prdx6  (Proteintech)

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 -depleted SRA-hLECs were highly vulnerable to H 2 O 2 - or UVB-induced cell damage: ( A ) photomicrograph representing stably infected SRA-hLECs with LV ShControl and LV ShPrdx6 ; ( B , C ) expression assays showing the Prdx6 -depleted SRA-hLECs. Total protein and RNA were isolated from stably selected SRA-hLECs infected with LV ShControl and LV ShPrdx6 . Prdx6 protein and mRNA expression were measured using a specific probe. All histograms are presented as mean ± SD values derived from three separate experiments ( n = 3). LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( D ) Prdx6 depletion enhanced the expression of Nlrp3 inflammasome and its target genes in SRA-hLECs. Total protein was isolated from LV ShControl or LV ShPrdx6 infected SRA-hLECs and subjected to Western blot, as indicated in the figure. β-actin/tubulin was used as a loading control; ( E – H ) depletion of Prdx6 showed reduced cell viability and increased accumulation of ROS under oxidative stress. SRA-hLECs infected with LV ShControl and LV ShPrdx6 were distributed into a 96-well plate, 12–14 h later, these cells were exposed to different concentrations of H 2 O 2 or UVB. 24 h later, cell viability ( E , G ); and ROS levels were examined at 6 h ( F , H ). All histograms are presented as mean ± SD values derived from three independent experiments ( n = 3). Statistical significance: LV ShControl vs. LV ShPrdx6 with H 2 O 2 -treatement, and LV ShControl vs. LV ShPrdx6 with UVB-treatment; # p < 0.05, * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Stable Transfection, Infection, Expressing, Isolation, Derivative Assay, Western Blot, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 -depleted SRA-hLECs showed higher sensitivity to H 2 O 2 or UVB-induced oxidative stress and bore Nlrp3 inflammasome and its inflammatory signaling: ( A , D ) Prdx6 -depleted SRA-hLECs and cells facing oxidative stress showed elevated levels of Caspase-1. LV ShControl and LV ShPrdx6 infected SRA-hLECs were cultured and exposed to H 2 O 2 or UVB stress, as indicated in the figure. Forty-eight hours later, a cellular extract was prepared, and an equal amount of cellular extract was used to quantify the levels of Caspase-1; ( B , C , E , F ) mature IL-1β and IL-18 secretion were increased in Prdx6 -depleted SRA-hLECs and the cells facing oxidative stress. LV ShControl and LV ShPrdx6 -infected SRA-hLECs were cultured and exposed to H 2 O 2 or UVB stress. After 48 h, the supernatant(s) were collected and IL-1β ( B , E ) and IL-18 ( C , F ) levels were measured; and ( G ) total RNA was isolated from LV ShControl and LV ShPrdx6 SRA-hLECs facing oxidative stress and subjected to RT-qPCR to assess the expression of Nlrp3 inflammasome and its downstream genes, mRNA, using their specific primers, as indicated. Data represent the mean ± SD values calculated from four independent experiments ( n = 4). Significant values: 0 vs. 100 µM H 2 O 2 in LV ShControl, 0 vs. 100 µM H 2 O 2 in LV ShPrdx6, and LV ShControl vs. LV ShPrdx6 treated with H 2 O 2 ; 0 vs. 200 J/m 2 UVB in LV ShControl, 0 vs. 200 J/m 2 in LV ShPrdx6 , and LV ShControl vs. LV ShPrdx6 exposed to UVB; # p < 0.05, * p < 0.001 as indicated in figures.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection, Cell Culture, Isolation, Quantitative RT-PCR, Expressing

