Journal: Frontiers in Marine Science

Article Title: Molecular characterization of a short neuropeptide F signaling system in the swimming crab, Portunus trituberculatus, and its role in ovarian development

doi: 10.3389/fmars.2024.1451544

Figure Lengend Snippet: FIGURE 1 Expression of PtsNPFR in HEK293T cells and ligand-receptor activation of signaling. (A) Cells expressing PtsNPFR/pEGFP-N1 fusion protein were stained with a nuclei probe (DAPI) and a membrane plasma probe (Dil). (B) HEK293T transiently co-transfected with PtsNPFR/pEGFP-N1, pCRE-luc and pRL-TK were treated with different doses of PtsNPF1, PtsNPF2, and PtsNPF3 and the concentration of cAMP was determined. Values are plotted as mean ± SEM from three biological replicates.

Article Snippet: The transfected cells were fixed with 4% paraformaldehyde (PFA) for 20 min, and then stained with the cell membrane probe Dil (Beyotime, China) at 37°C for 10 min. After removing the Dil solution, the cells were washed three times with PBS and further incubated with a nuclear dye DAPI (Beyotime, China) at 37°C for 10 min. After removing the DAPI solution, the cells were washed three times with PBS, mounted in an antifade mounting medium (Solarbio, China), and imaged using a Zeiss laser scanning confocal microscope (LSM880, 294 Carl Zeiss, Oberkochen, Germany).

Techniques: Expressing, Activation Assay, Staining, Membrane, Clinical Proteomics, Transfection, Concentration Assay

Journal: Advanced Science

Article Title: Endothelium‐Derived Engineered Extracellular Vesicles Protect the Pulmonary Endothelial Barrier in Acute Lung Injury

doi: 10.1002/advs.202306156

Figure Lengend Snippet: Uptake and distribution of LET‐EVs and EVs in vivo and in vitro. A) Fluorescence microscope of cellular uptake of DIL‐labeled LET‐EVs and EVs after 2 and 24 h of incubation with HPMVECs. The stains used were as follows: DIL‐labeled EVs (Orange), F‐actin (Green), and DAPI (Blue) (Scale bar: 25 µm). B) Quantification of EVs integrated fluorescence density based on ImageJ analysis. The uptake efficiency of DIL‐labeled LET‐EVs was higher than that of DIL‐EVs. C) Imaging of ALI mice after 0, 2, and 48 h of administration of DIR, DIR‐EVs, and DIR‐LET‐EVs. Compared with the DIR and DIR‐EVs groups, the fluorescence signal of LET‐EVs was mainly accumulated in the epigastric region after 2 h. D) Fluorescence imaging of tissues from ALI mice in the DIR, DIR‐EVs, and DIR‐LET‐EVs groups after 48 h. Compared with the DIR and DIR‐EVs groups, the signal in the DIR‐LET‐EVs groups was mainly aggregated in lung tissues. E) Quantitative analysis of mean fluorescence intensities in different tissues following administration of EVs and LET‐EVs. F) The clearance of EVs in lung tissues was calculated by dividing average lung signals by average liver signals. G) Uptake of DIL‐EVs and DIL‐LET‐EVs by endothelial cells in lung tissues. Immunofluorescence staining was performed in lung sections using an antibody against CD31 (endothelial cell marker, green). Nuclei were stained with DAPI (Scale bar: 100 µm). All the data are presented as the mean ± SD ( n = 3). * p < 0.05, *** p < 0.001 compared with the EVs group by unpaired Student's t ‐tests.

Article Snippet: Purified EVs were incubated with 10 μ m of the cell membrane dye DIL (Solarbio, Beijing, China) for 30 min. Unbound DIL dye was removed with three washes with PBS buffer (pH 7.4, 3000 g, 20 min per wash) in a 10 kDa MWCO Amicon centrifugal filter at 4 °C.

