cacna1e  (Proteintech)

Journal: The Journal of Experimental Medicine

Article Title: Discovery of potent and selective inhibitors of human NLRP3 with a novel mechanism of action

doi: 10.1084/jem.20242403

Figure Lengend Snippet: BAL-0028 inhibits primate NLRP3 but is a poor inhibitor of NLRP3 from other mammals. Comparison of BAL-0028 and MCC950 in IL-1β release assays from cells stimulated with LPS and nigericin. (A–I) J774A.1 mouse macrophage cell line (A), Wistar rat PBMCs (B), Beagle CD14 + monocytes (C), New Zealand white rabbit PBMCs (D), African green monkey ( C. sabaeus ) PBMCs (E) and CD14 + monocytes (F), cynomolgus monkey ( M. fascicularis ) CD14 + monocytes (G), WT 129S6 iBMDM (H), and 129S6-human promoter NLRP3 iBMDM (I). (A, H, and I) Graph symbols show average IL-1β values relative to vehicle control ± SEM from N = 3 independent experiments performed in triplicate. (B–G) Graph symbols show average IL-1β values relative to vehicle control ± SD from one experiment performed in duplicate (C, D, and F) or triplicate (B, E, and G). IC 50 curves were fitted by nonlinear regression analysis.

Article Snippet: Cryopreserved Beagle canine CD14 + , cynomolgus monkey CD14 + cells, New Zealand white rabbit PBMCs, and Wistar rat PBMCs were obtained from IQ Biosciences.

Techniques: Comparison, Control

Journal: The Journal of Experimental Medicine

Article Title: Discovery of potent and selective inhibitors of human NLRP3 with a novel mechanism of action

doi: 10.1084/jem.20242403

Figure Lengend Snippet: BAL-0598 inhibits the activation of human and monkey NLRP3 and is a non-covalent inhibitor . Related to and . (A) BAL-0598 in IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and MSU. (B) BAL-0598 effect on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C and D) Comparison of BAL-0598, BAL-0028, and MCC950 in an LDH release assay in primary peritoneal macrophages isolated from (C) WT 129S6 or (D) 129S6 mouse promoter- NLRP3 mice stimulated with LPS and ATP. Graph symbols show average LDH values ± SEM from N = 2 independent experiments performed in duplicate. (E and F) BAL-0598 in IL-1β release assays from cells stimulated with LPS and nigericin. African green monkey CD14 + monocytes (E) and PBMCs (F). (A, B, E, and F) Graph symbols show average values relative to vehicle control ± SEM (A, N = 2) or SD (B, E, and F, N = 1) from independent experiments performed in duplicate (E) or triplicate (A, B, and F); IC 50 curve was fitted by nonlinear regression analysis. (G) Comparison of BAL-0028, BAL-0598, and MCC950 in an IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and nigericin. Cells were treated with compounds before nigericin stimulation, and compounds were left on or were washed out for 1 min before nigericin addition. The graph shows average IL-1β values ± SD from one experiment performed in triplicate. (H) Schematic illustration of U937 NLRP3 and NLRP3-AID mutant cell model.

Article Snippet: Cryopreserved Beagle canine CD14 + , cynomolgus monkey CD14 + cells, New Zealand white rabbit PBMCs, and Wistar rat PBMCs were obtained from IQ Biosciences.

Techniques: Activation Assay, Release Assay, Fluorescence, Microscopy, Comparison, Lactate Dehydrogenase Assay, Isolation, Control, Mutagenesis

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: Frontiers in Immunology

Article Title: Characterization of cancer-related fibroblasts in bladder cancer and construction of CAFs-based bladder cancer classification: insights from single-cell and multi-omics analysis