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 knockdown in mLECs led to the increased oxidative load, which was directly linked to increased expression of Nlrp3, ASC, Caspase-1, pro-inflammatory cytokines, and GSDMD: ( A ) photomicrograph showing stably infected mLECs expressing either LV ShControl or LV ShPrdx6 ; ( B ) Prdx6 -depleted mLECs demonstrated increased levels of ROS. ROS intensity was observed using CellROX deep red reagent. Data represent the mean ± SD values from three independent experiments ( n = 3). LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C ) Prdx6 -depleted mLECs showed elevated levels of Caspase-1. LV ShControl and LV Sh Prdx6-infected mLECs were cultured, and 48 h later, cellular extract was prepared. Caspase-1 activity was measured and compared using an equal amount of cellular extract; ( D , E ) Prdx6 -depleted mLECs displayed increased secretion of mature IL-1β and IL-18. LV ShControl and LV ShPrdx6 -infected mLECs were cultured, and 48 h later, supernatant was collected to measure IL-1β ( D ) and IL-18 ( E ) levels; and ( F , G ) total protein and RNA were isolated from mLECs stably infected with LV ShControl and LV ShPrdx6 and subjected to Western blot and mRNA analyses to examine the expression of Nlrp3, Caspase-1, ASC, inflammatory cytokines IL-1β and IL-18, and pyroptosis executor GSDMD, using their specific probes, as indicated. The data represent the mean ± SD values derived from four independent experiments ( n = 4). LV ShControl vs. LV ShPrdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Expressing, Stable Transfection, Infection, Cell Culture, Activity Assay, Isolation, Western Blot, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 delivery subsided the aberrant expression and activation of Nlrp3, bioactive Caspase-1, IL-1β, IL-18, and GSDMD in Prdx6 −/− mLECs: ( A ) total protein was isolated from Prdx6 +/+ and Prdx6 −/− mLECs with or without TAT-HA-Prdx6 and subjected to Western blot, as shown in the figure; ( B ) TAT-HA-Prdx6 delivery to Prdx6 −/− mLECs displayed reduced levels of Caspase-1. Cellular extract was prepared and Caspase-1 status was measured; ( C , D ) increased secretion of bioactive IL-1β or IL-18 observed in Prdx6 −/− mLECs was inhibited by Prdx6 delivery. Prdx6 +/+ and Prdx6 −/− mLECs with or without TAT-HA-Prdx6 were cultured. Forty-eight hours later, supernatant(s) were collected and measured for IL-1β ( C ) and IL-18 ( D ) levels; and ( E ) total RNA was isolated from Prdx6 +/+ and Prdx6 −/− mLECs transduced with or without TAT-HA-Prdx6 and subjected to qPCR, as indicated. The data represent the mean ± SD values of four independent replicates ( n = 4). Prdx6 +/+ vs. Prdx6 −/− and Prdx6 −/− vs. Prdx6 −/− with TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Activation Assay, Isolation, Western Blot, Cell Culture, Transduction

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 delivery to Prdx6 -deficient mLECs suppressed elevated ROS levels and enhanced cell viability in response to UVB- or H 2 O 2 -induced cell death: ( A , C ) TAT-HA-Prdx6 protein delivery resisted against UVB- or H 2 O 2 -induced cell death in Prdx6 −/− mLECs. Prdx6 +/+ and Prdx6 −/− mLECs cells were cultured in 96-well plates. Prdx6 −/− mLECs were transduced with TAT-HA-Prdx6 (10 μg/mL) protein. 3 h later, these cells were exposed to different concentrations of UVB or H 2 O 2 . Twenty-four hours later, cell viability was measured, and ( B , D ) TAT-HA-Prdx6 protein delivery inhibited the elevated ROS level induced by UVB or H 2 O 2 exposure in Prdx6 −/− mLECs. Prdx6 +/+ and Prdx6 −/− mLECs were cultured in 96-well plates. Prdx6 −/− mLECs were transduced with TAT-HA-Prdx6 (10 μg/mL) protein. After 3 h, these cells were exposed to different concentrations of UVB or H 2 O 2 and subjected to ROS measurement at 6 h. The data reflect mean ± SD values derived from four independent experiments ( n = 4). 