Techniques: In Vivo, In Vitro, Fluorescence, Microscopy, Labeling, Incubation, Imaging, Immunofluorescence, Staining, Marker

Journal: bioRxiv

Article Title: The Rab32 small GTPase is required for efficient cross-priming of CD8 + T cells against cell-associated antigens by XCR1 + type 1 DCs in vivo

doi: 10.1101/2025.05.03.652057

Figure Lengend Snippet: Rab32 is highly expressed in cDC1s but does not control their differentiation. a. Expression of Rab GTPases in various DC subsets in mice (microarray data from Immgen database). A group of Rabs – 7b, 19, 39a, 32 and 43 is differentially and highly expressed in cDC1s. b. The Rab32 tm1a have a promoter driven cassette present between the exon 1 and 2 or Rab32, which disrupts Rab32 protein expression. The tm1a mice were crossed with a germ line cre mice to create genetically deleted Rab32 KO mice (Rab32 cdel). c. Verification of the absence of Rab32 in the pre-Cre deletion Rab32 KO (tm1a) and post-Cre deletion Rab32 KO (cdel) mice by western blot. Analysis on tma1 and cdel performed on BMDCs and sorted splenic CD11c+XCR1+ cDC1s, respectively, with corresponding littermate control and actin loading control. d. FACS analysis of cDC populations in the spleens of naïve WT and KO mice. The Lin- consists of non-cDC markers including TER119, NK1.1, TCRb, GR-1 and CD19. Bar graphs depict the Quantification of XCR1+ cDC1s and SIRPa+ cDC2s populations. Data from 4 independent experiments. Each point represents an individual mouse. (WT n=11, KO n=13). Confocal microscopy projections of CD11c+ XCR1+ cDC1s sorted from the spleen of Rab32 WT mice, fixed and stained with antibodies against Rab32 along with various intracellular membrane markers- giatin (cis-Golgi), TGN38 (trans-Golgi), calnexin (Endoplasmic reticulum), COX5B (Mitochondria), H-2Kb and Lamp1 (Lysosomes). Scale bar - 7um.

Article Snippet: The membranes were stained with respective primary antibodies in TBS.T for 1.5h at RT - Rab32 (400 dil, host mouse, Santa Cruz 390178) or Actin (20,000 dil, host mouse, Sigma A1978).

Techniques: Control, Expressing, Microarray, Western Blot, Confocal Microscopy, Staining, Membrane

Journal: bioRxiv

Article Title: The Rab32 small GTPase is required for efficient cross-priming of CD8 + T cells against cell-associated antigens by XCR1 + type 1 DCs in vivo

doi: 10.1101/2025.05.03.652057

Figure Lengend Snippet: Rab32 does not control the cross-presentation of cell-associated antigens by cDC1s in vitro. a. FACS plots depict comparison MHC-I, MHC-II and activation marker (CD40 and CD80) expression on Rab32 WT and KO cDC1s. Bar graphs represent quantified data from multiple experiments. MHC-I, MHC-II and CD40 data from 2 independent experiments, CD80 data from 3 independent experiments. b. Spleen sorted CD11c+ XCR1+ cDC1s were co-cultured ex-vivo with CTV labelled and purified OT-I cells in the presence of different concentrations SIINFEKL (high affinity for OT-I TCR) or SIIQFEKL (mutated peptide, low affinity for OT-I TCR). FACS plot depicts OT-I divisions (CTV dilutions) in response to SIINFEKL peptide presentation by WT or KO cDC1s. OT-I divisions in response to SIINFEKL or SIIQFEKL presentation by WT or KO cDC1s were quantified. Data from 1 experiment with 3 individual cDC1 sortings from WT or KO mice each. c. In the same experiment, IL-2 (day1) and IFNγ (day3) secretions were quantified by ELISA. d. Experimental design for the ex-vivo cross-presentation assay. IL-2 and IFNγ secretions were measured as readout for cross-presentation. IL-2 and IFNγ secretions in presence of increasing Kbm1_OVA:cDC1 ration were quantified by ELISA. Data from 4 individual cDC1 sortings from WT or KO mice each.

Article Snippet: The membranes were stained with respective primary antibodies in TBS.T for 1.5h at RT - Rab32 (400 dil, host mouse, Santa Cruz 390178) or Actin (20,000 dil, host mouse, Sigma A1978).

Techniques: Control, In Vitro, Comparison, Activation Assay, Marker, Expressing, Cell Culture, Ex Vivo, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The Rab32 small GTPase is required for efficient cross-priming of CD8 + T cells against cell-associated antigens by XCR1 + type 1 DCs in vivo

doi: 10.1101/2025.05.03.652057

Figure Lengend Snippet: Rab32 controls the cross-priming against cell-associated antigens independently of its expression in CD8+ T cells. a. Experimental design for the OT-I adoptive transfer experiment. b. FACS panels depict a comparison of OT-I proliferation between WT and KO mice injected with 0.1x106 Kbm1_OVA. c. Quantification of the percentage of OT-I in the CD8+ T cell population in the spleens of WT and KO mice, for different numbers of injected Kbm1_OVA. Each point represents an individual mouse, and each line represents an individual experiment (*p<0.05, two-way ANOVA test). d. FACS panels depict a comparison of OT-I proliferation (CTV dilutions) and gain of effector function (loss of CD62L) between WT and KO mice. Quantification of the percentage of CTVlowCD62Llow cells in OT-I population in the spleens of WT and KO mice, for different numbers of injected Kbm1_OVA (*p<0.05, ***p<0.001, two-way ANOVA test).