doi: 10.3389/fimmu.2025.1580986

Figure Lengend Snippet: Machine learning algorithms to obtain hub targets of C3 subtype. (A) The identification of potential targets using the randomForest and Lasso algorithm in C3 subtype. (B) Survival analysis for AHANK2, SLC9B2, and MN1. The relative expression of different stages and grades for AHANK2, SLC9B2, and MN1, respectively. (C) GSEA enrichment analysis for AHANK2, SLC9B2, and MN1 based on hallmark gene set. (D) Correlation between 22 infiltrating immune cells and AHANK2, SLC9B2, and MN1, respectively. (E) WB and the relative expression of AHANK2, SLC9B2, and MN1 proteins in different tumor stages. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Among them, primary antibodies included: FAT4 (1:1000; PA5-72970, Invitrogen, China), RPL37P1 (1:1000; ab228542, Abcam, China), FGFR1 (1:1000; R381166, Zenbio, China), RNASEH1 (1:1000; 82771-1-RR, Proteintech, China), AHNAK2 (1:1000; 680021, Zenbio, China), SLC9B2 (1:1000; 24065-1-AP, Proteintech, China), MN1 (1:1000; 24697-1-AP, Proteintech, China), TTLL3 (1:1000; PA5-70598, Invitrogen, China), FABP6 (1:1000; 126828, Zenbio, China), TBC1D3 (1:1000; DF3346, Affinity, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: XIST regulates MN1 expression through miR-15a-5p. (A) Volcano plot illustrating the differentially expressed genes between XIST-shRNA and control groups in T24 cells. (B, C) KEGG pathway analysis of (B) upregulated and (C) downregulated genes following XIST knockdown. (D) Venn diagram showing the overlap between XIST ceRNA target genes and downregulated genes after XIST knockdown. (E) Expression levels of MN1, SH3PXD2A, and TFP1 genes in male and female bladder cancer patients. (F) Prognostic significance of MN1, SH3PXD2A, and TFP1 gene expression in bladder cancer patients. (G) Prognostic impact of MN1 expression in male and female bladder cancer patients. (H) Effect of XIST overexpression on MN1 mRNA and protein levels in T24 cells. (I) Effect of XIST overexpression on MN1 mRNA and protein levels in RT4 cells. (J) Volcano plot illustrating the differentially expressed miRNAs between XIST-shRNA and control groups in T24 cells. (K) Correlation analysis between MN1 and miR-15a expression. (L) Effect of XIST knockdown on miR-15a-5p expression in T24 and RT4 cells. (M, N) Effect of miR-15a-5p overexpression on MN1 mRNA and protein levels in (M) T24 and (N) RT4 cells. (O) Dual-luciferase reporter assay demonstrating the interaction between XIST and miR-15a-5p. (P, Q) Effect of miR-15a-5p on XIST expression in (P) T24 and (Q) RT4 cells. (R) Dual-luciferase reporter assay demonstrating the interaction between MN1 and miR-15a-5p. (S, T) Effect of miR-15a-5p on MN1 expression in (S) T24 and (T) RT4 cells. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques: Expressing, shRNA, Control, Knockdown, Gene Expression, Over Expression, Luciferase, Reporter Assay

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: The impact of miR-15a-5p and MN1 on the proliferation and metastasis of bladder cancer cells. (A) RT-PCR analysis of miR-15a-5p expression in T24 cells following transfection with miR-15a-5p-mimic. (B–D) Impact of miR-15a-5p transfection on the (B) proliferation, (C) migration, and (D) invasion capabilities of T24 cells. (E) RT-PCR analysis of miR-15a-5p expression in RT4 cells following transfection with miR-15a-5p-mimic. (F–H) Impact of miR-15a-5p transfection on the (F) proliferation, (G) migration, and (H) invasion capabilities of RT4 cells. (I) Western blot analysis of MN1 expression in T24 cells following MN1 overexpression. (J–L) Impact of MN1 overexpression on the (J) proliferation, (K) migration, and (L) invasion capabilities of T24 cells. (M) Western blot analysis of MN1 expression in RT4 cells following MN1 overexpression. (N–P) Impact of MN1 overexpression on the (N) proliferation, (O) migration, and (P) invasion capabilities of RT4 cells. (Q, R) Impact of MN1 overexpression on the (Q) proliferation and (R) metastasis capabilities of T24 cells in the zebrafish model. (S, T) Impact of MN1 overexpression on the (S) proliferation and (T) metastasis capabilities of RT4 cells in the zebrafish model. *p<0.05, **p<0.01, ***p<0.001. ns, p>0.05.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Migration, Western Blot, Over Expression

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: The impact of XIST on the proliferation and metastasis of bladder cancer cells is related to MN1. (A) Western blot analysis of MN1 expression in T24 cells after transfection with XIST-shRNA and MN1-OS. (B–D) Impact of XIST-shRNA and MN1-OS transfection on the (B) proliferation, (C) migration, and (D) invasion capabilities of T24 cells. (E) Western blot analysis of MN1 expression in RT4 cells after transfection with XIST-shRNA and MN1-OS. (F–H) Impact of XIST-shRNA and MN1-OS transfection on the (F) proliferation, (G) migration, and (H) invasion capabilities of RT4 cells. *p<0.05, **p<0.01, ***p<0.001. ns, p>0.05.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques: Western Blot, Expressing, Transfection, shRNA, Migration

