Journal: Scientific Reports

Article Title: Elevated SLC4A11 expression promotes OV progression via interaction with EGFR

doi: 10.1038/s41598-025-10255-z

Figure Lengend Snippet: SLC4A11 is linked to ovarian cancer. ( a ) Fifty-two genes whose expression was elevated in ovarian cancer tissue compared with healthy tissue are displayed in a Venn diagram. Data were obtained from the GSE40505 , GSE18520 , GSE66057 , TCGA and GTEx datasets. ( b ) SLC4A11 mRNA expression levels in 88 nontumor tissues and 426 OV tissues from the GEPIA database. ( c ) SLC4A11 expression is used to predict the overall prognosis of ovarian cancer patients. Data obtained from GEPIA. ( d ) The degree of SLC4A11 expression in the GEO database ( GSE40595 , 63 OV, and 14 nontumor tissue samples; GSE18520 , 53 OV and 10 nontumor tissue samples; GSE66957 , 57 OV and 13 nontumor tissue samples). Chi-square test: * p < 0.05, ** p < 0.01, *** p < 0.001. ( e ) SLC4A11 expression in OV tissue microarrays at various disease stages and in normal tissues. Scale bar: 50 µM. ( f ) The number of samples expressing SLC4A11 in OV samples ( n = 117) and normal tissue samples ( n = 16) was determined by blinded IHC. Scoring is based on staining intensity: negative (no positive colouration): 0 points; weak positive (light yellow): 1 point; positive (brownish yellow): 2 points; and strong positive (tan): 3 points. A score greater than 1 is considered high.

Article Snippet: After deparaffinization, staining, and antigen retrieval, the samples were coated with an anti-SLC4A11 antibody (1:150, NBP1-46156, NOVUS) and then incubated.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Elevated SLC4A11 expression promotes OV progression via interaction with EGFR

doi: 10.1038/s41598-025-10255-z

Figure Lengend Snippet: SLC4A11 effects on the in vitro activities of OV cell lines. ( a ) SLC4A11 expression in ovarian cancer cell lines (relative to 18 S rRNA). ( b ) After the small interfering RNA was knocked down, SLC4A11, PCR, and Western blotting were used to verify the knockdown efficiency of the small interfering fragment. ( c ) The CCK-8 test was used to quantify the amount of cell proliferation in OV8 and SKOV3 cell lines following SLC4A11 knockdown(The dotted line represents the OD value of cell doubling). ( d ) The proliferation ability of SLC4A11 knockdown OV8 and SKOV3 cells was detected via a plate cloning assay, which was repeated 3 times, and the number of cell clones was quantified. Scale bar, 5 mm. ( e ) The migration capacity of SLC4A11 knockdown OV8 and SKOV3 cells was detected via Transwell assay in triplicate. The number of migrating cells was quantitatively analyzed. Scale bar, 50 μm. ( f ) qRT‒PCR and Western blotting are commonly used to assess the overexpression efficiency of SLC4A11. ( g ) The proliferation ability of HEY cell lines overexpressing SLC4A11 was detected via a CCK-8 assay(The dotted line represents the OD value of cell doubling). ( h ) Colony formation assay to evaluate the proliferation capacity of HEY cells after SLC4A11 overexpression and quantify the number of cell clones. Scale bar, 5 mm. There were three assay repetitions. ( i ) Transwell assays were used to assess cell migration in the HEY cell line following SLC4A11 overexpression, and the number of migrating cells was quantified. Scale bar, 50 μm. There were three assay repetitions. The values are the means ± SDs, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t-test).

Article Snippet: After deparaffinization, staining, and antigen retrieval, the samples were coated with an anti-SLC4A11 antibody (1:150, NBP1-46156, NOVUS) and then incubated.