Article Snippet: The membranes were stained with respective primary antibodies in TBS.T for 1.5h at RT - Rab32 (400 dil, host mouse, Santa Cruz 390178) or Actin (20,000 dil, host mouse, Sigma A1978).

Techniques: Expressing, Adoptive Transfer Assay, Comparison, Injection

Journal: bioRxiv

Article Title: The Rab32 small GTPase is required for efficient cross-priming of CD8 + T cells against cell-associated antigens by XCR1 + type 1 DCs in vivo

doi: 10.1101/2025.05.03.652057

Figure Lengend Snippet: Rab32 is required for the efficient cross-priming of CD8+ T cells against cell- associated antigens in-vivo. a. Rab32 WT or KO mice were i.p. injected with 20ug polyI:C. 14h later, splenic cDC1s from the injected mice and non-injected WT controls were examined by FACS. The maturation phenotype of cDC1s was assessed by MHCI, MHCII, CD80 and CD40 levels. b. FACS plots depict the profile of endogenous CD8+ T cells in the spleens of Rab32 WT and KO mice, 10 days following immunisation with Kbm1_OVA+polyI:C. The upper panel depicts the OVA-specific (Tetramer+CD62Llow) CD8+ T cells, while lower panel depicts the effector (CD62LlowCD44hi) CD8+ T cells in the same spleen samples. c,d. Quantification of the percentages of Tetramer+ CD62Llow and CD44hi CD62Llow endogenous CD8+ T cells in the spleens of mice in each group. Data from 5 individual experiments. (Kbm1 control n=8, WT n=27, KO n=32, **p<0.01, Mann-Whitney test). e, f, g. Same as in b,c,d. but in the blood. Data from 3 individual experiments. (Kbm1 control n=4, WT n=12, KO n=18, *p<0.05, **p<0.01, unpaired t-test).

Article Snippet: The membranes were stained with respective primary antibodies in TBS.T for 1.5h at RT - Rab32 (400 dil, host mouse, Santa Cruz 390178) or Actin (20,000 dil, host mouse, Sigma A1978).

Techniques: In Vivo, Injection, Control, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

doi: 10.1167/iovs.19-27656

Figure Lengend Snippet: scRNA-seq identifies Pbk as preferentially expressed in mature TA cells in the corneal epithelium. (A) t-SNEs colored by the normalized log-transformed expression of Pbk. (B) Immunofluorescence staining with PBK antibody in a frozen section of human limbal and corneal epithelium. PBK expression was dramatically lower in limbal epithelium than in the corneal epithelium. Cor, cornea; K15, Keratin 15; Lim, limbus. Scale bar: 20 μm. Dotted lines mark the basement membrane. n = 3. (C, D) hTCEpi cells were transfected with two different siRNAs recognizing PBK transcripts (siPBK no. 1 and no. 3) or sicontrol. Immunoblotting showed that siPBK transfection dramatically reduced PBK protein expression (C). Cell cycle distribution was analyzed by flow cytometry, showing that a loss of PBK induced a G2/M phase arrest (D). n = 4.

Article Snippet: The following antibodies were used: PBK (Catalog #16110–1-AP-1; 1:1000 dil; Proteintech), ATF3 (Catalog #33593 - 1:1000 dil; Cell Signaling Technologies, Danvers, MA, USA), GAPDH (sc-32233–1:1000 dil; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transformation Assay, Expressing, Immunofluorescence, Staining, Membrane, Transfection, Western Blot, Flow Cytometry

Journal: Investigative Ophthalmology & Visual Science

Article Title: Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

doi: 10.1167/iovs.19-27656

Figure Lengend Snippet: scRNA-seq identifies a reduction of PBK expression in Beclin 1 +/− mouse corneal epithelial cells. Immunofluorescence staining with PBK antibody in a frozen section of mouse limbal and corneal epithelium. In wild-type mice, PBK is preferentially expressed in the corneal epithelium (B) compared with the limbal epithelium (A). PBK expression is significantly decreased in the corneal and limbal epithelium from Beclin 1 +/− mice (C, D). PC, peripheral cornea. Scale bar: 50 μm. Dotted lines mark the basement membrane. n = 4.