0 vs. 80, 160 and 320 J/m 2 UVB treatment, and Prdx6 +/+ vs. Prdx6 −/− and Prdx6 −/− vs. Prdx6 −/− supplemented with TAT-HA-Prdx6 exposed to UVB; 0 vs. 25, 50 and 100 µM H 2 O 2 treatment, and Prdx6 −/− vs. Prdx6 −/− with TAT-HA-Prdx6 under H 2 O 2 treatment; # p < 0.05, * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Cell Culture, Transduction, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 attenuated Nlrp3 inflammasome and its inflammatory genes ASC, Caspase-1, IL-1β, and IL-18 secretion by inhibiting ROS accumulation in Prdx6 −/− mLECs: ( A ) photomicrograph representing stably infected Prdx6 −/− mLECs with LV GFP-Control or GFP-tagged Prdx6 activation linked with lentiviral (LV GFP-Prdx6); ( B ) total protein was isolated from LV-GFP-Control or LV GFP-Prdx6 infected Prdx6 −/− mLECs and subjected to Western blot, as indicated in the figure; ( C ) elevated levels of Caspase-1 were inhibited by Prdx6 overexpression in Prdx6 −/− mLECs. LV GFP-Control and LV GFP-Prdx6-infected Prdx6 −/− mLECs were cultured and 48 h later, cellular extract was prepared. Caspase-1 levels were measured using an equal amount of cellular extract; ( D , E ) Prdx6 overexpression normalized the increased secretion of mature IL-1β and IL-18 in Prdx6 −/− mLECs. LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs were cultured, and supernatant was collected after 48 h to evaluate IL-1β ( D ) and IL-18 ( E ) levels; and ( F ) total RNA was isolated from LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs and subjected to q-PCR to assess the expression of Nlrp3, Caspase-1, ASC, cytokines, such as IL-1β and IL-18, and pyroptosis executor GSDMD mRNA expression using their specific probes, as indicated. Results represent the mean ± SD values from three independent replicates ( n = 3). LV GFP-Control vs. LV GFP-Prdx6 in Prdx6 −/− mLECs; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Stable Transfection, Infection, Control, Activation Assay, Isolation, Western Blot, Over Expression, Cell Culture, Expressing

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A , B ) Prdx6 delivery abated the H 2 O 2 - or UVB-driven oxidative load and increased expression of Nlrp3 inflammasome-related inflammatory components in SRA-hLECs. SRA-hLECs were pretreated with TAT-HA-Prdx6 for 3 h and followed by H 2 O 2 ( A ) or UVB ( B ) exposure for 24 h in serum-free DMEM medium and thereafter treated for 3 days. Total RNA was isolated to assess the expression status of Nlrp3, ASC, Caspase-1, IL-1β, IL-18, and GSDMD mRNA expression using qPCR with their specific probes, as indicated in the figure. β-actin was used as a control. The data represent the mean ± SD values calculated from three independent experiments ( n = 3). Statistical analyses: 0 vs. 100µM H 2 O 2 treatment, and H 2 O 2 vs. H 2 O 2 plus TAT-HA-Prdx6; 0 vs. 200 J/m 2 UVB treatment, and UVB vs. UVB plus TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Isolation, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 regulation of the transcriptional activity of Nlrp3: ( A , B ) Prdx6 -depleted LECs displayed increased Nlrp3 transcription activity. Redox-active Prdx6 -depleted SRA-hLECs ( A ) or mLECs ( B ) were transiently transfected with pGL4-mNlrp3 (−1866/+166). Forty-eight hours later, luciferase activity was measured. The data are shown as mean ± SD from three different experiments. LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C ) UVB-exposed mLECs showed a significant increase in Nlrp3 transcription. mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166); 24 h later, these cells were exposed to different concentrations of UVB. Luciferase activity was measured after 24 h of UVB exposure. The results are shown as mean ± SD from four independent experiments ( n = 4). 