbols represents a statistically significant difference from the control group ( p < 0.05). A Plan-Apochromat ×40/1.3 Oil objective with wide-field fluorescent microscope was utilized. Scalebar = 25 microns.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Labeling, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Arvcf is required for organization of N-cadherin and β-catenin in pAJs. (A–B) Whole lens fiber cells immunofluorescently co-labeled for Arvcf (blue) and N-cadherin (green). The squared region in A is the region magnified in panel (B) . The arrows represent N-cadherin nanoclusters that appear to be overlapping with Arvcf protein. Note that this is a common occurrence. (C–F) Whole lens fiber cells from Arvcf +/+ (C–D) or Arvcf −/− (E–F) mice immunofluorescently co-labeled for β-catenin (blue) and N-cadherin (green) and imaged when focused on a z-plane within the bicellular membrane of two neighboring cells (C,E) or focused on a z-plane through the cytoplasm of a single fiber cell ∼1.8 μm from the membrane (D,F) . Note that the cytoplasmic signal is relatively low in control but greater in the mutant lens fiber cells. (G–J) Magnified regions outlined by the squared areas in (C–F) showing both the overlapping and individual signals. (K–P) Attributes including the density (K,N) , total area coverage (L,O) , and individual size (M,P) of N-cadherin (K–M) and β-catenin (N–P) puncta were quantified from immunofluorescently labeled Arvcf +/+ and Arvcf −/− fiber cells and their means and standard error are depicted in the corresponding graphs. The asterisks symbols represents a statistically significant difference from the control group ( p < 0.05). A Plan-Apochromat ×63/1.40 oil objective was utilized with a Zeiss Airyscan equipped microscope. All scalebars represent 2 microns.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Labeling, Mutagenesis, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Arvcf is required for lens fiber cell interlocking protrusion morphology. (A–B) Whole lens fiber cells immunofluorescently co-labeled for Arvcf (blue) and N-cadherin (green). The box in A is magnified in (B) . The asterisks mark interlocking protrusions with lateral margins that are enriched for both Arvcf and N-cadherin. A Plan-Apochromatic CS2 ×100/1.4 oil objective was utilized with a Leica TCS SP8 STED equipped microscope. (C–D) Whole lens fiber cells immunofluorescently co-labeled for β-catenin (blue) and N-cadherin (green). The box in C is magnified in (D) . The arrowheads mark regions of discontinuous signal of both β-catenin and N-cadherin in Arvcf −/− fiber cells (D) . Also note that interlocking protrusions are not well defined by β-catenin and N-cadherin in Arvcf −/− fiber cells. A Plan-Apochromat ×63/1.40 oil objective was utilized with a Zeiss Airyscan equipped microscope. (E–P) SEM images of lens fiber cells from 1 month old (E–N) and 7 month old (M–P) Arvcf +/+ and Arvcf −/− lenses located at the indicated depths from the lens surface. Note that while N-cadherin/β-catenin do not delineate interlocking protrusion borders, they are still present in abundance but are often elongated and misshapen. Asterisks mark regions where the characteristic paddle structures appear missing. Lens fiber cells in (M–N) are shaded to view morphology. Scalebars in (E–F) = 10 microns, (G–P) = 1 micron.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Labeling, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts

doi: 10.3389/fcell.2022.840129

Figure Lengend Snippet: Arvcf is required for N-cadherin/β-catenin localization within interlocking protrusions. (A–L) Representative individual images of similarly sized, interlocking protrusions from Arvcf +/+ (A,G) and Arvcf −/− (B,H) lenses immunofluorescently labeled with N-cadherin (A–B) or β-catenin (G–H) were aligned and used to measure the fluorescent intensity of each pixel. The mean intensity for each pixel was calculated and used to generate an artificial image that represents the average signal intensity for N-cadherin (C–D) or β-catenin (I–J) in Arvcf +/+ (C,I) and Arvcf −/− (D,J) fiber cell interlocking protrusion. (E,K) represent an artificial image where the fold change at each pixel position is indicated by a heatmap. Regions where Arvcf +/+ interlocking protrusions have more signal than Arvcf −/− protrusions have warmer (redder) colors and regions where differences are less pronounced have cooler colors (bluer). (F,L) represent an artificial image indicating where statistically significant differences in pixel intensity are located. Note that the tips of interlocking protrusions have the greatest and statistically significant differences ( p < 0.05). (M–P) The mean intensity and standard error of pixels from individual interlocking protrusion images along the lines drawn parallel to the distal-proximal or left-right axes from each experimental group are depicted in the graphs. Note the depressed N-cadherin and β-catenin intensity along both axes Arvcf −/− protrusions. All images were taken with a Plan-Apochromat ×63/1.40 oil objective and a Zeiss Airyscan equipped microscope was utilized.

