hela tetoff cells  (TaKaRa)

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: Engineering FLuc for detecting a disulfide bond formation defect in the ER (A) (i) Rationale for detecting a disulfide bond formation detect in the ER using a prospective. FLuc-based reporter. (ii) Renilla luciferase (RLuc) serves as an internal control. (B) Development of a FLuc-based reporter. (C) Cysteine residues in FLucs and their close homologs. See Table S1 for the sources of amino acid sequences aligned using ClustalW. Residue numbers correspond to P. pyralis FLuc. Cysteines occurring more than twice at each position are listed. Proteins from Phengodidae (glowworm), Lampyridae (firefly), and Elateridae (click beetle) families are denoted in blue, black, and magenta, respectively. They belong to insect luciferases except for Agrypnus binodulus fatty acyl-CoA synthetase ( asterisk ). . (D) Effect of DTT treatment on FLuc activity in cells expressing Calr (17 aa)-FLuc-KDEL. HeLa cells, cultured in 6-well plates, were transfected with 95 ng of a plasmid expressing Calr (17 aa)-FLuc-KDEL and 5 ng of pRL-SV40 expressing RLuc. After 24 h, cells lysates were subjected to a dual luciferase assay. Where indicated, 1 mM DTT was added to the culture 2 h before harvest. The data represent the means ± SD from four independent samples. Statistical analysis utilized a two-tailed Student’s t test. p values are indicated in the graph. (E) Effects of cysteine substitutions on the activity of FLuc produced in the cytosol. HeLa cells, grown in 6-well plates, were transfected with 380 ng of a plasmid expressing the indicated FLuc variant in the cytosol, and 20 ng of pRL-SV40. After 24 h, cell lysates were subjected to a dual luciferase assay. The data represent the means ± SD from three independent samples. (F) Effect of introducing eight cysteine substitutions (V61C, L115C, V128C, S163C, A222C, I282C, S314C, and N463C) into Calr (17 aa)-FLuc-KDEL on Fluc activity. (G) Effect of DTT treatment on FLuc activity in cells expressing Calr (17 aa)-FLuc-KDEL with eight cysteine substitutions. Where indicated, 1 mM DTT was added to the culture 2 h before harvest. In (F) and (G), the data represent the means ± SD from four independent samples. Statistical analysis was performed as described in (D).

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Luciferase, Control, Residue, Activity Assay, Expressing, Cell Culture, Transfection, Plasmid Preparation, Two Tailed Test, Produced, Variant Assay

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: Reporter sensitivity is greatly improved by enhancing the ER translocation of FLuc∗ (A) Modifications implemented in the reporter. (B) Effect of incorporating tandemly duplicated N-glycosylation sites and a triple FLAG tag on the reporter’s response. HeLa cells expressing Calr (17 aa)-FLuc∗ were cultured, treated with or without 1 mM DTT for 2 h, lysed, and subjected to a dual luciferase assay. (C) Detection of the reporter proteins in HeLa cells (6.0 μg) expressing Calr (17 aa)-FLuc∗ or Calr (40 aa)-FLuc∗. Where indicated, lysates were treated with endoglycosidase H (Endo H) to identify the glycosylated ( Gly. ), and non-glycosylated ( Non-gly. ) forms of FLuc∗. Closed arrowheads, glycosylated FLuc∗; open arrowhead, non-glycosylated FLuc∗. (D) Effect of inserting the early mature region of calreticulin on FLuc activity. HeLa cells expressing either Calr (17 aa)-FLuc∗ or Calr (40 aa)-FLuc∗ were lysed and subjected to a dual luciferase assay. (E) Effect of DTT treatment on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗. Where indicated, 1 mM DTT was added to the culture 2 h before harvest. In (B–E), the data represent the means ± SD from four independent samples. Statistical analysis was performed using a two-tailed Student’s t test.

