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Proteintech upf1 primary antibody
The tRF-Leu-AAG promoted PC development by suppressing <t>UPF1.</t>
Upf1 Primary Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The biological behavior of tRNA-derived fragment tRF-Leu-AAG in pancreatic cancer cells"

Article Title: The biological behavior of tRNA-derived fragment tRF-Leu-AAG in pancreatic cancer cells

Journal: Bioengineered

doi: 10.1080/21655979.2022.2064206

The tRF-Leu-AAG promoted PC development by suppressing UPF1.
Figure Legend Snippet: The tRF-Leu-AAG promoted PC development by suppressing UPF1.

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a Schematic of strategy used for assembling luciferase based minigene splicing reporters. b Bar graph of lucMAPT-30D reporter readout following co-transfection with FLAG NC, RBFOX1-MCP fusion (RBFOX1), and RBFOX1 lacking an MCP fusion (RBFOX1 NoMS2) (mean ± s.d., n = 3 replicate transfections). c Western blots for validation of <t>UPF1</t> shRNA constructs qualitatively showing decreased UPF1 protein levels for each of four UPF1 shRNA constructs tested in HEK293T cells. d qPCR for validation of SMG7 shRNA constructs showing decreased SMG7 expression levels as quantified using the delta-delta Ct method in RNA extracted from MDAMB231 and MCF10A cells stably expressing the constructs (n = 2 biological replicates (1 replicate/line), n = 2 technical replicates). e Bar graph of reporter readouts in HEK293T cells stably expressing a non-targeting shRNA (NT), a UPF1-targeting shRNA (sh302), and two SMG7-targeting shRNAs (sh65 and sh88), co-transfected with reporter plasmids and FLAG NC (mean ± s.d., n = 6 replicate transfections). P-value is calculated by two-tailed independent two-sample t -test. f Layout of 96-well transfections used throughout the screens. g Agarose gels of RNA-level validation of hits from the splicing screen. All hits were tested for lucMAPT-30D (top) and lucMAPT-30U (bottom). Numbers along the top correspond to lane number in - . n = 2 replicate transfections.
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Knockdown validations for all shRNAs used. ( A ) Top: western blot validating <t>UPF1,</t> UPF2, and UPF3B knockdowns; middle: western blot validating SMG6 knockdown; bottom: qPCR validation of SMG1 knockdown. ( B ) Top left: western blot for CUL2 knockdown validation; top right: western blot for ABCE1 knockdown validation; middle left: western blot for EIF5A knockdown validation; bottom left: qPCR validation of ZNF598 knockdown; bottom right: qPCR validation for EIF3J knockdown. GAPDH was the loading control for all western blots. RPL27 was the housekeeping gene for all qPCRs. qPCRs were performed in triplicate.
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Abnormal expression of <t>UPF1</t> in psoriasis. (A) Paired sample t-test for 186 paired psoriasis samples from GEO datasets (GDS5392, GDS4602, GDS2518, GDS3539 and GDS4600). (B) Student's t-test for UPF1 mRNA levels in 10 psoriasis scales and 8 healthy cornified epidermal layer samples. Among the samples, four paired tissues were indicated with black lines. The UPF1 expression increased in the psoriasis group; 3/4 paired samples showed an upregulated expression level of UPF1 . (C) Mice received a daily topical IMQ cream to induce psoriasis-like skin. The expression of UPF1 increased in all four psoriasis-like tissues, as compared with the paired normal skin tissue samples. (D) The expression of UPF1 protein distributed throughout the cytoplasm in mouse skin was detected by IHC. The white arrows indicate positive UPF1 protein staining, and the red arrows indicate skin hyperkeratosis in IMQ-treated samples. (E) IHC staining was suantified using ImageJ software. The UPF1 protein decreased in 2/4 paired samples. (F) The mRNA expression of three NMD substrates, NIK , TBL2 and NAT9 , were higher in psoriasis-like skin compared with normal tissue. Data are presented as the mean ± SD from three independent experiments. * P<0.05 and *** P<0.001. n.s., not significant; UPF1 , up-frameshift suppressor 1 homolog; IMQ, imiquimod; IHC, immunohistochemistry; NMD, nonsense-mediated RNA decay; NIK , NF-κ-B-inducing kinase; TBL2 , transducing β like 2; NAT9 , N-Acetyltransferase 9; PS, psoriasis or IMQ-treated psoriasis-like skin; PN, normal cornified epidermal layer.
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a Schematic of strategy used for assembling luciferase based minigene splicing reporters. b Bar graph of lucMAPT-30D reporter readout following co-transfection with FLAG NC, RBFOX1-MCP fusion (RBFOX1), and RBFOX1 lacking an MCP fusion (RBFOX1 NoMS2) (mean ± s.d., n = 3 replicate transfections). c Western blots for validation of UPF1 shRNA constructs qualitatively showing decreased UPF1 protein levels for each of four UPF1 shRNA constructs tested in HEK293T cells. d qPCR for validation of SMG7 shRNA constructs showing decreased SMG7 expression levels as quantified using the delta-delta Ct method in RNA extracted from MDAMB231 and MCF10A cells stably expressing the constructs (n = 2 biological replicates (1 replicate/line), n = 2 technical replicates). e Bar graph of reporter readouts in HEK293T cells stably expressing a non-targeting shRNA (NT), a UPF1-targeting shRNA (sh302), and two SMG7-targeting shRNAs (sh65 and sh88), co-transfected with reporter plasmids and FLAG NC (mean ± s.d., n = 6 replicate transfections). P-value is calculated by two-tailed independent two-sample t -test. f Layout of 96-well transfections used throughout the screens. g Agarose gels of RNA-level validation of hits from the splicing screen. All hits were tested for lucMAPT-30D (top) and lucMAPT-30U (bottom). Numbers along the top correspond to lane number in - . n = 2 replicate transfections.

