cacna1e  (Proteintech)

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: Identification of CACNA1E as an m 6 A-hypermethylated and upregulated gene in OS. A Starplot illustrating the distribution of genes with differential expression (up/down; |log2fold change|≥ 1 and P < 0.05) and differential m 6 A (hyper/hypo; |log2fold change|≥ 1 and P < 0.05) in OS tissues with adjacent normal tissues. B Heatmap showing the top5 up-/down-regulated genes in OS tissues versus adjacent normal tissues. C CACNA1E mRNA level examined by qRT-PCR in OS and adjacent normal tissues. D CACNA1E mRNA level examined by qRT-PCR in osteoblast cells (hFOB1.19) and OS cells (SJSA-1, MNNG, U2OS, and SAOS2). E Kaplan–Meier survival curves of OS patients ( n = 85) stratified by high and low CACNA1E expression, with log-rank test for comparing survival rate. F Multivariate Cox regression analysis for determining CACNA1E as an independent prognostic factor after adjusting for clinical variables (age, gender, and metastasis status) based on the TARGET database. G - I qRT-PCR and Western blot assays for verifying the stable knockdown of CACNA1E in MNNG and SJSA-1 cells. J , K CCK-8 and ( L , M ) colony formation assays for determining the effect of CACNA1E on OS cell proliferation. N , O Wound healing assay for examining the migration of shNC- and shCACNA1E-transfected OS cells. Photographs are displayed at 0, 6, 12, and 24 h. Scale bar, 100 μm. P - R Analysis of OS cell migration and invasion by Transwell assays. Scale bar, 100 μm. S - U Western blot for examining the effect of CACNA1E on EMT markers in OS cells. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 derived from Student’s t-test or one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Quantitative Proteomics, Quantitative RT-PCR, Expressing, Western Blot, Knockdown, CCK-8 Assay, Wound Healing Assay, Migration, Transfection, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: Targeted inhibition of CACNA1E effectively hinders OS growth and lung metastasis in vivo. A - D Photographs of subcutaneous tumor models and dissected tumors. The models were established by subcutaneous injection of shNC- and shCACNA1E-transfected ( A , B ) MNNG and ( C , D ) SJSA-1 cells ( n = 6) into nude mice. E – G Measurement of tumor volume and weight. H Representative H&E-stained images and IHC staining of CACNA1E, Ki-67, and EMT markers in subcutaneous tumor sections. Scale bar, 100 μm. I , J Quantification of IHC staining intensity of CACNA1E, Ki-67, and EMT markers. K Representative bioluminescence images of nude mice that received intravenous injection of shNC- or shCACNA1E-transfected MNNG and SJSA-1 cells labeled with luciferase. L Quantification analysis of bioluminescent images of lung metastases. M Photographs of dissected lungs from mouse tumor metastasis models through injection of shNC- and shCACNA1E-transfected MNNG and SJSA-1 cells via the tail vein. N Representative H&E-stained images of lung sections for showing metastatic cancer nodules. Scale bar, 100 μm. ** P < 0.01; **** P < 0.0001 derived from Student’s t-test or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Inhibition, In Vivo, Injection, Transfection, Staining, Immunohistochemistry, Labeling, Luciferase, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: METTL3-mediated m 6 A modification stabilizes CACNA1E mRNA in OS. A IGV plot visualizing m 6 A peaks within CACNA1E transcript in OS and corresponding normal tissues based on MeRIP-seq. B Prediction of m 6 A motifs within CACNA1E. C Analysis of significant confidence levels of putative m 6 A motifs within CACNA1E. D Relative enrichment of CACNA1E m 6 A level normalized to input in OS cells detected by MeRIP-qPCR. E Pearson correlation analysis between the mRNA levels of METTL3 and CACNA1E in TCGA OS samples. F FISH for detecting CACNA1E mRNA and METTL3 protein in OS cells. Scale bar, 50 μm. G qRT-PCR for measuring the stable knockdown of METTL3 in OS cells. H , I MeRIP-qPCR for detecting CACNA1E m 6 A level in control and METTL3-knockdown OS cells. J Schematic representation of the m 6 A motif (WT) in the 3’ UTR of CACNA1E from MeRIP-seq as well as the mutant m 6 A motif (Mut). K Relative luciferase activity in 293 T cells co-transfected with CACNA1E m 6 A WT or Mut luciferase reporter vector and oe-METTL3 or NC plasmid. L Measurement of CACNA1E mRNA level by qRT-PCR in control and METTL3-knockdown OS cells. M – O Detection of METTL3 and CACNA1E expression by Western blot in control and METTL3-knockdown OS cells. P FISH for detecting CACNA1E mRNA and METTL3 protein in shNC- and shMETTL3-transfected OS cells. Scale bar, 50 μm. Q , R Actinomycin D assay for analyzing the effect of METTL3 silence on the mRNA stability of CACNA1E. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, P > 0.05 derived from Student’s t-test or one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Modification, Quantitative RT-PCR, Knockdown, Control, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: METTL3 boosts OS progression by mediating m 6 A modification of CACNA1E. A , B Cell viability of shNC- and shMETTL3-transfected OS cells determined by CCK-8. C , D Colony-forming analysis of OS cells. E , F Migratory ability of OS cells evaluated by wound healing assay. Photographs are displayed at 0, 6, 12, and 24 h. Scale bar, 100 μm. G - I Migratory and invasive potential of OS cells detected by Transwell assays. Scale bar, 100 μm. J , K CCK-8 assay of METTL3-knockdown or/and CACNA1E-overexpressing OS cells. L , M Colony formation of METTL3-knockdown or/and CACNA1E-overexpressing OS cells. N , O Wound healing assay of METTL3-knockdown or/and CACNA1E-overexpressing OS cells. Photographs are displayed at 0, 6, 12, and 24 h. Scale bar, 100 μm. P - R Migration and invasion of OS cells measured by Transwell assays. Scale bar, 100 μm. