ub flag  (Proteintech)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: QKI6 modulates the ubiquitination levels of PD-L1 in NSCLC. A–B: qPCR analysis of A QKI6 expression and B PD-L1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells. C and D Flow cytometry analysis for detecting the phenotypic expression of PD-L1 in A549 QKI6-overexpressing and QKI6-knockdown cell lines. E and F WB assay for PD-L1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells. G PD-L1 ubiquitination analysis was performed on different cell fractions using Co-IP. The following cell groupings were used: (1) A549 + overexpressing NC + HA-Ub + Flag-PD-L1; (2) A549 + overexpressing NC + HA-Ub + Flag-PD-L1 + MG132; (3) A549 + overexpressing QKI6 + HA-Ub + Flag-PD-L1 + MG132. H WB assay for STUB1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Ubiquitin Proteomics, Expressing, Knockdown, Flow Cytometry, Co-Immunoprecipitation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: Interaction between hsa_circ_0074158 and QKI6. A CircRNAs binding to QKI6 in A549 cells were detected by RIP-seq. A total of 284 circRNAs showed increased expression, while 1951 circRNAs exhibited decreased expression. B–D Expression levels of hsa_circ_0074158, hsa_circ_0001470, and hsa_circ_0099287 were measured by qPCR. Hsa_circ_0074158 was significantly more expressed in QKI6 high-expression cell lines ( P < 0.001), and its levels were reduced in QKI6-knockdown cell lines ( P < 0.01). E Schematic illustrating the mechanism of hsa_circ_0074158. F GO enrichment analysis of hsa_circ_0074158. G and H KEGG enrichment analysis of hsa_circ_0074158. I rip-qPCR results of circ_0074158. J EMSA confirming that QKI6 binds to hsa_circ_0074158

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Binding Assay, Expressing, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: Circ_0074158 overexpression reduces proliferation, invasion, and migration in NSCLC. A and B qPCR assay for hsa_circ_0074158 expression in A549 cell lines with knockdown and overexpression. A549-shRNA-1 showed the most effective knockdown efficiency ( P < 0.0001). C Cell proliferation inhibition of A549 + circ_0074158 was significantly greater than the control ( P < 0.0001). The proliferation inhibition rate of A549 + shcirc_0074158 was significantly lower than the control ( P < 0.0001). Low expression of QKI6 significantly enhances cell proliferation. High expression of QKI6 increased the inhibition rate of cell proliferation ( P < 0.001). D and E Apoptosis level of A549 + circ_0074158 was significantly higher than the control in flow cytometry analysis ( P < 0.0001). High expression of QKI6 increased apoptosis levels ( P < 0.0001). F and G Cell invasion and migration ability of A549 + circ_0074158 was significantly lower than the control ( P < 0.01), whereas A549 + shcirc_0074158 had significantly higher invasion and migration ability ( P < 0.01). High expression of QKI6 reduced cell invasion and migration ( P < 0.0001). H and I Cell migration ability assessed by wound healing assay in A549 + circ_0074158 was significantly lower than the control ( P < 0.0001), whereas A549 + shcirc_0074158 exhibited significantly higher migration ( P < 0.01). High expression of QKI6 reduced cell migration ( P < 0.01). J WB detection of E-cadherin and N-cadherin expression in A549, A549 + circ_0074158, and A549 + shcirc_0074158 cells

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Over Expression, Migration, Expressing, Knockdown, shRNA, Inhibition, Control, Flow Cytometry, Wound Healing Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: CircRNA activates CD8 + T cells in PBMCs and inhibits immune escape. A–D PBMCs were co-cultured with A549 + NC cells, A549 + circ_0074158-overexpressing cells, A549 + sh-NC cells, and A549 + circ_0074158-knockdown cells. ELISA assays for granzyme B (Gra B) (A ) , Perforin (B), IFN-γ (C), and TNF-α (D) secretion were significantly increased in the circ_0074158-overexpressing group ( P < 0.001) and significantly inhibited in the circ_0074158-knockdown group ( P < 0.001). E, G: Circ_0074158 overexpression inhibited cell proliferation as detected by EdU assays (E), while its knockdown restored cell proliferation ability (G) ( P < 0.0001). F, H TUNEL assays showed that apoptosis levels in the circ_0074158-overexpression group were significantly higher than in the control group ( P < 0.0001), while apoptosis levels were significantly lower in the circ_0074158-knockdown group ( P < 0.0001). I and J Flow cytometry analysis of the co-culture system revealed that the ratio of CD3 + CD8 + T cells was significantly higher in the circ_0074158-overexpression group compared to the control ( P < 0.001), whereas the opposite was observed for the circ_0074158-knockdown group ( P < 0.01)