Techniques: In Vitro, Expressing, Small Interfering RNA, Western Blot, Knockdown, CCK-8 Assay, Cloning, Clone Assay, Migration, Transwell Assay, Over Expression, Colony Assay

Journal: Scientific Reports

Article Title: Elevated SLC4A11 expression promotes OV progression via interaction with EGFR

doi: 10.1038/s41598-025-10255-z

Figure Lengend Snippet: The effect of SLC4A11 on ovarian cancer has been demonstrated in vivo. ( a ) Cell clones were selected for puromycin resistance after SKOV3 cells were infected with either the control vector (shNC) or the lentivirus-producing SLC4A11 short hairpin RNA (shSLC4A11). The SLC4A11 knockdown efficiency was detected via qPCR. ( b ) Cell proliferation capacity after SLC4A11 knockdown in OV8 and SKOV3 cell lines was detected by a CCK-8 assay(The dotted line represents the OD value of cell doubling). ( c ) Ten nude mice were randomly divided into a control group and SLC4A11-knockdown group, with 5 mice in each group, and then inoculated with shNC and shSLC4A11 ovarian cancer stably transfected strains in the right axilla, with approximately 1*10 6 cells inoculated at each site. After 5 weeks, the nude mice were sacrificed, and pictures of the tumors were taken. ( d ) The volume of subcutaneous xenograft tumors in nude mice was recorded weekly, n = 5, V = π/6*a*b*c. ( e ) Measured volume of removed xenografts. ( f ) Xenograft weight was measured. ( g ) Immunohistochemistry was used to analyze the expression of SLC4A11 and Ki67 in tumors. Scale bar: 100 μm. ( h ) Quantification of the positivity rate of Ki67 in ( g ). ( i ) Ten nude mice were randomly divided into a control group and SLC4A11 overexpression group, with 5 mice in each group, and then inoculated with the VE or OE-SLC4A11 ovarian cancer stably transfected strains in the right armpit; approximately 1*10 6 cells were inoculated at each site. After 5 weeks, the nude mice were sacrificed, and pictures of the tumors were taken. ( j ) The volume of subcutaneous xenograft tumors in nude mice was recorded weekly. ( k ). Vectors of the removed xenografts were measured. ( l ). Xenograft weight was measured. m. Immunohistochemistry was used to analyze the expression of SLC4A11 and Ki67 in tumors. Scale bar: 100 μm. n. Quantification of the positivity rate of Ki67 in ( m ). The mean value was taken ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 (t-test).

Article Snippet: After deparaffinization, staining, and antigen retrieval, the samples were coated with an anti-SLC4A11 antibody (1:150, NBP1-46156, NOVUS) and then incubated.

Techniques: In Vivo, Clone Assay, Infection, Control, Plasmid Preparation, shRNA, Knockdown, CCK-8 Assay, Stable Transfection, Transfection, Immunohistochemistry, Expressing, Over Expression

Journal: Scientific Reports

Article Title: Elevated SLC4A11 expression promotes OV progression via interaction with EGFR

doi: 10.1038/s41598-025-10255-z

Figure Lengend Snippet: SLC4A11 interacts with EGFR to initiate the PI3K/AKT signaling pathway. ( a ) GSEA was carried out via the iconic gene set to compare the two different expression groups for SLC4A11 in the TCGA database. The DEGs analyzed via RNA-seq were used for GSEA. ( b ) Association between SLC4A11 and EGFR in the GEPIA dataset. ( c ) Kaplan‒Meier analysis according to EGFR expression was used to evaluate the progression-free survival of OV patients. The TCGA dataset provided the data. ( d ) Coimmunoprecipitation of endogenous SLC4A11 and endogenous EGFR in OV cells. ( e ) Coimmunofluorescence, which uses green and red fluorescence to visualize SLC4A11 and EGFR expression in OV cells, was used in conjunction with blue fluorescence to visualize DAPI-stained cell nuclei. ( f ) The hallmark gene set was subjected to GSEA on the basis of the expression levels of SLC4A11 in the database. ( g ) Western blotting analysis of EGFR and total and phosphorylated PI3K and AKT in both cell lines after treatment with SLC4A11-siRNA. ( h ) Quantitative analysis of the WB results.

Article Snippet: After deparaffinization, staining, and antigen retrieval, the samples were coated with an anti-SLC4A11 antibody (1:150, NBP1-46156, NOVUS) and then incubated.

