Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: HEPACAM2 expression in SCLC cells and tumors. (A) HEPACAM2 mRNA levels in cancer cell lines are based on RNA‐seq data from the CCLE. (B) HEPACAM2 mRNA levels were validated by qPCR and are presented as log10 of fold change relative to the β‐actin mRNA expression level (symbols show an average of three replicates per cell line). We used the RNA‐seq value for each HEPACAM2 level, log2(RPKM), downloaded from the CCLE data set found at http://cBioPortal.org , to compare our qPCR data. (C) HEPACAM2 mRNA levels in tumor samples were detected by RNA‐seq comparing SCLC ( n = 29) with other cancers in TCGA; **** P value <0.0001. (D) HEPACAM2 mRNA levels in tumor samples were detected by RNA‐seq comparing SCLC ( n = 29) to normal lung ( n = 25) and both typical ( n = 17) and atypical ( n = 13) pulmonary carcinoid tumors. BRCA indicates breast cancer; CCLE, Cancer Cell Line Encyclopedia; CR, colorectal cancer; GBM, glioblastoma; LUAD, lung adenoma; LUSC, lung squamous NSCLC; mRNA, messenger RNA; NK, natural killer; NOS, not otherwise specified; NSCLC, non–small cell lung cancer; PRAD, prostate adenoma cancer; qPCR, quantitative polymerase chain reaction; RNA‐seq, RNA sequencing; RPKM, reads per kilobase per million mapped reads; SCLC, small cell lung cancer; SKCM, skin melanoma cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Localization of protein expression of HEPACAM2. (A) Immunofluorescence staining of HEPACAM2 in SCLC cell lines revealed that most protein expression localized to the plasma membrane. A ProSci antibody was used to detect HEPACAM2 (pseudo green) and counterstained with Hoechst (pseudo blue). (B) Immunofluorescence staining of overexpressed HEPACAM2 (pseudo green) in an NSCLC cell line, H1299, revealed that most protein expression localized to the plasma membrane (yellow arrows), as evidenced by counterstaining (pseudo pink) with a plasma membrane marker (CellBrite) ( Right ). The scale bar represents 10 µm. (C) Western blots of identical protein lysates of control and HEPACAM2‐overexpressing H1299 cells were detected with both FLAG and ProSci antibodies. Black arrowheads indicate the major ∼70‐kDa band. (D) Western blots of HEPACAM2 expression in identical protein lysates of control, vehicle‐treated and tunicamycin‐treated HEPACAM2‐overexpressing H1299 cells were detected by both FLAG and ProSci antibodies. Black arrowheads indicate the major ∼70‐kDa band; red arrowheads indicate a ∼50‐kDa band. (E) Western blots of HEPACAM2 expression in biotin pull‐down control and HEPACAM2‐overexpressing H1299 cells were detected with a ProSci antibody after streptavidin pull‐down. The black arrowhead indicates the major ∼70‐kDa band. C indicates control; Con, control; IB, immunoblotting; kDa, kilodalton; NSCLC, non–small cell lung cancer; OE, overexpressing; SCLC, small cell lung cancer; T , tunicamycin‐treated; V , vehicle‐treated.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Marker, Western Blot, Control

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: HEPACAM2 expression detected in EVs of SCLC cell lines. (A) mRNAs of HEPACAM2 could be detected in EVs of SCLC cell lines (H526, H209, and H510A) but not NSCLC cell lines (PC9, H1299, HCC15, and A427) and a normal cell line (BEAS2B). For better visualization, HEPACAM2 mRNA is shown as the ratio of log2‐transformed H2:β‐actin. An unpaired t ‐test with a Welch correction was performed with GraphPad Prism, version 10.1.0. (B) HEPACAM2 protein was also found via liquid chromatography–tandem mass spectrometry in the secretomes of NE‐specific SCLC cell lines. Results are expressed as the mean values of the data as bars, representing sample values and error bars representing the SEM. EV indicates extracellular vesicle; mRNA, messenger RNA; NE, neuroendocrine; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Transformation Assay, Liquid Chromatography, Mass Spectrometry