Article Snippet: Antibodies: Arvcf (1:500 for sections; 1:250 for whole lens fiber cells, Thermofisher, PA5-64129), N-cadherin (1:500 BD Biosciences, 610,921), αN-catenin (1:250, ABclonal A15269), δ-catenin (1:500, Thermofisher, PA5-53275), p120-catenin (1:200, BD Biosciences, BDB610133), β-catenin (1:500, BD biosciences, BDB610153), β-catenin (1:200, GeneTex, GTX101435), Aquaporin-0 (1:200, Alpha Diagnostics International, AQP01-A), αB-crystallin (1:500, DSHB, CPTC-CRYAB-3).

Techniques: Labeling, Microscopy

Journal: Molecular cancer therapeutics

Article Title: Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma

doi: 10.1158/1535-7163.MCT-20-0319

Figure Lengend Snippet: Val-083 cytotoxicity is exerted independently of MMR or MGMT expression. A, Viability of parental U-251 MG, T98G, U-87 MG, LN-18, and SF268 cell lines upon exposure to different concentrations of Val-083 measured by MTT. Values are represented as percentage of cell viability compared with DMSO control. B, Cell-cycle profile of U-251 MG or T98G cells treated with different doses of TMZ or Val-083 for 48 hours. C, Competition assay of U-251 MG cells expressing shRNAs targeting MSH6 (shMSH6.3908), PMS2 (shPMS2.946), and MSH2 (shMSH2.357) after treatment with TMZ or VAL-083. The plotted value is the fold enrichment of the shRNA-expressing cells (eGFP+) compared with the control cells (mCherry+) and normalized to the DMSO control. Statistical test: Two-way ANOVA (P values: *P < 0.05, **P < 0.01, compared with DMSO treated). D, CFA of U-251 MG cells expressing control shRNA or shMSH6 after treatment with TMZ or Val-083 for 9 days. E, Cell-cycle profile of U-251 MG cells expressing control shRNA (shRen.660) or shMSH6.3908 after treatment with TMZ or Val-083 for 48 hours. F, Quantification of mean γH2AX fluorescence in nuclei of cells treated with vehicle, TMZ, or Val-083 for 48 hours. Values are normalized to the DMSO control, and fold increase in H2AX signal is plotted. Statistical test: Two-way ANOVA (P values: ***, P < 0.001; ****, P < 0.0001, compared with DMSO treated).

Article Snippet: Different concentrations of TMZ (Merck, Catalog No. T2577), Val-083 (Adooq Bioscience, Catalog No. A15269), or DMSO (Sigma-Aldrich) were added.

Techniques: Expressing, Competitive Binding Assay, shRNA, Fluorescence

Journal: Molecular cancer therapeutics

Article Title: Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma

doi: 10.1158/1535-7163.MCT-20-0319

Figure Lengend Snippet: TMZ and Val-083 combination treatment show synergistic cytotoxic effect. A, Table showing the percentage of cell viability (as compared with DMSO, top number in each cell) and SD (bottom number). B, Plot of synergy score calculated with the HSA method of U-251 MG cells treated with different doses of TMZ and Val-083. C, Table showing the maximum synergy scores of different adherent cell lines (U-251 MG, T98G, U-87 MG, SF268, LN-18) and tumorspheres grown in suspension (H543, H516, H676), as well as the doses of TMZ and Val-083, which rendered said score. MGMT expression is also indicated. D, CFA of U-251 MG cells incubated with low doses of TMZ, Val-083, or the combination for 9 days. E, Cell-cycle profile of U-251 MG cells incubated for 48 hours with TMZ, Val-083, or the combination of both. F, Representative pictures of γH2AX staining of U-251 MG cells treated for 48 hours with DMSO, TMZ, Val-083, or the combination of the last two. G, Quantification of γH2AX signal intensity in nuclei of U-251 MG cells treated for 48 hours with TMZ, Val-083, or the combination of both. Statistical test (P values: ***, P < 0.001). H, CFA of T98G cells incubated with high and low doses of TMZ, Val-083, or the combination for 9 days. I, Quantification of γH2AX signal intensity in nuclei of T98G cells treated for 48 hours with TMZ, Val-083, or the combination of both. Statistical test: t test (P values: *, P < 0.05; ***, P < 0.001).