Journal: Nature biotechnology

Article Title: Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion

doi: 10.1038/s41587-023-02014-0

Figure Lengend Snippet: a Schematic of strategy used for assembling luciferase based minigene splicing reporters. b Bar graph of lucMAPT-30D reporter readout following co-transfection with FLAG NC, RBFOX1-MCP fusion (RBFOX1), and RBFOX1 lacking an MCP fusion (RBFOX1 NoMS2) (mean ± s.d., n = 3 replicate transfections). c Western blots for validation of UPF1 shRNA constructs qualitatively showing decreased UPF1 protein levels for each of four UPF1 shRNA constructs tested in HEK293T cells. d qPCR for validation of SMG7 shRNA constructs showing decreased SMG7 expression levels as quantified using the delta-delta Ct method in RNA extracted from MDAMB231 and MCF10A cells stably expressing the constructs (n = 2 biological replicates (1 replicate/line), n = 2 technical replicates). e Bar graph of reporter readouts in HEK293T cells stably expressing a non-targeting shRNA (NT), a UPF1-targeting shRNA (sh302), and two SMG7-targeting shRNAs (sh65 and sh88), co-transfected with reporter plasmids and FLAG NC (mean ± s.d., n = 6 replicate transfections). P-value is calculated by two-tailed independent two-sample t -test. f Layout of 96-well transfections used throughout the screens. g Agarose gels of RNA-level validation of hits from the splicing screen. All hits were tested for lucMAPT-30D (top) and lucMAPT-30U (bottom). Numbers along the top correspond to lane number in - . n = 2 replicate transfections.

Article Snippet: Primary antibodies for UPF1 (Cell Signaling Technology, D15G6, 1:1,000), PRPF39 (Invitrogen, PA5-21627, 1:1,000) and GAPDH (Millipore, MAB374, 1:10,000) were diluted in 5% milk in TBST and probed overnight at 4 °C.