Article Snippet: The following antibodies were used: PBK (Catalog #16110–1-AP-1; 1:1000 dil; Proteintech), ATF3 (Catalog #33593 - 1:1000 dil; Cell Signaling Technologies, Danvers, MA, USA), GAPDH (sc-32233–1:1000 dil; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

doi: 10.1167/iovs.19-27656

Figure Lengend Snippet: scRNA-seq identifies an increase in Atf3 expression in beclin 1 +/− mouse corneal epithelial cells. (A) Immunofluorescence staining with ATF3 antibody in frozen sections of mouse limbal and corneal epithelium. In wild-type mice, ATF3 is preferentially expressed in the limbal epithelium compared with the corneal epithelium. (B) Bar graph shows a significant increase in ATF3-expressing cells in the limbal epithelium from beclin 1 +/− mice as calculated from immunofluorescence images using ImageJ ( http://imagej.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) (ATF3+ cells, n = 4). Scale bar: 50 μm. Dotted lines mark the basement membrane. (C) Immunoblotting showed that ATF3 expression was significantly higher in human limbal epithelial cells (HLECs) than in human corneal epithelial cells (HCECs). n = 3. (D) Atf3 mRNA expression in beclin 1 +/− mice corneal epithelium as compared with its littermate controls (wild-type) shows an increase in Atf3 expression. Values are fold change over wild-type. n = 4. (E) hTCEpi cells were transfected with siATF3 or sicontrol (siCTRL). Cell proliferation was analyzed by WST-1 assay, showing that loss of ATF3 increased proliferation. Immunoblotting showed that siATF3 transfection dramatically reduced ATF3 protein expression. n = 3. (F) Model proposing how autophagy regulates epithelial proliferation via increased PBK and H2AX expression and decreased ATF3 expression, resulting in stem/TA cell activation.

Article Snippet: The following antibodies were used: PBK (Catalog #16110–1-AP-1; 1:1000 dil; Proteintech), ATF3 (Catalog #33593 - 1:1000 dil; Cell Signaling Technologies, Danvers, MA, USA), GAPDH (sc-32233–1:1000 dil; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Western Blot, Transfection, WST-1 Assay, Activation Assay

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A Western blot of HEK293T cell lysates transfected with Dcc-pHluorin (Dcc-pH) and JamC-HALO. Immunoprecipitation was performed using GFP or negative control IgG antibodies. Blots were probed for Dcc and HALO-tag, with the yellow arrowhead indicating the expected JamC-HALO band size. B Schematic depicting interactions between Dcc, JamC, and the polarity protein Pard3. Pard3 recruits JamC to the membrane through its PDZ1 domain, which binds the Class 2 PDZ motif at JamC’s C-terminus. JamC interacts with Dcc’s extracellular domain, while Pard3’s PDZ3 the PDZ 3 domain is predicted to interact with a Class 1 PDZ binding motif (X-S/T-X-ϕ COOH ) , , present on Dcc intracellular domain. C , D Airyscan confocal imaging of CGNs nucleofected with Dcc-pHluorin (cyan), JamC-SNAP (yellow), and Halo-Pard3 (magenta). Phluorin and the SNAP dye used here are both pH sensitive highlighting membrane-bound proteins. C Single focal plane showing overlap of Dcc with JamC and Pard3 at the proximal dilation of a CGN (white arrowheads). D Maximum projection of two CGNs forming an adhesion, showing Dcc clustering at the adhesion site before and after Ntn1 addition (200 ng/L). Dcc co-localized with JamC/Pard3 at the adhesion (white arrowhead) and accumulated at the adhesion periphery (hollow arrowhead). Five minutes after the addition of Ntn1 at 200 ng/L, the number of bright Dcc clusters (blue arrowhead) at the membrane surface increased and some newly formed clusters were recruited to the periphery of the JamC/Pard3/Dcc-positive adhesion (white arrowhead). Proximity Labelling Assay (PLA) using Duolink™ fluorescence protocol on fixed dissociated granule neurons plated on laminin and cultured for 24 h, using 2 pairs of primary antibodies: Rabbit against Dcc extracellular domain (ECD) and a Goat against JamC ECD ( E ), and Mouse against Dcc intracellular domain (ICD) and a Rabbit against Pard3 ( F ). Duolink™ staining with no primary, only one or both primaries were compared. Bar graphs represent the ratio of PLA staining intensity (Gray) against Dapi (Cyan) intensity normalized to the negative control without primary antibody (E: n = 4, F: n = 4), replicated 4 (E) and 3 (F) times with similar results. Scale bars: (C, D) 5 µm, (E-F) 10 µm. Error bars represent SEM. See Source Data File.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Western Blot, Transfection, Immunoprecipitation, Negative Control, Membrane, Binding Assay, Imaging, Fluorescence, Cell Culture, Staining