0 vs. 80, 160, 320 J/m 2 UVB treatment; # p < 0.05, * p < 0.001; ( D – F ) Prdx6 delivery suppressed the elevated Nlrp3 transcription in redox-active Prdx6 −/− mLECs and mLECs facing oxidative stress. Prdx6 −/− mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166). Twenty-four hours later, these transfectants were transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h and luciferase activity was measured ( D ). Control vs. TAT-HA-Prdx6; * p < 0.001. mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166), and 24 h later, these cells were pretreated with TAT-HA-Prdx6 protein for 3 h, followed by H 2 O 2 or LPS ( E ) or UVB ( F ) exposure, as indicated in the figure; then, Luciferase activity was measured after 24 h. Results reflect mean ± SD values derived from four separate experiments ( n = 4). 0 vs. H 2 O 2 , 0 vs. LPS, H 2 O 2 vs. H 2 O 2 + TAT-HA-Prdx6, LPS vs. LPS + TAT-HA-Prdx6; 0 vs. UVB and UVB vs. UVB + TAT-HA-Prdx6; * p < 0.001; ( G – I ) elevated Nlrp3 transcription was inhibited by Prdx6 overexpression in Prdx6 −/− mLECs and mLECs under oxidative stress conditions. Prdx6 −/− mLECs and mLECs infected with LV GFP-Control and/or LV GFP-Prdx6 were transfected with pCAT-mNlrp3 (−1866/+166). 24 h later, these transfected mLECs were exposed to H 2 O 2 or LPS ( H ) or UVB ( I ). CAT activity was measured at 48 h. Prdx6 −/− mLECs ( G ), and mLECs exposed to 24 h of H 2 O 2 or LPS ( H ), or UVB ( I ). All measurements are shown as mean ± SD from four separate experiments ( n = 4). LV-GFP-Vector vs. LV-GFP-Prdx6, Prdx6 +/+ vs. Prdx6 −/− mLECs, 0 vs. H 2 O 2 or LPS and/or UVB, LV-GFP-Vector (H 2 O 2 or LPS or UVB) vs. LV-GFP-Prdx6 (H 2 O 2 or LPS or UVB); * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Transfection, Luciferase, Transduction, Control, Derivative Assay, Over Expression, Infection, Plasmid Preparation

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 overexpression resulted in a reduction in NF- ĸ B and phosphorylated I ĸ Bα expression in Prdx6 -deficient LECs: ( A , B , D , E ) Prdx6 −/− mLECs and/or Prdx6 -depleted SRA-hLECs showed increased expression of NF- ĸ B and phosphorylated I ĸ Bα. Total RNA and nuclear and cytosol protein extracts were isolated and subjected to RT-qPCR ( A , B ) and Western analysis ( D , E ) of NF- ĸ B, I ĸ Bα and phospho-I ĸ Bα with specific probes, respectively. Tubulin was used as an internal loading control. Values are expressed as mean ± SD from four separate experiments ( n = 4). Prdx6 +/+ vs. Prdx6 −/− mLECs, LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C , F ) increased NF- ĸ B and phospho-I ĸ Bα in Prdx6 −/− mLECs were suppressed by Prdx6 overexpression. Total RNA, as well as nuclear and cytosol protein extracts, were isolated and subjected to RT-qPCR ( C ) and Western analysis ( F ) of NF- ĸ B and phospho-I ĸ Bα with specific probes, respectively. Tubulin was used as an internal control. Values are expressed as mean ± SD from four independent experiments ( n = 4). LV GFP-control vs. LV-GFP-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Over Expression, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Redox-active transcription factor NF- ĸ B was activated in Prdx6 -deficient LECs and LECs facing oxidative stress but its activity was normalized by Prdx6 overexpression: ( A ) HIV-1 LTR-CAT and its NF- ĸ B mutant construct; ( B , C ) Prdx6 deficiency displayed an active form of redox-active transcription factor NF- ĸ B. Prdx6 +/+ , Prdx6 −/− , and Prdx6 -depleted SRA-hLECs were transiently transfected with HIV-1 LTR-CAT; 48 h later, NF- ĸ B transcription activity was measured. The data represent the mean ± SD, from four independent repeats ( n = 4). Prdx6 +/+ vs. Prdx6 −/− and LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( D ) SRA-hLECs were transfected with HIV-1 LTR-CAT and its NF- ĸ B mutant construct; 24 h after post-transfection, these cells were exposed to H 2 O 2 or LPS or UVB, as indicated in the figure. The transactivation assay was performed after 24 h of oxidative stress exposure and NF- ĸ B activity was shown; and ( E ) LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs were transfected with HIV-1 LTR-CAT, and NF- ĸ B transcription activity was measured at 48 h. Each value represents the mean ± SD from four independent repeats. Untreated vs. H 2 O 2 /LPS/UVB treatment and LV-GFP-Control vs. LV-GFP-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Over Expression, Mutagenesis, Construct, Transfection, Transactivation Assay, Control, Infection