Techniques: Luciferase, Cotransfection, Transfection, Western Blot, shRNA, Construct, Expressing, Stable Transfection, Two Tailed Test

Knockdown validations for all shRNAs used. ( A ) Top: western blot validating UPF1, UPF2, and UPF3B knockdowns; middle: western blot validating SMG6 knockdown; bottom: qPCR validation of SMG1 knockdown. ( B ) Top left: western blot for CUL2 knockdown validation; top right: western blot for ABCE1 knockdown validation; middle left: western blot for EIF5A knockdown validation; bottom left: qPCR validation of ZNF598 knockdown; bottom right: qPCR validation for EIF3J knockdown. GAPDH was the loading control for all western blots. RPL27 was the housekeeping gene for all qPCRs. qPCRs were performed in triplicate.

Journal: bioRxiv

Article Title: Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

doi: 10.1101/2024.01.10.575080

Figure Lengend Snippet: Knockdown validations for all shRNAs used. ( A ) Top: western blot validating UPF1, UPF2, and UPF3B knockdowns; middle: western blot validating SMG6 knockdown; bottom: qPCR validation of SMG1 knockdown. ( B ) Top left: western blot for CUL2 knockdown validation; top right: western blot for ABCE1 knockdown validation; middle left: western blot for EIF5A knockdown validation; bottom left: qPCR validation of ZNF598 knockdown; bottom right: qPCR validation for EIF3J knockdown. GAPDH was the loading control for all western blots. RPL27 was the housekeeping gene for all qPCRs. qPCRs were performed in triplicate.

Article Snippet: Primary antibodies include anti-UPF1 (Abcam, ab109363), anti-UPF2 (Cell Signaling, 11875S), anti-UPF3B (Abcam, ab134566), anti-SMG6 (ABclonal, A10141), anti-CUL2 (Bethyl Laboratories, A302-476A), anti-ABCE1 (Abcam, ab185548), anti-EIF5A (Abcam, ab32407), and anti-GAPDH (Abcam, ab9484).

Techniques: Western Blot

The tRF-Leu-AAG promoted PC development by suppressing UPF1.

Journal: Bioengineered

Article Title: The biological behavior of tRNA-derived fragment tRF-Leu-AAG in pancreatic cancer cells

doi: 10.1080/21655979.2022.2064206

Figure Lengend Snippet: The tRF-Leu-AAG promoted PC development by suppressing UPF1.

Article Snippet: Subsequently, the proteins were blocked with a quick-blocking solution for 30 min. UPF1 primary antibody (1:1000; 23,379-1-AP, Proteintech) or β-Actin primary antibody (1:5000; 60,008-1-Lg, Proteintech) was overnight incubated at 4°C.

Techniques:

Abnormal expression of UPF1 in psoriasis. (A) Paired sample t-test for 186 paired psoriasis samples from GEO datasets (GDS5392, GDS4602, GDS2518, GDS3539 and GDS4600). (B) Student's t-test for UPF1 mRNA levels in 10 psoriasis scales and 8 healthy cornified epidermal layer samples. Among the samples, four paired tissues were indicated with black lines. The UPF1 expression increased in the psoriasis group; 3/4 paired samples showed an upregulated expression level of UPF1 . (C) Mice received a daily topical IMQ cream to induce psoriasis-like skin. The expression of UPF1 increased in all four psoriasis-like tissues, as compared with the paired normal skin tissue samples. (D) The expression of UPF1 protein distributed throughout the cytoplasm in mouse skin was detected by IHC. The white arrows indicate positive UPF1 protein staining, and the red arrows indicate skin hyperkeratosis in IMQ-treated samples. (E) IHC staining was suantified using ImageJ software. The UPF1 protein decreased in 2/4 paired samples. (F) The mRNA expression of three NMD substrates, NIK , TBL2 and NAT9 , were higher in psoriasis-like skin compared with normal tissue. Data are presented as the mean ± SD from three independent experiments. * P<0.05 and *** P<0.001. n.s., not significant; UPF1 , up-frameshift suppressor 1 homolog; IMQ, imiquimod; IHC, immunohistochemistry; NMD, nonsense-mediated RNA decay; NIK , NF-κ-B-inducing kinase; TBL2 , transducing β like 2; NAT9 , N-Acetyltransferase 9; PS, psoriasis or IMQ-treated psoriasis-like skin; PN, normal cornified epidermal layer.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: Abnormal expression of UPF1 in psoriasis. (A) Paired sample t-test for 186 paired psoriasis samples from GEO datasets (GDS5392, GDS4602, GDS2518, GDS3539 and GDS4600). (B) Student's t-test for UPF1 mRNA levels in 10 psoriasis scales and 8 healthy cornified epidermal layer samples. Among the samples, four paired tissues were indicated with black lines. The UPF1 expression increased in the psoriasis group; 3/4 paired samples showed an upregulated expression level of UPF1 . (C) Mice received a daily topical IMQ cream to induce psoriasis-like skin. The expression of UPF1 increased in all four psoriasis-like tissues, as compared with the paired normal skin tissue samples. (D) The expression of UPF1 protein distributed throughout the cytoplasm in mouse skin was detected by IHC. The white arrows indicate positive UPF1 protein staining, and the red arrows indicate skin hyperkeratosis in IMQ-treated samples. (E) IHC staining was suantified using ImageJ software. The UPF1 protein decreased in 2/4 paired samples. (F) The mRNA expression of three NMD substrates, NIK , TBL2 and NAT9 , were higher in psoriasis-like skin compared with normal tissue. Data are presented as the mean ± SD from three independent experiments. * P<0.05 and *** P<0.001. n.s., not significant; UPF1 , up-frameshift suppressor 1 homolog; IMQ, imiquimod; IHC, immunohistochemistry; NMD, nonsense-mediated RNA decay; NIK , NF-κ-B-inducing kinase; TBL2 , transducing β like 2; NAT9 , N-Acetyltransferase 9; PS, psoriasis or IMQ-treated psoriasis-like skin; PN, normal cornified epidermal layer.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Staining, Immunohistochemistry, Software

Somatic mutations of UPF1 transcripts in patients with psoriasis vulgaris. (A) Sequencing traces corresponding to UPF1 insA (GenBank ID, MH183202) in patient A and UPF1 del (GenBank ID, MK089816) in patient B. The red frame indicates the insertion or shared sequence for splicing. The sequencing primers used were F4/R4 and F3/R3 from Table SIII. (B) Schematic representation of the aberrant mRNAs and proteins. The mutations and PTCs are indicated in red, while the EJs and Ters are presented in black in the mRNA schematic. The functional domains of UPF1 are shown in the protein schematic, and the gray lines indicate that the amino acid sequence was changed until the Ter. (C) Primers specific to UPF1 mutants or UPF1 WT were designed to detect the expression of UPF1 transcripts in the 2 patients. (D) The portions of UPF1 insA and UPF1 del were evaluated by reverse transcription-quantitative PCR. Mutated UPF1 transcripts were only detectable in the psoriasis scales. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. UPF1 , up-frameshift suppressor 1 homolog ; PTC, premature termination codon; EJ, exon-exon junction; Ter, termination codon; WT, wild-type; PS, psoriasis scales; PN, normal cornified epidermal layer; SQ, serine-glutamine; CH, calponin homology.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: Somatic mutations of UPF1 transcripts in patients with psoriasis vulgaris. (A) Sequencing traces corresponding to UPF1 insA (GenBank ID, MH183202) in patient A and UPF1 del (GenBank ID, MK089816) in patient B. The red frame indicates the insertion or shared sequence for splicing. The sequencing primers used were F4/R4 and F3/R3 from Table SIII. (B) Schematic representation of the aberrant mRNAs and proteins. The mutations and PTCs are indicated in red, while the EJs and Ters are presented in black in the mRNA schematic. The functional domains of UPF1 are shown in the protein schematic, and the gray lines indicate that the amino acid sequence was changed until the Ter. (C) Primers specific to UPF1 mutants or UPF1 WT were designed to detect the expression of UPF1 transcripts in the 2 patients. (D) The portions of UPF1 insA and UPF1 del were evaluated by reverse transcription-quantitative PCR. Mutated UPF1 transcripts were only detectable in the psoriasis scales. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. UPF1 , up-frameshift suppressor 1 homolog ; PTC, premature termination codon; EJ, exon-exon junction; Ter, termination codon; WT, wild-type; PS, psoriasis scales; PN, normal cornified epidermal layer; SQ, serine-glutamine; CH, calponin homology.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Sequencing, Functional Assay, Expressing, Real-time Polymerase Chain Reaction