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression correlates with NE carcinoma in tumors. (A) Boxplots showing mRNA levels of HEPACAM2 in NSCLC and SCLC with a two‐tailed ANOVA test. The graph displays the mean values as bars, with individual data points representing sample values and error bars indicating the SEM. (B) Spatially resolved expression of HEPACAM2 ( Top Left ) is specific to the region of SCLC histology, which expresses high NE genes (i.e., SYP and ASCL1), but not the NSCLC region, with features of adenocarcinoma (i.e., NAPSA) in a combined NSCLC/SCLC tissue sample; performed by 10x Genomics. The image was generated with the Loupe Browser, version 6.4.1. (C) Heatmap of transcription factors ordered by HEPACAM2 and z scaled by row, which allowed comparison between samples. ANOVA indicates analysis of variance; mRNA, messenger RNA; NE, neuroendocrine; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; SEM, standard error of the mean; TPM, transcript per million.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Two Tailed Test, Generated, Comparison

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression and association with NE carcinoma in tumors. (A–C) In patient samples, no change in HEPACAM2 level of expression was found to be associated with different stages of disease (A), biopsy location (B), and patient response (C). (D) High levels of HEPACAM2 expression were negatively correlated with PFS and OS in our SCLC cohort. LS, limited stage; NE indicates neuroendocrine; OS, overall survival; PFS, progression‐free‐survival; SCLC, small cell lung cancer; TPM, transcript per million.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression in treatment‐emergent prostatic small cell carcinoma. A positive trend in HEPACAM2 ( Left ), SEZ6 ( Middle ), and INSM1 ( Right ) mRNA expression is shown as cancer transitions from adenocarcinoma to adenocarcinoma with NE features to small cell carcinoma of the prostate. The graph displays the mean values as bars, with individual data points representing sample values and error bars indicating the SEM (cBioPortal; metastatic prostate adenocarcinoma; SU2C/PCF Dream Team ²⁵ ). FPKM indicates fragments per kilobase per million; mRNA, messenger RNA; NE, neuroendocrine; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression is not associated with other targets for antibody–drug conjugate therapies. Heatmaps showing the relative expression of some common antibody–drug conjugate targets versus HEPACAM2 in two examined SCLC cohorts. The Dowlati cohort represents late‐stage SCLC; George et al. ²⁴ represents early‐stage SCLC. NE indicates neuroendocrine; SCLC, small cell lung cancer.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Functional effects of HEPACAM2 knockdown in vitro. (A) Western blots of identical protein lysates of control and HEPACAM2 knockdown (H2‐1) H69 cells were detected with a ProSci antibody. (B) HEPACAM2 knockdown in H69 cells attenuated mRNA expression of ASCL1 and Myc. The graph displays the mean values as bars, with individual data points representing three independent experiments and error bars indicating the SEM. p < 0.050. gRNA indicates guide RNA; kDa, kilodalton; mRNA, messenger RNA; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Functional Assay, Knockdown, In Vitro, Western Blot, Control, Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Positive correlation between ASCL1 and HEPACAM2. (A) ASCL1 is positively associated with HEPACAM2 on the basis of Pearson analysis. (B) The ENCODE database indicated that ASCL1 binds the HEPACAM2 promoter in several SCLC cell lines. ChIP‐seq indicates chromatin immunoprecipitation sequencing; ENCODE, Encyclopedia of DNA Elements; SCLC, small cell lung cancer.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: ChIP-sequencing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Functional effects of HEPACAM2 knockdown in SCLC cells. HEPACAM2 knockdown in H1694 cells resulted in a decrease in cell growth as measured by CCK8 proliferation assay up to 5 days (A), a decrease in migration rate as measured by Transwell assay (B), and an increase in cell–cell interaction as indicated by cell attachment counts (C). Results are expressed as mean values, with individual data points representing three independent experiments and error bars indicating the SEM. gRNA indicates guide RNA; SCLC, small cell lung cancer; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Functional Assay, Knockdown, Proliferation Assay, Migration, Transwell Assay, Cell Attachment Assay