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 derived from one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Modification, Transfection, CCK-8 Assay, Wound Healing Assay, Knockdown, Migration, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: IGF2BP2 recognizes and enhances METTL3-mediated m 6 A modification of CACNA1E in OS. A Analysis of the interactions between FLAG-CACNA1E and m 6 A “readers” (especially IGF2BP1/2/3, YTHDF1, and YTHDC1) by Co-IP assay in 293 T cells. B Kaplan–Meier plots of overall survival in high and low IGF2BP2 expression patients with OS. C , D RIP experiment for validating the interactions between METTL3 and CACNA1E as well as between IGF2BP2 and CACNA1E. E Relative luciferase activity in 293 T cells co-transfected with CACNA1E m 6 A wild-type (WT) or mutant (Mut) luciferase reporter vector and oe-IGF2BP2 or NC plasmid. F , G IGF2BP2 and CACNA1E mRNA levels measured by qRT-PCR in shNC- and shIGF2BP2-transfected OS cells. H , I Detection of the mRNA stability of CACNA1E by qRT-PCR in shNC- and shIGF2BP2-transfected OS cells after exposure to actinomycin D for indicated intervals. J , K CCK-8, ( L , M ) colony formation experiment, ( N , O ) wound healing assay, and ( P - R ) Transwell migration and invasion assays of shNC- and shIGF2BP2-transfected OS cells. Scale bar, 100 μm. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, P > 0.05 derived from Student’s t-test or one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Modification, Co-Immunoprecipitation Assay, Expressing, Luciferase, Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Migration, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: CACNA1E facilitates OS progression through modulating WNT7B expression. A Volcano plots for differentially expressed transcripts between shCACNA1E- and shNC-transfected OS cells based on RNA-seq data. B KEGG enrichment plots for signaling pathways enriched by the differentially expressed transcripts. C Proteins interacting with WNT7B and CACNA1E using the GeneMANIA database. D CACNA1E and WNT7B mRNA levels in OS and normal tissues. E WNT7B mRNA level examined by qRT-PCR in osteoblast cells (hFOB1.19) and OS cells (SJSA-1, MNNG, U2OS, SAOS2). F – H WNT7B mRNA and protein levels detected by qRT-PCR and Western blot in shCACNA1E- and shNC-transfected OS cells. I WNT7B mRNA level examined by qRT-PCR in shMETTL3- and shNC-transfected OS cells. J , K Cell viability of METTL3-knockdown or/and WNT7B-overexpressing OS cells determined by CCK-8. L , M Colony-forming potential of OS cells. N , O Migratory ability of OS cells assessed by wound healing assay. Photographs are displayed at 0, 6, 12, and 24 h. Scale bar, 100 μm. P - R Migratory and invasive potential of OS cells measured by Transwell assays. Scale bar, 100 μm. S , T Kaplan–Meier plots of overall survival and disease-free survival in OS patients stratified by WNT7B expression. These data are presented as the means ± SD of three independent experiments. ** P < 0.01; *** P < 0.001; **** P < 0.0001 derived from one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Expressing, Transfection, RNA Sequencing, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Wound Healing Assay, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: CACNA1E affects the WNT7B-mediated non-canonical Wnt/Ca 2+ signaling pathway by transcriptionally regulating WNT7B. A , B Fluo-8 AM calcium assay for determining intracellular Ca 2+ level in shNC- and shCACNA1E-transfected OS cells. Scale bar, 100 μm. C - G CAMK2A, CAMK2N, PLCB2, and PLCB4 mRNA and protein levels detected by qRT-PCR and Western blot in shNC- and shCACNA1E-transfected OS cells. H , I Analysis of intracellular Ca 2+ level by Fluo-8 AM calcium assay in OS cells co-transfected with shCACNA1E and WNT7B overexpression plasmid. Scale bar, 100 μm. J - N CAMK2A, CAMK2N, PLCB2, and PLCB4 mRNA and protein levels assessed by qRT-PCR and Western blot in OS cells co-transfected with shCACNA1E and WNT7B overexpression plasmid. O Schematic diagram of the truncated WNT7B binding motifs. P Relative luciferase activity in 293 T cells transfected with the truncated WNT7B luciferase reporter vectors. Q ChIP-PCR analysis for verifying the binding of CACNA1E with WNT7B promoter. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, P > 0.05 derived from one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Calcium Assay, Transfection, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: Targeted inhibition of CACNA1E improves MTX sensitivity and overcomes MTX resistance through WNT7B-mediated calcium signaling pathway. A - C P-gp and CACNA1E protein levels measured by Western blot in parental and MTX-resistant OS cells. D , E Cell viability of shNC-/shCACNA1E-transfected and MTX-treated OS cells. F , G Apoptosis of shNC-/shCACNA1E-transfected and MTX-treated OS cells analyzed by flow cytometry. H , I Colony-forming potential of shNC-/shCACNA1E-transfected and MTX-treated MTX-resistant OS cells. J - M IC50 value of MTX in shNC-/shCACNA1E-transfected MTX-resistant OS cells. N - Q Representative Chou-Talalay plots and CI-Fa plots showing the synergistic effects between CACNA1E knockdown and MTX analyzed using the CompuSyn software. R , S Apoptosis of shNC-/shCACNA1E-transfected and MTX-treated MTX-resistant OS cells measured by TUNEL staining. Scale bar, 100 μm. T , U Intracellular Ca 2+ level in shNC-/shCACNA1E-transfected and MTX-treated MTX-resistant OS cells detected by Fluo-8 AM calcium assay. Scale bar, 100 μm. V , W WNT7B protein level measured by Western blot in the above cells. These data are presented as the means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 derived from Student’s t-test or one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Inhibition, Western Blot, Transfection, Flow Cytometry, Knockdown, Software, TUNEL Assay, Staining, Calcium Assay, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: Targeted inhibition of CACNA1E and MTX synergistically prevent tumor growth in vivo. A - D Photographs of subcutaneous tumors of nude mice after subcutaneous infection of shNC-/shCACNA1E-transfected ( A , B ) MNNG and ( C , D ) SJSA-1 cells and intraperitoneal administration of MTX ( n = 6). E , F The size of subcutaneous tumors at different time points. G - J The size and weight of subcutaneous tumors. K Representative images of H&E-stained subcutaneous tumors and IHC images of Ki-67, CACAN1E, and WNT7B. Scale bar, 100 μm. L , M Quantification of Ki-67, CACAN1E, and WNT7B expression. (N, O) IHC assay for detecting the proteins levels of CACNA1E and WNT7B in MTX-sensitive and -resistant OS tissues. Scale bar, 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, P > 0.05 derived from Student’s t-test or one- or two-way analysis of variance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Inhibition, In Vivo, Infection, Transfection, Staining, Expressing, Derivative Assay