Techniques: Expressing, RNA Sequencing, Fluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: Elevated SLC4A11 expression promotes OV progression via interaction with EGFR

doi: 10.1038/s41598-025-10255-z

Figure Lengend Snippet: SLC4A11 enhances the resistance of ovarian cancer cells to EGFR inhibitors. ( a ) Erlotinib (0 µM or 20 µM) was added to SLC4A11 OV8 ovarian cancer cells, and the proliferative activity of the cells was detected with a CCK8 kit(The dotted line represents the OD value of cell doubling). ( b ) Sensitivity of ovarian cancer cells in the no-load group and SLC4A11-overexpressing groups to erlotinib, as determined by cell viability. ( c , d ). Cells transfected with SLC4A11-overexpressing lentivirus or SLC4A11-overexpressing lentivirus were treated with 0 µM or 40 µM erlotinib, respectively, and the migration ability of the cells was detected via a transwell assay. The number of migrated cells was quantitatively analyzed (t-test). ( e , f ). Erlotinib at 0 µM and 40 µM was added to cells transfected with no-load or SLC4A11-overexpressing lentivirus, the migration ability of the cells was detected by a wound healing assay, and the number of migrating cells was quantitatively analyzed (t-test). ( g ). The cells were divided into the no-load group, SLC4A11 overexpression group, no-load + erlotinib group, and erlotinib overexpression + erlotinib group, and the expression levels of different proteins and their phosphorylated proteins in the EGFR/PI3K/AKT pathway were detected via western blotting. ( h ). Quantitative analysis of the WB results.

Article Snippet: After deparaffinization, staining, and antigen retrieval, the samples were coated with an anti-SLC4A11 antibody (1:150, NBP1-46156, NOVUS) and then incubated.

Techniques: Activity Assay, Transfection, Migration, Transwell Assay, Wound Healing Assay, Over Expression, Expressing, Western Blot

Journal: Cell Reports Medicine

Article Title: Targeted AAV6 gene therapy restores corneal endothelial function in three hereditary corneal dystrophies

doi: 10.1016/j.xcrm.2026.102649

Figure Lengend Snippet: Therapeutic evaluation of gene therapy in FECD and CHED models (A) Slit lamp and OCT images of adult COL8A2 Q455K mice before and at 1 and 3 months after intracameral injection of AAV-Con or AAV-Col8a2. (B and C) (B) Changes in corneal opacity area in adult Q455K mice treated with AAV-Con or AAV-Col8a2. n = 4. ∗∗∗ p < 0.001 (C) IVCM images of the corneal endothelium in adult Q455K mice in different treatment groups. Multiple guttae (red arrows) were observed in the AAV-Con group. Scale bar, 100 μm. (D) Alizarin red staining of corneas from AAV-Con and AAV-Col8a2 groups. Scale bars, 100 μm. (E) AAV-Col8a2 treatment significantly reduced guttae numbers in Q455K adult mice. n = 4. ∗∗∗ p < 0.001. (F) Slit lamp and OCT images of neonatal Q455K mice before and at 1 and 3 months after intracameral injection of AAV-Con or AAV-Col8a2. (G) Percentage of neonatal Q455K eyes that maintained corneal transparency after AAV-Con or AAV-Col8a2 treatment. n = 16. ∗∗∗ p < 0.001. (H) In vivo confocal microscopy images of the corneal endothelium in neonatal Q455K mice in different treatment groups. Multiple guttae (red arrows) were observed in the AAV-Con group. Scale bar, 100 μm. (I) Alizarin red staining of corneas from AAV-Con and AAV-Col8a2 groups. Scale bars, 100 μm. (J) AAV-Col8a2 treatment significantly reduced guttae numbers in neonatal Q455K mice. n = 4. ∗∗∗ p < 0.001. (K) In Slc4a11 KO mice, corneal edema resolved at 1 week after intracameral injection of AAV-Slc4a11, and transparency was maintained throughout the 8-week observation period. (L) CCT returned to near-normal levels within 1 week after intracameral injection of AAV-Slc4a11 and remained stable thereafter. n = 4, ∗∗ p < 0.01. Data are represented as mean ± SEM.

Article Snippet: SLC4A11 knockout mice , Cyagen Biosciences , Cat#KOCMP-269356-Slc4a11-B6N-VA.