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Translocation Assay, FLAG-tag, Expressing, Cell Culture, Luciferase, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: Modulating the Ero1α activity has significant impacts on the redox environment of the ER (A) Structure of Bisphenol A (BPA) ( left ), an inhibitor of the Ero1α-dependent oxidation of PDI ( right ). . (B) Effect of BPA treatment on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗. Before assay, the cultures were treated with the indicated concentrations of BPA for 2.5 h. The data represent the means ± SD from four independent samples. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. (C) Effect of BPA treatment on the oxidative state of FLuc∗. HeLa cells expressing Calr (40 aa)-FLuc∗ were treated with ( BPA ) or without ( Mock ) 200 μM BPA for 2.5 h, subjected to alkylation with Mal-PEG 2000 ( Mal-PEG 2K ), separated by reducing SDS-PAGE, and detected with anti-FLAG antibody. Lane 1, mock-transfected cells. Lane 2, no alkylation. Lane 3, proteins fully reduced in a buffer (100 mM Tris-HCl [pH 8.0], 100 mM DTT and 1% SDS) before alkylation of free cysteines with Mal-PEG 2000. An asterisk on lane 4 indicates the fully reduced form of FLuc∗ appearing upon treatment of cells with BPA. (D) Effect of overexpressing Ero1α and its hyperactive mutant on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗. (E) Effect of overexpressing Ero1α or its hyperactive mutant on the oxidative states of indicated FLuc∗ reporter proteins assessed as described in panel C. (F) Structures of Calr (40 aa)-FLuc∗ and Calr (40 aa)-FLuc∗ (−2 Cys) reporters. (G) Effect of overexpressing Ero1α and its hyperactive mutant on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗ (−2 Cys). For comparison, the activities were normalized with that of cells expressing Calr (40 aa)-FLuc∗. In (D) and (G), the data represent the means ± SD from five independent samples. Statistical analysis utilized one-way ANOVA followed by Tukey’s test.

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Activity Assay, Expressing, SDS Page, Transfection, Mutagenesis, Comparison

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: Detection of a protein targeting defect using a simple enzyme assay (A) Structure of IL6R (40 aa)-FLuc∗. (B) Confirmation of siRNA-mediated knockdown of SRP54. HeLa cells, cultured in 6-well plate, were transfected with 20 nM of either a control siRNA ( Mock ), or SRP54-specific siRNAs #2, or #3 using Lipofectamine RNAiMAX. After 24 h, cells were further transfected with 95 ng of a plasmid expressing ILR6R (40 aa)-FLuc∗, 5 ng of pSV40-RLuc, and 100 ng of an empty vector (pcDNA3.1 + ) ( Knockdown ) or a rescue plasmid expressing siRNA-resistant SRP54 ( Rescue ) using Effectene. After additional 24 h, cell lysates (3.5 μg) were subjected to reducing SDS-PAGE, and immunoblotting with an antibody against SRP54 ( upper ) or GAPDH ( lower ) for knockdown confirmation. (C and E) Detection of the reporter protein in cell lysates (4.0 μg) obtained as described in (B). Cell lysates were treated with or without Peptide - N -Glycosidase F (PNGase F) to identify the glycosylated ( Gly. ) and non-glycosylated ( Non-gly .) forms of FLuc∗. Closed arrowheads, glycosylated FLuc∗; open arrowheads, non-glycosylated FLuc∗. Note that SRP54 knockdown resulted in the accumulation of FLuc∗ that failed to enter the ER (open arrowheads in C). This protein export defect was rescued by co-transfection of a plasmid expressing siRNA-resistant SRP54 as indicated by the disappearance of the non-glycosylated form (E). (D) Effect of SRP54 knockdown on FLuc activity in cells expressing ILR6R (40 aa)-FLuc∗ (see B for the knockdown experiments). The data represent the means ± SD from three independent samples. Statistical analysis utilized one-way ANOVA followed by Tukey’s test. “ns” denotes not significant ( p > 0.05).