Journal: bioRxiv

Article Title: Membrane Curvature Promotes ER-PM Contact Formation via Junctophilin-EHD Interactions

doi: 10.1101/2024.06.29.601287

Figure Lengend Snippet: a , An illustration of the overlapping between the PM targeting and the curvature sensing candidates in JPH2 interactome. b , Representative images of GFP-JPH3 on nanobars shRNA knockdown of scramble, Clathrin, CAVIN1, CAV1/2, or EHD1/2/4. ShRNAs sequences were fused with fluorescence proteins to identify knockdown cells. Bright field and the fluorescent protein expression are shown on the left. Zooms are the enlarged view of the region in yellow boxes. Averaged nanobar images from multiple cells are shown on the right. Cell number for average: scramble = 28, shCLTC (Clathrin heavy chain) = 23, shCAV1/2 = 53, shCAVIN1 = 30, shEHD1/2/4 = 55. Scale bar 10 µm in whole cell images, 5 µm in zoom-in images. c , Quantifications of GFP-JPH3 end-to-side ratios upon shRNA knockdowns. Conditions and cell numbers are the same as in b. ****P<0.0001, NS(shClathrin) P>0.9999, NS(shCAV1shCAV2) P>0.9999, NS(shCAVIN1) P=0.0592. d , The distribution of GFP-JPH3 on nanobars before and after 10 mM MβCD treatment at 37°C for 30 min. Scale bar 5 µm. Right: averaged images of all bars in a single cell. e , Quantifications of the nanobar end-to-side ratios for GFP-JPH3 in U2OS cells before and after the MβCD treatment. n = 15 cells for each group. f , Representative images of U2OS cells co-expressing EHD4-GFP/mCherry with indicated JPH3 mutants. Enlarged views are zoomed from the yellow boxes in whole cell image on the left. Scale bar 10 µm in whole cell images, 5 µm in zoom-in images. g , Pearson’s correlation coefficients (PCC) between coexpressed EHD4-GFP/mCherry and JPH3 mutants shown in ( f ). Cell number: mCherry-8MORN_LCR = 21, GFP-8MORN_LCR-αHelix = 17, mCherry-LCR = 19, GFP-8MORN-αHelix = 19. ****P < 0.0001, **P = 0.0024 (8MORN_LCR-αHlx vs. 8MORN-αHlx), **P = 0.0075 (8MORN_LCR vs. 8MORN-αHlx), *P = 0.0174 (8MORN-αHlx vs. LCR), NS P= 0.9995 (8MORN_LCR-αHlx vs. 8MORN_LCR). h , PCC for colocalization between mCherry-8MORN_LCR and EHD4-GFP or between mCherry-8MORN_LCR and anti-clathrin. n = 21 cells for each group. ****P<0.0001. i , Co-immunoprecipitation (IP) from HEK cells expressing EHD4-GFP and mCherry-8MORN_LCR or mCherry-LCR. GFP was pulled via GFP-antibody beads, and the immunoprecipitates were blotted with mCherry antibody. mCherry-8MORN_LCR, but not mCherry-LCR, was pulled together with EHD4-GFP. All experiments were independently replicated at least three times. All error bars represent STD. Kruskal–Wallis test corrected with Dunn’s multiple-comparison test was used for ( c ). Unpaired two-tailed Welch’s t test was used to assess significance for ( e , h ). Brown-Forsythe and Welch ANOVA tests was used to assess significance for ( g ).

Article Snippet: Anti-EHD4 antibody (50-172-7111) was purchased from Fisher Scientific.

Techniques: shRNA, Knockdown, Fluorescence, Expressing, Immunoprecipitation, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Membrane Curvature Promotes ER-PM Contact Formation via Junctophilin-EHD Interactions