Article Snippet: Different concentrations of TMZ (Merck, Catalog No. T2577), Val-083 (Adooq Bioscience, Catalog No. A15269), or DMSO (Sigma-Aldrich) were added.

Techniques: Expressing, Incubation, Staining

Journal: Molecular cancer therapeutics

Article Title: Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma

doi: 10.1158/1535-7163.MCT-20-0319

Figure Lengend Snippet: Treatment with combination of TMZ and Val-083 increases survival in mice. A, Quantification of the ratio of luminescence emission by U251-MG in ex vivo-treated brain slices after 7 days as compared with the initial. Plots represent the fold change compared with the DMSO control. Statistical test: t test (P values: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignficant); P values on top of the boxes are comparison over the DMSO control. B, Kaplan–Meier curve showing the survival proportions of mice transplanted with U-251 MG cells and treated with TMZ, Val-083, or the combination of both. Log-rank P values: **, P < 0.01; ns, nonsignificant). C, Kaplan–Meier curve showing the survival proportions of mice transplanted with H676 cells and treated with TMZ, Val-083, or the combination of both. Log-rank P values: **, P < 0.01; ns, nonsignificant).

Article Snippet: Different concentrations of TMZ (Merck, Catalog No. T2577), Val-083 (Adooq Bioscience, Catalog No. A15269), or DMSO (Sigma-Aldrich) were added.

Techniques: Ex Vivo

Journal: Molecular cancer therapeutics

Article Title: Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma

doi: 10.1158/1535-7163.MCT-20-0319

Figure Lengend Snippet: Mechanisms of action of TMZ and Val-083. Expression of MGMT effectively removes the O6-guanine methylation induced by TMZ exposure, which allows cell survival. Lack of MGMT allows the formation of mismatches in the next replication round, which leads to the generation of double DNA-strand breaks mediated by the MMR machinery. The accumulation of double-strand breaks ultimately lead to cell death. However, defects in the MMR pathway paves the way for the accumulation of unresolved mismatches, which allows cell survival and a hypermutation phenotype. In the case of Val-083, there is a formation of interstrand adducts, that are not resolved by MGMT, and lead to cytotoxicity independently of MMR status.

Article Snippet: Different concentrations of TMZ (Merck, Catalog No. T2577), Val-083 (Adooq Bioscience, Catalog No. A15269), or DMSO (Sigma-Aldrich) were added.

Techniques: Expressing, Methylation

Journal: Frontiers in Neuroscience

Article Title: Differences in DNA Methylation Reprogramming Underlie the Sexual Dimorphism of Behavioral Disorder Caused by Prenatal Stress in Rats

doi: 10.3389/fnins.2020.573107

Figure Lengend Snippet: Effect of PS on DNMT and demethylase in adolescence. (A) DNMT3a protein expression was markedly upregulated (* P < 0.05, n = 6) while DNMT1 level was marginally increased in PS females. No significant difference in Tet-2 expression was observed between PS and control females. (B) Tet-2 level was decreased in PS males (* P < 0.05, n = 6), whereas DNMT3a and DNMT1 levels were similar in PS and control males. * P < 0.05, * P < 0.01 vs. corresponding control group. Data represent mean ± SEM.

Article Snippet: Total protein was extracted from hippocampal tissue samples and quantified, and equal amounts were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, United States) that was blocked in 3% non-fat milk for 1 h at room temperature followed by overnight incubation at 4°C with primary antibodies against GR (Cell Signaling Technology, Danvers, MA, United States; 12041S, 1:1,000), DNMT1 (ab188453, 1:1,000) and DNMT3a (ab188470, 1:1,000) (both from Abcam, Cambridge, MA, United States), Tet-2 (ABclonal, Woburn, MA, United States; A1526, 1:1,000), and β-actin (Proteintech, Chicago, IL, United States; 66,009–1, 1:2,000).

Techniques: Expressing