Journal: Cancers

Article Title: RARβ Expression in Keratinocytes from Potentially Malignant Oral Lesions: The Functional Consequences of Re-Expression by De-Methylating Agents

doi: 10.3390/cancers13164064

Figure Lengend Snippet: Baseline expression of RARβ2 mRNA and total RARβ protein levels in PMOL cell lines and tissues. ( A ) Assessment of baseline RARβ2 mRNA expression by qPCR demonstrated significantly higher expression in mPMOL cells (D6 and D30) compared to the iPMOL cells and FNB6TERT cells ( p < 0.03). The mRNA expression of cell cycle regulators, CDKN1A and CDKN2A, was also higher in the mPMOL cells when compared to iPMOL cells ( p < 0.002) and higher expression of cRBP1 and differentiation markers ITGB1 and IVL was also observed ( p < 0.05, Kruskal–Wallis test). ( B ) Western blotting of total RARβ, CDKN1A, and CDKN2A with β-actin as a loading control. HeLa cells and i3T3 cells were added as experimental controls. These show a similar pattern of expression to that seen in the qPCR, albeit the Western blot shows total RARβ, rather than RARβ2. ( C ) Immunohistochemical expression of total RARβ in the FFPE biopsy tissues corresponding D6, D30, D19, D20, D34, and D38 showed lower RARβ expression in tissues from which iPMOLs were derived compared to those from which mPMOLs were generated. ( D ) Immunohistochemical expression of RARβ in the extended clinical cohort ( n = 10) demonstrated variable proportions of total RARβ expression, which was not related to the grade of epithelial dysplasia. ( E ) Immunohistochemical expression of RARβ in 3D TEM models constructed using iPMOL cells showed variable staining for RARβ, but in every case, higher than in the matched biopsy material ( C).

Article Snippet: Quantitative real-time PCR (qPCR) was performed using ABI Prism 7900 Fast Sequence Detection Instrument (Applied Biosystems, Foster, CA, USA) for the relative gene expression of RARβ2 (Hs00977141_mH), CDKN2A, (Hs99999189_m1), CDKN1A (Hs00355782_m1), IVL (Hs00846307_s1), ITGB1 (Hs05351551_g1), and CRBP1 (Hs01011512_g1), with β-2-microglobulin (B2M) (Assay ID: Hs99999907_m1) as a housekeeping gene using Taqman Assays procured from Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, Control, Immunohistochemical staining, Derivative Assay, Generated, Construct, Staining