Aberrant UPF1 transcripts disrupt the function of the NMD pathway. (A) The expression level of NIK , TBL2 and NAT9 showed an increase in psoriasis scales from patients A and B compared with PN samples. (B) 293T cells were transfected with the sh UPF1 construct, and with the UPF1 WT, UPF1 insA or UPF1 del vector 24 h later. UPF1 transcript expression was quantified 48 h after transfection. The expression levels of UPF1 insA and UPF1 del were lower than that of UPF1 WT following transfection with the respective constructs. (C) Western blot analysis was used to measure the expression of UPF1 protein isoforms, and GAPDH was used as a control. The UPF1 insA and UPF1 del generated minor protein bands, the predicted molecular weights are indicated. (D) mRNA expression levels of NMD substrates NIK , TBL2 and NAT9 were increased in UPF1 -depleted 293T cells, as detected by reverse transcription-quantitative PCR. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. n.s., not significant. UPF1 , up-frameshift suppressor 1 homolog; NMD, nonsense-mediated RNA decay; NIK , NF-κ-B-inducing kinase; TBL2 , transducing β like 2; NAT9 , N-Acetyltransferase 9; WT, wild-type; PS, psoriasis scales; PN, normal cornified epidermal layer.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: Aberrant UPF1 transcripts disrupt the function of the NMD pathway. (A) The expression level of NIK , TBL2 and NAT9 showed an increase in psoriasis scales from patients A and B compared with PN samples. (B) 293T cells were transfected with the sh UPF1 construct, and with the UPF1 WT, UPF1 insA or UPF1 del vector 24 h later. UPF1 transcript expression was quantified 48 h after transfection. The expression levels of UPF1 insA and UPF1 del were lower than that of UPF1 WT following transfection with the respective constructs. (C) Western blot analysis was used to measure the expression of UPF1 protein isoforms, and GAPDH was used as a control. The UPF1 insA and UPF1 del generated minor protein bands, the predicted molecular weights are indicated. (D) mRNA expression levels of NMD substrates NIK , TBL2 and NAT9 were increased in UPF1 -depleted 293T cells, as detected by reverse transcription-quantitative PCR. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. n.s., not significant. UPF1 , up-frameshift suppressor 1 homolog; NMD, nonsense-mediated RNA decay; NIK , NF-κ-B-inducing kinase; TBL2 , transducing β like 2; NAT9 , N-Acetyltransferase 9; WT, wild-type; PS, psoriasis scales; PN, normal cornified epidermal layer.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Western Blot, Generated, Real-time Polymerase Chain Reaction

Expression of AREG is regulated by the NMD pathway. (A) Schematic representation of AREG mRNA. The EJ in the 3′UTR was presumed to be an NMD pathway triggering feature. (B) AREG expression was increased in the psoriasis scales from patients A and B compared with their respective PN samples. (C) Keratinocytes were transfected with sh UPF1 or UPF1 WT constructs. The AREG mRNA level was regulated by UPF1 expression in HEKα and HaCaT cells, as quantified 48 h after transfection. (D) 293T cells were transfected with UPF1 expression vectors after transfection with sh UPF1 , and AREG mRNA levels were quantified 48 h after transfection. (E) AREG protein expression levels were measured by western blotting, and GAPDH was used as a control. AREG levels were only decreased by UPF1 WT. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. AREG , amphiregulin; NMD, nonsense-mediated RNA decay; EJ, exon-exon junction; WT, wild-type; UPF1 , up-frameshift suppressor 1 homolog; n.s., not significant; PS, psoriasis scales; PN, normal cornified epidermal layer.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: Expression of AREG is regulated by the NMD pathway. (A) Schematic representation of AREG mRNA. The EJ in the 3′UTR was presumed to be an NMD pathway triggering feature. (B) AREG expression was increased in the psoriasis scales from patients A and B compared with their respective PN samples. (C) Keratinocytes were transfected with sh UPF1 or UPF1 WT constructs. The AREG mRNA level was regulated by UPF1 expression in HEKα and HaCaT cells, as quantified 48 h after transfection. (D) 293T cells were transfected with UPF1 expression vectors after transfection with sh UPF1 , and AREG mRNA levels were quantified 48 h after transfection. (E) AREG protein expression levels were measured by western blotting, and GAPDH was used as a control. AREG levels were only decreased by UPF1 WT. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. AREG , amphiregulin; NMD, nonsense-mediated RNA decay; EJ, exon-exon junction; WT, wild-type; UPF1 , up-frameshift suppressor 1 homolog; n.s., not significant; PS, psoriasis scales; PN, normal cornified epidermal layer.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Transfection, Construct, Western Blot