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A , B Results of ex vivo slice culture assays under different conditions. In each case, the top curve shows the entire distribution of the radial distances of H2B-positive electroporated nuclei from the edge of the slice in replicates. Below this is a plot of the average radial distance from the edge among replicates, and below this is a micrograph representative of the nuclear distribution after 48 h in culture. All are displayed on the same scale, representing a distance from 0 to 300 µm. In addition to H2B-Cherry, the following constructs were electroporated: in ( A ), LacZ+Mir30 shLuc (control, n = 5); LacZ+Mir30 shPard3 ( n = 4); Atoh1::Ntn1+Mir30 shPard3 ( n = 4); Dcc at 0.25 µg+Mir30 shPard3(4); LacZ+Mir30 shJamC ( n = 5); Atoh1::Ntn1+Mir30 shJamC ( n = 5); and Dcc at 0.25 µg+Mir30 shJamC ( n = 3); in ( B ), LacZ+Mir30 shLuc (control, n = 5); Pard3+Mir30 shLuc ( n = 6); Pard3+Mir30 shDcc ( n = 4); JamC+Mir30 shLuc ( n = 3); and JamC+Mir30 shDcc ( n = 4). Each respective control is represented by a red dashed line in the distribution plot. C Chart representing the average variation across replicates in the endpoint nuclear displacement on the x-axis from an even probability of 50%:50%, with negative values representing an attraction to the source of the Ntn1 gradient (in red when statistically significant) and positive values representing repulsion (in blue when statistically significant). Unsorted dissociated CGNs were nucleofected and plated on laminin-coated channel microslides for 24 h, then Ntn1 was added unilaterally into the channel 10 min before the start of nuclear tracking for 2 h. Cells were nucleofected with H2B-mCherry, GPI-pHluorin, and the following: LacZ (control, n = 6), Pard3 ( n = 5), JamC ( n = 7), Mir30 shLuc (control, n = 6), Mir30 shPard3 ( n = 8), and Mir30 shJamC ( n = 6). Abbreviations: EGL external granule layer, ML molecular layer, IGL internal granule layer. In ( A ) through ( C ), error bars represent the SEM. Statistics: ˖ p ≤ 0.1, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, as assessed by an ANOVA followed by a Dunnett post hoc test against the respective controls in (A) and (B) and by a chi-square test in (C) against an even probability of 50%:50%. See Source Data File.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Ex Vivo, Construct, Control

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A , E Spinning-disk confocal live-cell imaging of dissociated granule neurons plated on laminin for 24 h after nucleofection with Dcc-pHlurorin (Dcc-pH) (cyan), GPI-TdTomato (magenta), and one of the following: in (A), LacZ (Ctrl, n = 10), Siah2 ( n = 14), Pard3 ( n = 14) or JamC ( n = 7); in ( E ), Mir30 shLuc (Ctrl, n = 9), Mir30 shSiah2 ( n = 10), Mir30 shPard3 ( n = 9), or Mir30 shJamC ( n = 9). Cells were tracked for a total of 1 h at 150 s intervals. Representative pictures for each condition show a maximum projection before (t: −1 m 15 s) and after (t: + 6 m 15 s) the addition of Ntn1 at 200 ng/mL. B Schematic representing the segmentation process for the analysis of the time-lapse images in ( A ) and ( E ). The Dcc-pH and GPI-tdTomato channels were segmented using Pixel classification with Ilastik. The resulting “clustered area fraction” is the ratio of the area of the segmented Dcc-pH regions (bright Dcc clusters) to the area of the membrane in each field of view for each time point. C , F Graphs representing the Dcc-pH area fraction over the membrane area, normalized to their respective controls. The different experimental conditions are the same as those in ( A ) and ( E ). A dashed-line marks the addition of 200 ng/mL of Ntn1 at t0. D , G Bar charts highlighting data presented in (C) and (F) for a time point before the addition of Ntn1 (t = −1 m 15 s) and for another time point shortly thereafter (t = +6 m 15 s). In D , G error bars represent the SEM. Statistics: ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, as assessed by an ANOVA followed by a Games–Howell post hoc test against the respective controls. See also Source Data File. Scale bars in (A) and (E) represent 10 µm.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Live Cell Imaging, Membrane