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: The role of RBP1 in the visual cycle and generation of Abca4 −/− /Rdh8 −/− /Rbp1 −/− mouse l ines. A , schematic representation of the visual cycle, illustrating the cellular and molecular components involved in visual chromophore regeneration. Proteins are color-coded based on their roles: enzymes ( blue ), transporters and receptors ( purple ), and retinoid carriers ( green ). Rhodopsin is shown in gray , pink , and yellow depending on its state, while RBP1 is highlighted in red . B , representative agarose gels showing genotyping results for Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice and their heterozygous and WT counterparts. C , western blot analysis of ocular tissues from Rbp1 −/− mice. Unlike in Abca4 −/− /Rdh8 −/− (DKO) controls, RBP1 is absent in eyeball homogenates of Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. D , immunofluorescence-based confirmation of RBP1 deficiency. Retina sections were probed with an anti-RBP1 antibody, revealing specific staining exclusively in the RPE cell layer of Abca4 −/− /Rdh8 −/− mice. No immunofluorescence signal was detected in the corresponding Rbp1 −/− mice on the Abca4 −/− /Rdh8 −/− (DKO) genetic background. The scale bar represents 50 μm. E , the retinal morphology of 4-month-old mice showed no signs of spontaneous retinal degeneration in either mouse line. The scale bar represents 150 μm. PR (photoreceptors), ONL (outer nuclear layer), OPL (outer plexiform layer), INL (inner nuclear layer), IPL (inner plexiform layer); GCL (ganglion cell layer). F , quantification of 11c-RAL in the retinas of Abca4 −/− /Rdh8 −/− (DKO) and Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. No statistically significant (ns) differences were observed in visual chromophore content, indicating comparable levels of rhodopsin (n = 6). G , the effect of RBP1 deficiency on the rate of dark adaptation. Scotopic ERG responses were recorded after 90 min of dark adaptation following exposure to a bright light flash that bleached ∼80% of the rod visual pigment. Delayed dark adaptation in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice was evidenced by reduced ERG a- and b-wave amplitudes compared to Abca4 −/− /Rdh8 −/− (DKO) mouse line. The absence of RDH8 and ABCA4 alone had a detectable, but much subtler effect on the regeneration of visual pigment, as observed for Abca4 +/+ /Rdh8 +/+ /Rbp1 +/+ mice, indicated as “WT”. Data represent mean ± S.D. (n = 6). Student’s t -test was used for two-group comparisons of individual experimental points (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005). 11c-RAL, 11-cis-retinal; ERG, electroretinogram; RBP1, cellular retinol-binding protein 1; RPE, retinal pigment epithelium.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Western Blot, Immunofluorescence, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Ocular phenotype of Rbp1 −/− mice on the Abca4 −/− /Rdh8 −/− genetic backgrounds. A , schematic representation of the experimental design. The light intensity and exposure duration were chosen to induce ∼80% reduction of the retinal ONL in Abca4 −/− /Rdh8 −/− mice. B , representative SLO images of retinas of 4-month-old mice after bright light exposure. Increased autofluorescence with a characteristic punctate pattern indicates immune cell infiltration into the damaged retina of Abca4 −/− /Rdh8 −/− (DKO) mice, a feature not observed in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. C , quantification of the SLO signal shown in panel ( B ) reveals a statistically significant difference (∗∗∗ p < 0.0005, unpaired t test) between DKO and TKO mice (9 ≤ n ≤ 14). D , retinal integrity assessment in light-exposed mice using OCT. ONL thickness was measured to quantify the degree of retinal degeneration. The scale bar represents 50 μm. E , quantification of retinal morphology changes based on OCT images. The data indicate partial protection against light-induced damage in RBP1-deficient mice ( Abca4 −/− /Rdh8 −/− /Rbp1 −/− labeled as TKO) compared to their Abca4 −/− /Rdh8 −/− (DKO) control counterparts. The prebleach data for DKO and TKO mice are depicted in gray and white triangles, respectively. Data represent mean ± S.D. (12 ≤ n ≤ 16). Asterisks indicate statistical significance between individual points (∗∗∗ p < 0.0005, unpaired t - test). F , representative histology images corresponding to the OCT data shown in panel ( D ) confirm the protective effect of the Rbp1 gene deletion. G , ERG-based functional assessment of retinal responses after exposure to damaging bright light. While Abca4 −/− /Rdh8 −/− mice exhibited diminished light responses, retinal function was largely preserved in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons at individual experimental points). The prebleach data represent combined ERG responses from both mouse lines. Data represent mean ± S.D. (n = 6). ERG, electroretinogram; OCT, optical coherence tomography; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Labeling, Control, Functional Assay, Tomography, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Deletion of the Rbp1 gene decreases light sensitivity of Balb/cJ albino mice. A , schematic representation of the experimental design. Balb/cJ mice were exposed to bright light for 55 min, resulting in ∼50% reduction in ONL thickness. B , SLO images of retinas from 4-month-old albino mice after bright light exposure. Increased autofluorescence was readily observed in Rbp1 + / + (Balb/cJ) mice but was greatly diminished in Rbp1 −/− (Balb/cJ) mice, indicating a decreased in immune cell infiltration. C , quantification of the SLO signal revealed a statistically significant difference between Rbp1 +/+ (Balb/cJ) and Rbp1 −/− (Balb/cJ) mice (∗∗∗ p < 0.0005, unpaired t test, n = 6). D , quantification of ONL thickness in light-exposed mice. The absence of RBP1 in the light-sensitive albino mice led to partial