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Cell Culture, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression, TUNEL Assay, Control, Flow Cytometry, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: QKI6 modulates the ubiquitination levels of PD-L1 in NSCLC. A–B: qPCR analysis of A QKI6 expression and B PD-L1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells. C and D Flow cytometry analysis for detecting the phenotypic expression of PD-L1 in A549 QKI6-overexpressing and QKI6-knockdown cell lines. E and F WB assay for PD-L1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells. G PD-L1 ubiquitination analysis was performed on different cell fractions using Co-IP. The following cell groupings were used: (1) A549 + overexpressing NC + HA-Ub + Flag-PD-L1; (2) A549 + overexpressing NC + HA-Ub + Flag-PD-L1 + MG132; (3) A549 + overexpressing QKI6 + HA-Ub + Flag-PD-L1 + MG132. H WB assay for STUB1 expression in A549 cells, QKI6-overexpressing A549 cells, and QKI6-knockdown A549 cells

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Ubiquitin Proteomics, Expressing, Knockdown, Flow Cytometry, Co-Immunoprecipitation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: Interaction between hsa_circ_0074158 and QKI6. A CircRNAs binding to QKI6 in A549 cells were detected by RIP-seq. A total of 284 circRNAs showed increased expression, while 1951 circRNAs exhibited decreased expression. B–D Expression levels of hsa_circ_0074158, hsa_circ_0001470, and hsa_circ_0099287 were measured by qPCR. Hsa_circ_0074158 was significantly more expressed in QKI6 high-expression cell lines ( P < 0.001), and its levels were reduced in QKI6-knockdown cell lines ( P < 0.01). E Schematic illustrating the mechanism of hsa_circ_0074158. F GO enrichment analysis of hsa_circ_0074158. G and H KEGG enrichment analysis of hsa_circ_0074158. I rip-qPCR results of circ_0074158. J EMSA confirming that QKI6 binds to hsa_circ_0074158

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Binding Assay, Expressing, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: Circ_0074158 overexpression reduces proliferation, invasion, and migration in NSCLC. A and B qPCR assay for hsa_circ_0074158 expression in A549 cell lines with knockdown and overexpression. A549-shRNA-1 showed the most effective knockdown efficiency ( P < 0.0001). C Cell proliferation inhibition of A549 + circ_0074158 was significantly greater than the control ( P < 0.0001). The proliferation inhibition rate of A549 + shcirc_0074158 was significantly lower than the control ( P < 0.0001). Low expression of QKI6 significantly enhances cell proliferation. High expression of QKI6 increased the inhibition rate of cell proliferation ( P < 0.001). D and E Apoptosis level of A549 + circ_0074158 was significantly higher than the control in flow cytometry analysis ( P < 0.0001). High expression of QKI6 increased apoptosis levels ( P < 0.0001). F and G Cell invasion and migration ability of A549 + circ_0074158 was significantly lower than the control ( P < 0.01), whereas A549 + shcirc_0074158 had significantly higher invasion and migration ability ( P < 0.01). High expression of QKI6 reduced cell invasion and migration ( P < 0.0001). H and I Cell migration ability assessed by wound healing assay in A549 + circ_0074158 was significantly lower than the control ( P < 0.0001), whereas A549 + shcirc_0074158 exhibited significantly higher migration ( P < 0.01). High expression of QKI6 reduced cell migration ( P < 0.01). J WB detection of E-cadherin and N-cadherin expression in A549, A549 + circ_0074158, and A549 + shcirc_0074158 cells

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Over Expression, Migration, Expressing, Knockdown, shRNA, Inhibition, Control, Flow Cytometry, Wound Healing Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Circ_0074158 and QKI6: A regulatory axis for PD-L1 ubiquitination and immune evasion in NSCLC

doi: 10.1007/s00262-025-04152-7

Figure Lengend Snippet: CircRNA activates CD8 + T cells in PBMCs and inhibits immune escape. A–D PBMCs were co-cultured with A549 + NC cells, A549 + circ_0074158-overexpressing cells, A549 + sh-NC cells, and A549 + circ_0074158-knockdown cells. ELISA assays for granzyme B (Gra B) (A ) , Perforin (B), IFN-γ (C), and TNF-α (D) secretion were significantly increased in the circ_0074158-overexpressing group ( P < 0.001) and significantly inhibited in the circ_0074158-knockdown group ( P < 0.001). E, G: Circ_0074158 overexpression inhibited cell proliferation as detected by EdU assays (E), while its knockdown restored cell proliferation ability (G) ( P < 0.0001). F, H TUNEL assays showed that apoptosis levels in the circ_0074158-overexpression group were significantly higher than in the control group ( P < 0.0001), while apoptosis levels were significantly lower in the circ_0074158-knockdown group ( P < 0.0001). I and J Flow cytometry analysis of the co-culture system revealed that the ratio of CD3 + CD8 + T cells was significantly higher in the circ_0074158-overexpression group compared to the control ( P < 0.001), whereas the opposite was observed for the circ_0074158-knockdown group ( P < 0.01)

Article Snippet: For IP, the following cell groups were used: A549 + OE-NC + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + HA-Ub + Flag-PD-L1 + MG132, A549 + OE-circRNA + si-NC + HA-Ub + Flag-PD-L1 + MG132, and A549 + OE-circRNA + si-QKI6 + HA-Ub + Flag-PD-L1 + MG132, where MG132 (MCE, HY-13259) acts as a proteasome inhibitor.

Techniques: Cell Culture, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression, TUNEL Assay, Control, Flow Cytometry, Co-Culture Assay