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: Expression of XIST/miR-15a-5p/MN1 in bladder cancer. (A, B) XIST expression levels in (A) 50 female and (B) 50 male bladder cancer patient specimens. (C, D) miR-15a-5p expression levels in (C) 50 female and (D) 50 male bladder cancer patient specimens. (E, F) MN1 mRNA expression levels in (E) 50 female and (F) 50 male bladder cancer patient specimens. (G, H) Immunohistochemical analysis of MN1 protein expression in (G) 50 female and (H) 50 male bladder cancer patient specimens. (I) Comparative analysis of MN1 protein expression in male and female bladder cancer patient specimens. *** p< 0.001. **p<0.01.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques: Expressing, Immunohistochemical staining

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: XIST/miR-15a-5p/MN1 regulates FZD2 expression. (A) Volcano plot illustrating differentially expressed genes in T24 cells transfected with MN1-shRNA compared to the control group. (B, C) KEGG pathway analysis of (B) upregulated and (C) downregulated genes following MN1 knockdown. (D) Venn diagram depicting the intersection of downregulated genes after XIST and MN1 knockdown. (E) Differentially expressed downregulated intersecting genes in male versus female bladder cancer. (F) Venn diagram showing the intersection of upregulated genes after XIST and MN1 knockdown. (G) Differentially expressed upregulated intersecting genes in male versus female bladder cancer specimens. (H) Prognostic analysis of FZD2 in female bladder cancer. (I, J) Effects of (I) XIST knockdown and (J) MN1 knockdown on FZD2 expression in T24 cells. (K, L) Effects of (K) XIST knockdown and (L) MN1 knockdown on FZD2 expression in RT4 cells. *p<0.05, **p<0.01.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques: Expressing, Transfection, shRNA, Control, Knockdown

Journal: Frontiers in Immunology

Article Title: The role of the LncRNA XIST/miR-15a-5p/MN1 signaling axis in gender disparities in bladder cancer prognosis

doi: 10.3389/fimmu.2025.1554829

Figure Lengend Snippet: A mechanistic diagram. In female bladder cancer, the XIST/miR-15a-5p/MN1/FZD2 axis is overactivated, thereby promoting the malignant progression of bladder cancer.

Article Snippet: The sections were then incubated overnight at 4°C with an anti-MN1 antibody at a 1:100 dilution (Proteintech, China).

Techniques:

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: The primer sequences for the target genes

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Sequencing

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: KRT14 expression levels in ovarian cancer cell lines SK-OV-3 and A2780 and their cisplatin-resistant variants SK-OV-3/DDP and A2780/DDP. (A) KRT14 mRNA expression levels detected using qRT-PCR. (B) KRT14 protein expression levels visualized using Western blot analysis. Right bar graph: KRT14 protein levels relative to GAPDH. *, statistical significance (P<0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KRT14, keratin 14; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: KRT14 knockdown reduces SK-OV-3/DDP and A2780/DDP cell viability. si-KRT14 was transfected into cells to achieve KRT14 knockdown, with cells transfected with si-NC and un-transfected cells (blank cells) serving as controls. (A) KRT14 mRNA levels. (B) KRT14 protein levels. Right bar graph: KRT14 protein levels relative to GAPDH. (C) Viability of SK-OV-3/DDP and A2780/DDP cells in the three groups after treatment with varying concentrations of cisplatin (0, 2.5, 5, 10, 20, 40, and 80 µM). (D) Fitting curves used to determine the IC 50 values of SK-OV-3/DDP and A2780/DDP cells in response to cisplatin. *, statistical significance (P<0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IC 50 , half-maximal inhibitory concentration; KRT14, keratin 14; mRNA, messenger RNA; si-KRT14, siRNA targeting KRT14; si-NC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Knockdown, Transfection, Concentration Assay, Negative Control, Small Interfering RNA

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: KRT14 knockdown increases apoptosis and decreases MDR-related protein expression in SK-OV-3/DDP and A2780/DDP cells. si-KRT14 was transfected into cells to achieve KRT14 knockdown, with cells transfected with si-NC and untransfected cells (blank cell) serving as controls. (A,B) Apoptotic cell rates assessed using flow cytometry and Hoechst 33258 staining, respectively. The magnification is 20× and the green arrows indicate the apoptotic cells. (C) P-gp and MRP1 protein levels. Right bar graph: P-gp and MRP1 protein levels relative to GAPDH. *, statistical significance (P<0.05). FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KRT14, keratin 14; MDR, multidrug resistance; MRP1, multidrug resistance-associated protein 1; P-gp, P-glycoprotein; si-KRT14, siRNA targeting KRT14; si-NC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Knockdown, Expressing, Transfection, Flow Cytometry, Staining, Negative Control, Small Interfering RNA