Journal: Molecular Cancer

Article Title: METTL3-mediated m 6 A modification of CACNA1E promotes osteosarcoma progression and chemoresistance by enhancing WNT7B-mediated Ca 2+ signaling

doi: 10.1186/s12943-025-02553-x

Figure Lengend Snippet: A graphic schematic of the mechanism by which CACNA1E m 6 A-modified by METTL3 in an IGF2BP2-dependent manner activates WNT7B-mediated non-canonical Wnt/Ca 2+ signaling and thus promotes OS progression and MTX resistance

Article Snippet: Antibodies used were as follows: antibodies of CACNA1E (24697–1-AP), GAPDH (10494–1-AP), IGF2BP1 (22803–1-AP), IGF2BP2 (24744–1-AP), IGF2BP3 (14642–1-AP), YTHDF1 (17479–1-AP), YTHDC1 (14392–1-AP), and FLAG (20543–1-AP) from Proteintech (Shanghai, China); antibodies of N-cadherin (ab76011), Vimentin (ab8069), E-cadherin (ab231303), METTL3 (ab195352), WNT7B (ab227607), and P-gp (ab129450) from Abcam (Cambridge, MA, USA); CAMK2A antibody (#11945) from CST (Danvers, MA, USA); antibodies of CAMK2N (SAB4300530) and PLCB2 (HPA041298) from Sigma-Aldrich (St. Louis, MO, USA); antibody of PLCB4 (PA5-100855) from ThermoFisher.