doi: 10.1101/2024.06.29.601287

Figure Lengend Snippet: a , EHD4, CAVIN1, Caveolin1, and Caveolin2 all show preferential accumulations at the nanobar ends. Quantifications of the end-to-side ratios are shown on the right. Cell number: n =14 cells for Caveolin2, n = 15 cells for all other groups. ***P=0.0001, **P=0.0015; ****P<0.0001. b , mRNA transcript levels in U2OS cells (shown as normalized transcripts per million, nTPM), obtained from protein atlas database. Genes highlighted in gray: not identified in the JPH2 interactome but are closely related either genetically or functionally. Genes including EHD3, CAVIN2, CAVIN3, and CAVIN4 were not selected as knockdown targets in KD experiments because of their low TPM level in U2OS cells. c , Western blots confirming the effective shRNA knockdown of CAV1, CAV2, EHD1, EHD2, CLTC (clathrin heavy chain), and BIN1. GAPDH was blotted as a loading control. d , Representative images of U2OS cells co-expressing EHD4-mCherry with EHD1-GFP (Top) or with EHD2-GFP (Bottom). Enlarged views are zoomed from the yellow boxes in the whole cell images. e , Representative images of U2OS cells expressing GFP-JPH3 (green) and EHD4-mCh (magenta) seeding on nanobars. Enlarged views are zoomed from the yellow boxes in whole cell images. f , Representative images of immunostaining of EHD2 or EHD4 in iPSC-CMs or rat embryonic CMs on nanopillars. Magnified images of the region in yellow boxes are shown in the bottom row. Scale bar 10 µm in the top row, 2.5 µm in the bottom row. g , Representative images of immunostaining of EHD4 in iPSC-CMs on nanobars. Magnified images of the region in yellow boxes are shown in the middle row. Averaged fluorescent signals of all nanobars from multiple cells were averaged and displayed in the bottom row. n = 16 cells for the average image. Bottom graph: quantification of the end to side ratios of EHD4 compared to that of BFP-CAAX expressed in iPSC-CM on nanobars. Cell number: CAAX = 23 (same data as in ), EHD4 = 16. ****P <0.0001. h , Representative images of immunostaining of JPH2 in iPSC cells transfected with shRNA of scramble (top) or EHD1/2/4 (bottom). From left to right: Bright field image, fluorescent protein expression indicating successful transfection of shRNA, and JPH2 staining. Averaged nanobar images of JPH2 staining from multiple cells are shown at the bottom of JPH staining panels. Quantifications of the end-to-side ratios are shown on the right. Cell number for both average and quantification: Scramble = 24, shEHD1/2/4 = 26. ****P <0.0001. Scale bars 10 µm in all whole cell images, 5 µm in all zoomed images unless otherwise mentioned. All experiments were independently replicated at least two times. All error bars represent STD. Kruskal–Wallis test corrected with Dunn’s multiple-comparison test was used to assess significance in ( a ). Unpaired two-tailed Welch’s t test was used to assess significance in ( g ). Two-tailed Mann-Whitney test was used to assess significance in ( h ).

Article Snippet: Anti-EHD4 antibody (50-172-7111) was purchased from Fisher Scientific.

Techniques: Knockdown, Western Blot, shRNA, Control, Expressing, Immunostaining, Transfection, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Membrane Curvature Promotes ER-PM Contact Formation via Junctophilin-EHD Interactions

doi: 10.1101/2024.06.29.601287

Figure Lengend Snippet: a , The distribution of EHD4-GFP on nanobars before and after 10 mM MβCD treatment at 37°C for 30 min. Magnified images of the yellow boxes are shown in the middle row. Averaged fluorescent signals of all nanobars from multiple cells were averaged and displayed in the bottom row. Quantifications of the end-to-side ratios are shown on the right. n = 15 cells for both conditions. ****P <0.0001. b , Representative images of GFP-ΔLCR expressed in EHD-1,-2,-4 triple knockdown U2OS cells (bottom) or scramble knockdown control U2OS cells (top). c , Representative images of U2OS cells expressing mCherry-8MORN_LCR (magenta) and immunostained with clathrin-heavy-chain antibody (green). Enlarged views are zoomed from the yellow boxes in whole cell images. Quantification of this data was shown in Fig.6h. Scale bar 10 µm in all whole cell images, 5 µm in all zoomed images. All experiments were independently replicated at least two times. All error bars represent STD. Unpaired two-tailed Welch’s t test was used to assess the significance in ( a ).

Article Snippet: Anti-EHD4 antibody (50-172-7111) was purchased from Fisher Scientific.

Techniques: Knockdown, Control, Expressing, Two Tailed Test