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A – C ) In silico analysis revealed the presence of NF- ĸ B binding sites in Klf9 promoter: aged mLECs and Prdx6 −/− mLECs, a model for aging, displayed increased Klf9 promoter activity; ( A ) schematic diagram showing the 5′-construct of mKlf9 promoter, spanning −5856/+71 bps linked to CAT reporter gene, with the position of NF- ĸ B binding sequence; ( B , C ) mLECs were transiently transfected with the Klf9 promoter, along with the GFP vector. Forty-eight hours post transfection, CAT activity was examined. The data show the mean ± SD from four independent experiments ( n = 4). 2M vs. 17M-old and Prdx6 +/+ vs. Prdx6 −/− mLECs; * p < 0.001; ( D ) Prdx6 −/− mLECs infected with LV GFP-Control or LV GFP-Prdx6 were transfected with Klf9 promoter, and CAT activity was measured at 48 h. The results represent the mean ± SD from four separate replicates ( n = 4). LV GFP-Control vs. LV-GFP-Prdx6; * p < 0.001; and ( E , F ) elevated Klf9 transcriptional activity in aging mLECs or mLECs facing oxidative stress was inhibited by TAT-HA-Prdx6. Aging mLECs and mLECs were transiently transfected with Klf9 promoter construct along with GFP plasmid; 24 h later, aging mLECs were transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h, and mLECs were transduced with TAT-HA-Prdx6 (10 µg/mL) for 3 h, followed by H 2 O 2 and/or UVB exposure for 24 h. CAT activity was measured after 24 h. Transfection efficiency was normalized using GFP O.D, measured at Ex485/Em530 nm. The results represent the mean ± SD from four independent experiments ( n = 4). 2M vs. 8M-old and 19M-old, control vs. TAT-HA-Prdx6, untreated control vs. H 2 O 2 vs. H 2 O 2 + TAT-HA-Prdx6, and untreated control vs. UVB vs. UVB + TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: In Silico, Binding Assay, Activity Assay, Construct, Sequencing, Transfection, Plasmid Preparation, Infection, Control, Transduction

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A – D ) Prdx6 −/− (a model for aging) and aging mLECs showed increased levels of TXNIP with reduced antioxidant gene TRX. Elevated TXNIP levels in Prdx6 −/− and aging mLECs were related to TRX repression. Total protein and RNA were isolated from Prdx6 +/+ , Prdx6 −/− mLECs and aging mLECs and subjected to Western blot and RT-qPCR analysis. Data represent the mean ± SD values of six independent experiments ( n = 6). Prdx6 +/+ vs. Prdx6 −/− mLECs samples; young (2-month-old) vs. Old (19-month-old) mLECs samples; * p < 0.001; ( E , F ) Prdx6 delivery inhibited the elevated TXNIP expression and enhanced the TRX expression in Prdx6 −/− mLECs. Total RNA was isolated from LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs (E) and/or Prdx6 −/− mLECs transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h. TXNIP and TRX mRNA expression levels were examined by qPCR. The data show the mean ± SD values derived from six separate experiments ( n = 6). Prdx6 +/+ vs. Prdx6 −/− mLECs and Prdx6 −/− control vs. Prdx6 −/− with TAT-HA-Prdx6 samples; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Isolation, Western Blot, Quantitative RT-PCR, Expressing, Control, Infection, Transduction, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6’s role in the protection