Techniques: Modification

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: a CYFIP1 RNA immunoprecipitation (RNA-IP) from DIV 3 WT cortical neurons. Histogram showing relative enrichment of the mRNAs over the non-specific IgG, measured by RT-qPCR of the eluate. The values were normalized for the input and mHprt1 mRNA and expressed as fold change over the non-specific IgG of each mRNA ( n = 4 embryos; mean ± SEM; One-Way ANOVA p < 0.0001; mMap1b mRNA p = 0.0390, mCacna1c mRNA p = 0.0054, mCacna1e mRNA p = 0.0078, mCacna1i mRNA p = 0.0009, mCacng2 mRNA p = 0.9997, mCacnb3 mRNA p = 0.9983). b Total mRNA levels of the Ca 2+ channels in DIV 3 WT and Cyfip1 +/- cortical neurons. Histograms represent mCacna1c , mCacna1e , mCacna1i , mCacng2, mCacnb3 and mCyfip1 mRNA levels, normalized to mH3f3 levels and expressed as a fold change over WT (WT n = 6/7 embryos, Cyfip1 +/- n = 7 embryos; mean ± SEM; Two-tailed Multiple Mann-Whitney test, mCacna1c mRNA p = 0.0766, mCacna1e mRNA p = 0.0435, mCacna1i mRNA p = 0.0202, mCacng2 mRNA p = 0.6282, mCacnb3 mRNA p = 0.5343, mCyfip1 mRNA p = 0.0034). c Left, representative Western Blot showing CYFIP1, Ca V 1.2 (CACNA1C), Ca V 2.3 (CACNA1E), Ca V 3.3 (CACNA1I), Ca V γ2 (CACNG2/Stargazin) and Ca V β3 (CACNB3) in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. The molecular weight of each protein is indicated in kDa. Right, histogram representing Ca V 1.2, Ca V 2.3, Ca V 3.3, Ca V γ2, Ca V β3 and CYFIP1 protein expression levels in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. Protein levels were normalized to Coomassie staining (WT n = 4 embryos, Cyfip1 +/- n = 7/8 embryos; mean ± SEM; Two-tailed Multiple unpaired t -test, Ca V 1.2 p = 0.0338, Ca V 2.3 p = 0.0281, Ca V 3.3 p = 0.0129, Ca V γ2 p = 0.2574, Ca V β3 p = 0.6259, CYFIP1 p = 0.0137). d–f Representative images from WT and Cyfip1 +/- DIV 3 cortical neurons stained for Ca V 1.2, Ca V 2.3, Ca V 3.3 (magenta) and βIII-Tubulin (green) (scale bar 20 μm). Histograms show the fluorescence intensity of each calcium channel normalized to βIII-Tubulin in the total neuron (left) and in the axon (right), expressed as a percentage over WT (Ca V 1.2: WT n = 4 embryos, Cyfip1 +/- n = 5 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.1111, axon p = 0.4127; Ca V 2.3: WT n = 5 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0635, axon p = 0.0159; Ca V 3.3: WT n = 4 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0286, axon p = 0.0286). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Molecular Weight, Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: CYFIP1, potentially interacting with the RNA binding proteins HuD and HuR (previously identified as CYFIP1 interactors), is implicated in regulating the mRNA stability of calcium channel subunits Cacna1c , Cacna1e and Cacna1i . In Cyfip1 +/- neurons, reduced CYFIP1 levels result in a decrease protein abundance of the regulated calcium channel subunits, consequently leading to a decrease in intracellular and mitochondrial calcium concentration. Low levels of calcium ions may affect mitochondria polarity and motility, both of which we found impaired in Cyfip1 +/- axons. The decreased calcium concentration and the mitochondrial defects concur in reducing axonal growth observed in Cyfip1 +/- neurons. By restoring the intracellular calcium homeostasis, both the axonal growth and mitochondrial defects are rescued. Created in BioRender. https://BioRender.com/xquv8cy .

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Binding Assay, Quantitative Proteomics, Concentration Assay