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: The left panel shows the various stages of CGNs and their relative layer occupancy (gray = GNPs, peach = newly differentiated CGN, blue = maturing CGN). The right panel shows the layer-specific response to Netrin-1 produced in the GZ. Siah2 degrades Dcc and Pard3 in GNPs rendering them unresponsive to Netrin-1. Newly differentiated CGNs are slightly attracted to Netrin-1, likely controlling how long these cells remain in the iEGL. Maturing CGNs express high levels of Pard3 which promotes exocytosis of JamC and Dcc and formation of larger structures at the membrane surface. The need of JamC adhesion for Dcc to exit the germinal zone in response to Netrin-1 is at the heart of the coincidence detection circuit.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Produced, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

doi: 10.1167/iovs.19-27656

Figure Lengend Snippet: scRNA-seq identifies a reduction in H2ax expression in beclin 1 +/− mouse corneal epithelial cells. (A–D) Immunofluorescence staining with H2AX antibody in a frozen section of mouse limbal and corneal epithelium. In wild-type mice, H2AX is preferentially expressed in the corneal epithelium (B) compared with limbal epithelium (A). H2AX expression is significantly decreased in the corneal epithelium from beclin 1 +/− mice (D). n = 4. Scale bar: 50 μm. (E, F) Immunofluorescence staining with H2AX antibody in a frozen section of human limbal (F) and corneal (E) epithelium. H2AX was primarily restricted to the corneal epithelium. Scale bar: 20 μm. Dotted lines mark the basement membrane. n = 3.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% goat serum in PBS and incubated overnight with the following primary antibodies: rabbit polyclonal antibodies against Kl67 (SP6- 1:500 dil; Sigma-Aldrich Corp., St. Louis, MO, USA), PBK (16110–1-AP-1:100dil; Proteintech, Rosemont, IL, USA), TXNIP (40–3700- 1:100 dil; Thermo Scientific, Waltham, MA, USA), H2AX (10856–1-AP- 1:50 dil; Proteintech), ATF3 (HPA001562–1:50 dil; Sigma-Aldrich Corp.) or a mouse monoclonal antibody against K15 (MA5–11244–1:100 dil; Thermo Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

doi: 10.1167/iovs.19-27656

Figure Lengend Snippet: scRNA-seq identifies an increase in Atf3 expression in beclin 1 +/− mouse corneal epithelial cells. (A) Immunofluorescence staining with ATF3 antibody in frozen sections of mouse limbal and corneal epithelium. In wild-type mice, ATF3 is preferentially expressed in the limbal epithelium compared with the corneal epithelium. (B) Bar graph shows a significant increase in ATF3-expressing cells in the limbal epithelium from beclin 1 +/− mice as calculated from immunofluorescence images using ImageJ ( http://imagej.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) (ATF3+ cells, n = 4). Scale bar: 50 μm. Dotted lines mark the basement membrane. (C) Immunoblotting showed that ATF3 expression was significantly higher in human limbal epithelial cells (HLECs) than in human corneal epithelial cells (HCECs). n = 3. (D) Atf3 mRNA expression in beclin 1 +/− mice corneal epithelium as compared with its littermate controls (wild-type) shows an increase in Atf3 expression. Values are fold change over wild-type. n = 4. (E) hTCEpi cells were transfected with siATF3 or sicontrol (siCTRL). Cell proliferation was analyzed by WST-1 assay, showing that loss of ATF3 increased proliferation. Immunoblotting showed that siATF3 transfection dramatically reduced ATF3 protein expression. n = 3. (F) Model proposing how autophagy regulates epithelial proliferation via increased PBK and H2AX expression and decreased ATF3 expression, resulting in stem/TA cell activation.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% goat serum in PBS and incubated overnight with the following primary antibodies: rabbit polyclonal antibodies against Kl67 (SP6- 1:500 dil; Sigma-Aldrich Corp., St. Louis, MO, USA), PBK (16110–1-AP-1:100dil; Proteintech, Rosemont, IL, USA), TXNIP (40–3700- 1:100 dil; Thermo Scientific, Waltham, MA, USA), H2AX (10856–1-AP- 1:50 dil; Proteintech), ATF3 (HPA001562–1:50 dil; Sigma-Aldrich Corp.) or a mouse monoclonal antibody against K15 (MA5–11244–1:100 dil; Thermo Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Western Blot, Transfection, WST-1 Assay, Activation Assay