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Enzymatic Assay, Knockdown, Cell Culture, Transfection, Control, Plasmid Preparation, Expressing, SDS Page, Western Blot, Cotransfection, Activity Assay

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: Creating an assay to evaluate the ability of a small molecule to inhibit virus receptor biosynthesis (A) Structure of CADA. . (B) CADA binds to the signal sequence of human CD4, leading to the inhibition of CD4 biosynthesis. . (C) Structures of FLuc∗ reporters, CD4 (40 aa)-FLuc∗ and bPRL (40 aa)-FLuc∗. (D) Effect of CADA treatment on FLuc activity of HeLa cells expressing CD4 (40 aa)-FLuc∗. HeLa cells were cultured in 6-well plates, transfected with 38 ng of a plasmid expressing CD4 (40 aa)-FLuc∗ and 2 ng of pSV40-RLuc. After 20 h, cells were treated with or without 2 μM CADA for 4 h, lysed, and subjected to a dual luciferase assay. (E) Effect of CADA treatment on the localization of CD4 (40 aa)-FLuc∗ or bPRL (40 aa)-FLuc∗. Cells lysates (7.5 μg) obtained as described in (D) and (F) were treated with or without PNGase F and subjected to reducing SDS-PAGE and immunoblotting with anti-FLAG antibody to identify the glycosylated ( Gly. ) and non-glycosylated ( Non-gly. ) forms of FLuc∗. Closed arrowheads, glycosylated FLuc∗; open arrowheads, non-glycosylated FLuc∗. (F) Effects of CADA treatment on FLuc activity of bPRL (40 aa)-FLuc∗ expressed in HeLa cells. The experiments were conducted as described in (D), except that bPRL (40 aa)-FLuc∗ was used as an FLuc∗ reporter. In (D) and (F), the data represent the means ± SD from three independent samples. Statistical analysis was performed using a two-tailed Student’s t test.

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Virus, Sequencing, Inhibition, Activity Assay, Expressing, Cell Culture, Transfection, Plasmid Preparation, Luciferase, SDS Page, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet: LMF1 participates in the redox control of the ER (A) Model illustrating the putative function of LMF1. (B) Effect of overexpressing LMF1 on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗. The data represent means ± SD from four independent samples. Statistical analysis was performed using a two-tailed Student’s t test. (C) Effect of siRNA-mediated knockdown of LMF1 on FLuc activity of HeLa cells expressing Calr (40 aa)-FLuc∗. HeLa cells were initially transfected with 20 nM of control ( Mock ) or LMF1-specific siRNA ( LMF1-KD ) using Lipofectamine RNAiMAX and grown in 6-well plates. After 24 h, cells were transfected with 38 ng of a plasmid expressing Calr (40 aa)-FLuc∗, and 2 ng of pRL-SV40. Where indicated, LMF1-knockdown cells were co-transfected with an indicated amount of a plasmid expressing an siRNA-resistant PA-LMF1 ( Rescue ). After 24 h, cells lysates were subjected to a dual luciferase assay. The data represent the means ± SD from four independent samples. Statistical analysis utilized one-way ANOVA followed by Tukey’s test. Note that PA-LMF1 is an LMF1 derivative fused with a PA tag (12 amino acid residues) at its N terminus for detection purposes. Expression of PA-LMF1 in the knockdown cells at levels comparable to endogenous LMF1 restored FLuc∗ activity ( Figures 6 and ), confirming that the decrease in FLuc∗ activity observed in LMF1-knockdown cells was not caused by secondary effects of the siRNA used. (D) Effect of LMF1 overexpression on the oxidative states of FLuc∗ assessed using Mal-PEG 2000 (Mal-PEG 2K) as described in Figure 5 C. (E) Effect of LMF1 overexpression on the N-glycosylation status of the reporter protein assessed as described in Figure 3 C and 3E.

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Control, Activity Assay, Expressing, Two Tailed Test, Knockdown, Transfection, Plasmid Preparation, Luciferase, Over Expression

Journal: iScience

Article Title: Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

doi: 10.1016/j.isci.2024.111189

Figure Lengend Snippet:

Article Snippet: HeLa cells (HeLa Tet-off; Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) Nichirei Biosciences) at 37°C in 5% CO 2 air.

Techniques: Virus, Recombinant, Mutagenesis, Western Blot, Control, Software

Journal: iScience

Article Title: RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading

doi: 10.1016/j.isci.2023.108216

Figure Lengend Snippet:

Article Snippet: HeLa , TakaraBio , Cat. No. 631156.

Techniques: Virus, Expressing, Recombinant, Modification, Saline, Bicinchoninic Acid Protein Assay, shRNA, Software