preservation of retinal morphology (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons for individual experimental points). The prebleach data represent combined ONL thickness from both Rbp1 +/+ (Balb/cJ) and Rbp1 −/− (Balb/cJ) mouse lines. The data represent mean ± SD (n = 10). E , preservation of retinal morphology in Rbp1 −/− mice, as shown in panel ( D ), correlates with sustained retinal function, particularly at higher light intensities, as assessed by scotopic ERG responses (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t - test for individual experimental points). Data represent mean ± S.D. (n = 6). ERG, electroretinogram; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Preserving, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Temporal accumulation of A2E in mouse eyes. A , experimental design. Abca4 −/− /Rdh8 −/− (DKO), Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO), and corresponding WT mice were maintained under a 12 h/12 h light/dark cycle for up to 8 months to assess the rate of A2E accumulation. B , LC/MS-based detection and quantification of A2E. Synthetic A2E-d10 was used as an internal standard to determine endogenous A2E levels. Single reaction monitoring mode was employed for the unambiguous identification of endogenous and synthetic A2E molecules in eye extracts. C , time-dependent increase of A2E levels in DKO, TKO, and WT mouse eyes. The calculated rate of A2E accumulation was approximately three times slower in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice compared to Abca4 −/− /Rdh8 −/− (DKO) controls. Data represent mean ± S.D. (4 ≤ n ≤ 7). D , Box-and-whisker plot illustrating the distribution of experimental data for samples collected from 4-month-old and 8-month-old mice, presented in panel ( C ) (∗∗∗ p < 0.0005, unpaired t - test for two-group comparisons). E , representative SLO images of 8-month-old mouse retinas, showing differences in autofluorescence. The increased SLO signal across the retina correlates with higher A2E accumulation in Abca4 −/− /Rdh8 −/− (DKO) mice compared to Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) animals. F , the quantification of the SLO signal is shown in panel ( E ) (n = 8, ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons). A2E, N -retinylidene- N -retinylethanolamine; LC/MS, liquid chromatography/mass spectrometry; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Liquid Chromatography, Mass Spectrometry, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Inhibitors of RBP1 protect against acute light-induced retinal degeneration in Abca4 −/− /Rdh8 −/− mice. A , experimental design. RBP1 inhibitors were administered intraperitoneally to dark-adapted mice 30 min before exposure to retinal-damaging light. B , chemical structures of the inhibitors used in this study. Apparent K i values highlight the difference in potency between abn-CBD and oxadiazol-derived compounds. C , effect of light exposure on retinal integrity, measured by ONL thickness. Pretreatment with abn-CBD, a potent RBP1 inhibitor, provided robust protection against acute retinal degeneration (∗∗∗ p < 0.0005, unpaired t test for two-group comparisons at each experimental point). Data represent mean ± S.D. D , retinal autofluorescence after light exposure, assessed by SLO in abn-CBD-treated and untreated (control) mice. White puncta in the untreated group indicate inflammatory changes in the retina. E , quantification of SLO brightness from panel ( D ), showing a statistically significant difference between treated and untreated groups (∗ p < 0.05, unpaired t - test, n = 6). F , scotopic ERG recordings reveal statistically significant preservation of retinal function in mice pretreated with abn-CBD (∗ p < 0.05, unpaired t test, n = 6). abn-CBD demonstrated efficacy under high-intensity light exposure, which caused ∼80% photoreceptor degeneration in untreated mice. Data represent mean ± S.D. G , comparison of the protective effects of alternative RBP1 inhibitors. The efficacy of the less potent inhibitors (1–3, panel B ) was evident only under conditions of reduced light exposure (ONL thickness reduction of ∼30%). Under these conditions, post hoc analysis showed significant differences between untreated animals and those injected with RBP1 inhibitors. Among the alternatives, inhibitor two exhibited the most profound retinal protective effect, based on statistical analysis (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for individual experimental points). Data represent mean ± S.D. (n = 4). abn-CBD, abnormal cannabidiol; ERG, electroretinogram; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Derivative Assay, Control, Preserving, Comparison, Injection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: The role of RBP1 in retinoid homeostasis and the mechanistic basis for the protective effect of its inhibitors. RBP1 facilitates the intracellular transport of at-ROL in RPE cells. Its absence delays the regeneration of the visual chromophore by slowing down at-ROL esterification by LRAT, leading to a transient accumulation of at-ROL compared to WT mice. An unforeseen consequence of RBP1 deficiency is diminished light sensitivity in mice susceptible to light-induced retinal damage, along with a slower accumulation of at-RAL-derived side metabolites. Pharmacological inhibition of RBP1 mimics the phenotype of Rbp1 −/− mice, offering a potential therapeutic strategy for modulating the visual cycle. Therefore, RBP1 inhibitors may provide an alternative approach to treating diseases associated with impaired clearance of at-RAL and excessive bis-retinoid accumulation. LRAT, lecithin:retinol acyltransferase; RBP1, cellular retinol-binding protein 1; RPE, retinal pigment epithelium.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Derivative Assay, Inhibition, Binding Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Methylation, Control, Molecular Weight, Marker