Techniques: Injection, Staining, In Vivo, Confocal Microscopy

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Bioinformatic analysis of human SLC4A11. ( A ) cDNA sequences of known human SLC4A11 transcripts were mapped onto a 15 kb segment of Human chromosome 20 genomic DNA to show transcript structure (coloured boxes), connected by introns (black lines). Each transcript variant (v1, v2 and v3) has a unique starting exon which results in unique N-terminal regions of the predicted protein and a common core region (red). ( B ) Magnified view of the 5′ end of the SLC4A11 gene. Each variant has a predicted start codon (M1; vertical black line). M36 (numbering based on predicted sequence of v2) marks the start of the common coding sequence for all transcripts. ( C ) Alignment of DNA sequences surrounding the predicted start codons in SLC4A11 v2 for multiple species. Kozak translational start sequence efficiency is indicated with bases matching the consensus shown in orange. For human SLC4A11, Kozak analysis is indicated for v2 start at Met 1 and 36 (v2-M1 and v2-M36). ( D ) Alignment of amino acid sequences for v2 SLC4A11 for the indicated mammals. Red Methionine (M) residue indicates the position corresponding to human v2-M36. Green Met indicates in frame Met upstream of v2-M36. Stop codons are indicated (*). ( E ) Amino acid sequences of SLC4A11 v1-v3 are aligned with sequences of peptides used to generate splice form-selective antibodies highlighted in red: SLC4A11-common, green: SLC4A11-v2-M1 and blue: SLC4A11-v3.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Variant Assay, Sequencing

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Expression of SLC4A11 transcripts in human cornea. ( A ) Plasmid-cloned SLC4A11 v1, v2 and v3 were each used as a template with specific forward primers for v1, v2 and v3, respectively and a common reverse primer. PCR reaction was set up for 40 cycles, and total reaction mixture was loaded on a 1% agarose gel. ( B,C ) RNA isolated from total cornea and micro-dissected endothelial layer were subjected to reverse transcription reaction with (+RT) or without (−RT) reverse transcriptase enzyme. PCR was performed using cDNA template generated from ( B ) total cornea and ( C ) isolated endothelium, with forward primers corresponding to v1, v2 and v3 along with common reverse primer. Expected amplicon sizes were 244, 233 and 233 bp for v1, v2 and v3, respectively. ( D ) qPCR was performed on cDNA from corneal endothelium and total cornea using iQ SYBR green mix along with forward and reverse primers for v1, v2 and v3 and GAPDH. Reactions were carried out for 40 cycles and fluorescence was recorded at each elongation step. Standard curve was constructed for each variant by using respective synthetic templates in the range of 2.5–2.5 × 10 4 copies and plotting Log 10 (template copies) against their C T values. The sample C T values were corrected to GAPDH for every experiment and number of copies of each variant was calculated, using their respective standard curves. Background (-RT control) values were subtracted from the samples. The average number of copies per ng RNA were plotted for each variant in corneal endothelium and total cornea. Data are presented as mean ± SEM from three biological replicates (Two-way ANOVA and Sidak’s multiple comparison test). *p = 0.02, ns = not significant.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Expressing, Plasmid Preparation, Clone Assay, Agarose Gel Electrophoresis, Isolation, Generated, Amplification, SYBR Green Assay, Fluorescence, Construct, Variant Assay