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: Effect of KRT14 knockdown on LRP11 expression. To achieve KRT14 knockdown, cells were transfected with si-KRT14, with cells transfected with si-NC used as a control. (A) Volcano plots of differentially expressed mRNAs identified by Illumina RNA sequencing following KRT14 knockdown. (B) Venn diagram of differentially expressed mRNAs in both SK-OV-3/DDP and A2780/DDP cells. (C) Expression levels of MTAP, LRP11, NUDT16, and RBM15 in KRT14 knockdown cells as measured by qRT-PCR. (D) Western blot results for LRP11 expression in KRT14 knockdown cells. Right bar graph: LRP11 protein levels relative to GAPDH. *, statistical significance (P<0.05). FDR, false discovery rate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KRT14, keratin 14; LRP11, lipoprotein receptor-related protein 11; mRNA, messenger RNA; MTAP, methylthioadenosine phosphorylase; NUDT16, nudix hydrolase 16; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RBM15, RNA binding motif protein 15; si-KRT14, siRNA targeting KRT14; si-NC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Knockdown, Expressing, Transfection, Control, RNA Sequencing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Polymerase Chain Reaction, RNA Binding Assay, Negative Control, Small Interfering RNA

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: Effects of LRP11 overexpression on cell viability in SK-OV-3/DDP and A2780/DDP cells with and without KRT14 knockdown. Cells were transfected with si-KRT14 for KRT14 knockdown and LRP11-OP for LRP11 overexpression, while cells transfected with si-NC and EP served as controls. (A,B) LRP11 and KRT14 protein levels, with quantification relative to GAPDH provided below the graphs. (C) Viability of SK-OV-3/DDP and A2780/DDP cells in each group after treatment with various cisplatin concentrations (0, 2.5, 5, 10, 20, 40, and 80 µM). (D) Fitting curves used to determine the IC 50 values for the cells in response to cisplatin. # and & , statistical significance (P<0.05). EP, empty plasmid pcDNA3.1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IC 50 , half-maximal inhibitory concentration; KRT14, keratin 14; LRP11, lipoprotein receptor-related protein 11; LRP11-OP, LRP11 overexpression plasmid; si-KRT14, siRNA targeting KRT14; si-NC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Concentration Assay, Negative Control, Small Interfering RNA

Journal: Translational Cancer Research

Article Title: KRT14 knockdown reduces cisplatin resistance by lowering LRP11 expression levels in cisplatin-resistant ovarian cancer cell lines

doi: 10.21037/tcr-24-1795

Figure Lengend Snippet: Effects of LRP11 overexpression on apoptosis and MDR-related protein expression in SK-OV-3/DDP and A2780/DDP cells with or without KRT14 knockdown. Cells were transfected with si-KRT14 for KRT14 knockdown and LRP11-OP for LRP11 overexpression, while cells transfected with si-NC and EP served as controls. (A) Apoptotic cell rates assessed using flow cytometry. (B) Apoptotic cell rates assessed using Hoechst 33258 staining. Magnification: 20×. The green arrows indicate the apoptotic cells. (C) P-gp and MRP1 protein levels. Right bar graph: P-gp and MRP1 protein levels relative to GAPDH. # and & , statistical significance (P<0.05). EP, empty plasmid pcDNA3.1; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KRT14, keratin 14; LRP11, lipoprotein receptor-related protein 11; LRP11-OP, LRP11 overexpression plasmid; MDR, multidrug resistance; MRP1, multidrug resistance-associated protein 1; P-gp, P-glycoprotein; si-KRT14, siRNA targeting KRT14; si-NC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: After washing the PVDF membrane with tris-buffered saline with Tween 20 (TBST), it was blocked with 5% non-fat milk at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4 °C with the primary antibodies against LRP11 (catalogue number: orb674844, Biorbyt, Cambridge, UK), KRT14 (orb1259684, Biorbyt), P-gp (catalogue number: orb11034, Biorbyt), and MRP1 (catalogue number: orb1150958, Biorbyt), following three washes with TBST for 8 min each.

Techniques: Over Expression, Expressing, Knockdown, Transfection, Flow Cytometry, Staining, Plasmid Preparation, Negative Control, Small Interfering RNA