AREG triggers the NMD pathway post-transcriptionally, depending on its 3′UTR. (A) 293T cells were treated with DRB, and AREG mRNA levels were evaluated by RT-qPCR at 0, 3, 5, 7 and 10 h. The t 1/2 represents the half-life of AREG mRNA. The AREG mRNA was stabilized by UPF1 depletion, as detected by RT-qPCR. (B) The structures represent the expression vector for AREG ORF and AREG -3′UTR based on pEGFP-N1. (C) 293T cells were transfected with sh UPF1 , and AREG expression vectors were transfected 24 h later. AREG mRNA levels were quantified 48 h after transfection. The 3′UTR region was required for NMD triggering by AREG . (D) Schematic representation of the dual-luciferase reporter system pEZX- AREG -3′UTR. (E) 293T cells co-transfected with pEZX- AREG -3′UTR plasmid together with sh UPF1 or UPF1 WT vector. Cells were collected and the FLuc activity was measured 48 h after transfection. RLuc activity was used as a control. The 3′UTR region was sufficient to trigger the NMD pathway. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. AREG , amphiregulin; NMD, nonsense-mediated RNA decay; DRB, 5,6-dichloro-1-β-D-ribofuranosylben zimidazole; UPF1 , up-frameshift suppressor 1 homolog; firefly luciferase, FLuc; Renilla luciferase, RLuc; ns, not significant; NC, normal cells; RT-qPCR, reverse transcription-quantitative PCR.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: AREG triggers the NMD pathway post-transcriptionally, depending on its 3′UTR. (A) 293T cells were treated with DRB, and AREG mRNA levels were evaluated by RT-qPCR at 0, 3, 5, 7 and 10 h. The t 1/2 represents the half-life of AREG mRNA. The AREG mRNA was stabilized by UPF1 depletion, as detected by RT-qPCR. (B) The structures represent the expression vector for AREG ORF and AREG -3′UTR based on pEGFP-N1. (C) 293T cells were transfected with sh UPF1 , and AREG expression vectors were transfected 24 h later. AREG mRNA levels were quantified 48 h after transfection. The 3′UTR region was required for NMD triggering by AREG . (D) Schematic representation of the dual-luciferase reporter system pEZX- AREG -3′UTR. (E) 293T cells co-transfected with pEZX- AREG -3′UTR plasmid together with sh UPF1 or UPF1 WT vector. Cells were collected and the FLuc activity was measured 48 h after transfection. RLuc activity was used as a control. The 3′UTR region was sufficient to trigger the NMD pathway. Data are presented as the mean ± SD from three independent experiments. *** P<0.001. AREG , amphiregulin; NMD, nonsense-mediated RNA decay; DRB, 5,6-dichloro-1-β-D-ribofuranosylben zimidazole; UPF1 , up-frameshift suppressor 1 homolog; firefly luciferase, FLuc; Renilla luciferase, RLuc; ns, not significant; NC, normal cells; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