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: SLC4A11 v2 and v3 expression in human corneal endothelium. Corneal endothelium was micro-dissected from six human corneas. Protein lysates from pooled human corneal endothelium (20 µg protein) along with HEK293 cells transfected with cDNA encoding SLC4A11 v2 (10 µg protein lysate), v3 (5, 10, 20 µg protein lysates, indicated) and empty vector (20 µg protein lysate) were processed for immunoblots. Blots were probed with ( A ) SLC4A11-common antibody and ( B ) SLC4A11-v3 antibody, along with GAPDH antibody as a loading control. ( C ) Densitometry was performed to quantify band intensities. Detection ratio was established between SLC4A11-common antibody and SLC4A11-v3 antibody (see Supplementary Fig. ). Data from each trial were normalized to SLC4A11 v2 abundance. Data represent mean ± SEM from three replicates (Two-tailed t-test). *p = 0.005 when compared to v3. Note that intervening lanes to the left of the human cornea lane in ( A ) and ( B ) have been removed and are marked by the black line.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Identification of SLC4A11 v2 translation start site in total human corneal lysates. HEK293 cells were transfected with cDNA encoding: SLC4A11 v2 starting at Met1 (SLC4A11 v2-M1) or SLC4A11 v2 starting at Met36 (SLC4A11 v2-M36). Whole human cornea samples were flash frozen with liquid nitrogen, crushed, solubilized and then combined with 2X SDS-Page sample buffer. Samples were electrophoresed on 7.5% acrylamide gels. Corresponding immunoblots were probed with antibodies ( A ) Anti-SLC4A11-v2-M1 and ( B ) Anti-SLC4A11-common.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Transfection, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Plasma membrane localization of SLC4A11 v2-M1 and v2-M36. HEK293 cells were transfected with cDNA encoding SLC4A11 v2-M1 or SLC4A11 v2-M36 as shown. ( A ) Cells were labeled with membrane-impermeant Sulfo-NHS-SS-biotin (SNSB) and the cell lysates were divided into two equal fractions. One was incubated with streptavidin resin to remove biotinylated proteins. The unbound fraction (U) and the total cell lysate (T) were probed with SLC4A11-common antibody on immunoblots. GAPDH was used as an internal control. ( B ) The fraction of SLC4A11 and GAPDH labeled by SNSB was calculated. Dashed line indicates the background for this assay, as indicated by labeling of GAPDH. Data represent mean ± SEM from five replicates (One-way ANOVA with Sidak’s multiple comparisons test). *p < 0.0001 when compared to SLC4A11 v2-M1, ns = not significant. ( C ) Cells were processed for immunofluorescence and images were collected by confocal microscopy (scale bars indicated in each row). Nuclei were detected with DAPI staining (blue). SLC4A11 was detected with SLC4A11-common antibody and chicken anti-rabbit IgG conjugated with Alexa Fluor 594 (red). Plasma membrane marker, Na + /K + -ATPase was detected with anti-Na + /K + -ATPase and chicken anti-mouse Alexa Fluor 488 (green). Overlay represents merged images from all three channels.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Transfection, Labeling, Incubation, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Marker

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Osmotically driven water flux activity of SLC4A11 v2-M1 and v2-M36 and v3. HEK293 cells were transiently transfected with cDNA encoding eGFP along with empty vector or SLC4A11 v2-M1 or SLC4A11 v2-M36. ( A ) After performing water flux assays, cells from the same dishes were lysed and probed on immunoblots, using SLC4A11-common antibody to detect the expression of SLC4A11 variants. GAPDH and eGFP were also detected using respective antibodies. ( B , D ) HEK293 cells transfected with v2-M1 or v2-M36 or v3 cDNA were perfused alternately with iso-osmotic (black bar) and hypo-osmotic (white bar) media. eGFP fluorescence (F) was normalized to F averaged for time 0–120 or 0–60 seconds (F 0 ) and F/F 0 plotted. ( C , E ) Rate of fluorescence change (a surrogate for cell volume change) was calculated. Data represent mean ± SEM from 4–8 independent coverslips with 12–18 cells measured per coverslip (One-way ANOVA with Sidak’s multiple comparisons test) *p < 0.0001 when compared to SLC4A11 v2-M1 or SLC4A11 v2-M36, ns = not significant.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

doi: 10.1038/s41598-019-46094-y

Figure Lengend Snippet: Homology model of SLC4A11 v2-M36 and SLC4A11 v3 cytoplasmic domains. Homology models of cytoplasmic domains of ( A ) SLC4A11 v2-M36 (M36-Y340) and ( B ) SLC4A11 v3 (M1-Y359) generated by I-TASSER using crystal structure of human erythrocyte Band 3 cytoplasmic domain (PDB: 1HYN) as the template. Unique N-terminal sequence of v3 is highlighted in pink. ( C ) v2-M36 and v3 models aligned in PyMOL (molecular graphics system version 2.0.7), using segments that were modelled with 100% confidence (R90-P306 for v2-M36, R109-P325 for v3). N v2 , N v3 and C v2 , C v3 are the N and C termini for v2-M36 and v3, respectively.

Article Snippet: Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech (Cambridge, MA USA).

Techniques: Generated, Sequencing