UPF1 depletion-induced AREG disturbs keratinocyte homeostasis. (A) Cell counting kit-8 assays were performed to measure cell viability 48 h after UPF1 or AREG knockdown in HEKα and HaCaT cells. UPF1 and AREG depletion both decreased cell viability of keratinocytes. (B) The expression of Ki67 decreased in keratinocytes with UPF1 or AREG knockdown, while the expression of apoptosis-related gene BAD increased in both groups. (C) mRNA expression level of K5 , FLG and K10 was qualified by reverse transcription-quantitative PCR in keratinocytes. The expression levels of these differentiation makers decreased with UPF1 depletion and were rescued by supplementary AREG depletion. (D) Representative human keratinocyte scratch wounds with cells treated with the sh UPF1 construct, or sh UPF1 combined with si AREG . The wound images at 0 and 24 h are presented, and the epidermal sheet edge indicated by the yellow irregular curve was identified by ImageJ software automatically. UPF1 depletion induced the migration of keratinocytes by stabilization of AREG . (E) Quantitative analysis of the measured wound coverage at 0, 6, 12, 24 and 48 h. *** P<0.001 vs. NC group. (F) The motility markers SNAI1 , COX-2 and MMP1 and two chemokines associated with nuclear factor κB activation, CCL20 and CXCL1 , were identified at the mRNA level in keratinocyte cells. Expression levels of the migration makers and chemokines increased with UPF1 depletion and were rescued by supplementary AREG depletion. The results are presented as the mean ± SD from three independent experiments. *** P<0.001. UPF1 , up-frameshift suppressor 1 homolog; AREG , amphiregulin; BAD , BCL2 associated agonist of cell death; K5 , keratin-5; FLG , filaggrin; K10 , keratin-10; COX-2 , cyclooxygenase-2; MMP1 , matrix metalloproteinase 1; CCL20 , chemokine (C-C motif) ligand 20; CXCL1 , human recombinant GRO-alpha; NC, normal cells; Ki67 , proliferation marker protein Ki67; n.s., not significant.

Journal: International Journal of Molecular Medicine

Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG

doi: 10.3892/ijmm.2020.4487

Figure Lengend Snippet: UPF1 depletion-induced AREG disturbs keratinocyte homeostasis. (A) Cell counting kit-8 assays were performed to measure cell viability 48 h after UPF1 or AREG knockdown in HEKα and HaCaT cells. UPF1 and AREG depletion both decreased cell viability of keratinocytes. (B) The expression of Ki67 decreased in keratinocytes with UPF1 or AREG knockdown, while the expression of apoptosis-related gene BAD increased in both groups. (C) mRNA expression level of K5 , FLG and K10 was qualified by reverse transcription-quantitative PCR in keratinocytes. The expression levels of these differentiation makers decreased with UPF1 depletion and were rescued by supplementary AREG depletion. (D) Representative human keratinocyte scratch wounds with cells treated with the sh UPF1 construct, or sh UPF1 combined with si AREG . The wound images at 0 and 24 h are presented, and the epidermal sheet edge indicated by the yellow irregular curve was identified by ImageJ software automatically. UPF1 depletion induced the migration of keratinocytes by stabilization of AREG . (E) Quantitative analysis of the measured wound coverage at 0, 6, 12, 24 and 48 h. *** P<0.001 vs. NC group. (F) The motility markers SNAI1 , COX-2 and MMP1 and two chemokines associated with nuclear factor κB activation, CCL20 and CXCL1 , were identified at the mRNA level in keratinocyte cells. Expression levels of the migration makers and chemokines increased with UPF1 depletion and were rescued by supplementary AREG depletion. The results are presented as the mean ± SD from three independent experiments. *** P<0.001. UPF1 , up-frameshift suppressor 1 homolog; AREG , amphiregulin; BAD , BCL2 associated agonist of cell death; K5 , keratin-5; FLG , filaggrin; K10 , keratin-10; COX-2 , cyclooxygenase-2; MMP1 , matrix metalloproteinase 1; CCL20 , chemokine (C-C motif) ligand 20; CXCL1 , human recombinant GRO-alpha; NC, normal cells; Ki67 , proliferation marker protein Ki67; n.s., not significant.

Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).

Techniques: Cell Counting, Expressing, Real-time Polymerase Chain Reaction, Construct, Software, Migration, Activation Assay, Recombinant, Marker