and survival of lens epithelial cells in aging and oxidant-induced oxidative stress. During aging and oxidative stress, the loss of antioxidant defense in LECs leads to excessive ROS accumulation, triggering Nlrp3 inflammasome activation through NF- κ B and Klf9 signaling. Activated Klf9 further amplifies oxidative stress by increasing ROS levels and suppressing antioxidant gene expression, while NF- κ B directly enhances Klf9 transcription, together promoting sustained inflammasome activation. Aberrantly activated Nlrp3 assembles with ASC and proCaspase-1, resulting in the activation of Caspase-1, maturation of IL-1β/IL-18, cleavage of GSDMD, and induction of pyroptotic cell death. The delivery of Prdx6 suppresses NF- κ B and Klf9 activation, reduces ROS accumulation, restores antioxidant defense, and inhibits Nlrp3 inflammasome activation, thereby promoting healthy ocular aging and delaying cataract formation.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activation Assay, Gene Expression

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 -depleted SRA-hLECs were highly vulnerable to H 2 O 2 - or UVB-induced cell damage: ( A ) photomicrograph representing stably infected SRA-hLECs with LV ShControl and LV ShPrdx6 ; ( B , C ) expression assays showing the Prdx6 -depleted SRA-hLECs. Total protein and RNA were isolated from stably selected SRA-hLECs infected with LV ShControl and LV ShPrdx6 . Prdx6 protein and mRNA expression were measured using a specific probe. All histograms are presented as mean ± SD values derived from three separate experiments ( n = 3). LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( D ) Prdx6 depletion enhanced the expression of Nlrp3 inflammasome and its target genes in SRA-hLECs. Total protein was isolated from LV ShControl or LV ShPrdx6 infected SRA-hLECs and subjected to Western blot, as indicated in the figure. β-actin/tubulin was used as a loading control; ( E – H ) depletion of Prdx6 showed reduced cell viability and increased accumulation of ROS under oxidative stress. SRA-hLECs infected with LV ShControl and LV ShPrdx6 were distributed into a 96-well plate, 12–14 h later, these cells were exposed to different concentrations of H 2 O 2 or UVB. 24 h later, cell viability ( E , G ); and ROS levels were examined at 6 h ( F , H ). All histograms are presented as mean ± SD values derived from three independent experiments ( n = 3). Statistical significance: LV ShControl vs. LV ShPrdx6 with H 2 O 2 -treatement, and LV ShControl vs. LV ShPrdx6 with UVB-treatment; # p < 0.05, * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Stable Transfection, Infection, Expressing, Isolation, Derivative Assay, Western Blot, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 -depleted SRA-hLECs showed higher sensitivity to H 2 O 2 or UVB-induced oxidative stress and bore Nlrp3 inflammasome and its inflammatory signaling: ( A , D ) Prdx6 -depleted SRA-hLECs and cells facing oxidative stress showed elevated levels of Caspase-1. LV ShControl and LV ShPrdx6 infected SRA-hLECs were cultured and exposed to H 2 O 2 or UVB stress, as indicated in the figure. Forty-eight hours later, a cellular extract was prepared, and an equal amount of cellular extract was used to quantify the levels of Caspase-1; ( B , C , E , F ) mature IL-1β and IL-18 secretion were increased in Prdx6 -depleted SRA-hLECs and the cells facing oxidative stress. LV ShControl and LV ShPrdx6 -infected SRA-hLECs were cultured and exposed to H 2 O 2 or UVB stress. After 48 h, the supernatant(s) were collected and IL-1β ( B , E ) and IL-18 ( C , F ) levels were measured; and ( G ) total RNA was isolated from LV ShControl and LV ShPrdx6 SRA-hLECs facing oxidative stress and subjected to RT-qPCR to assess the expression of Nlrp3 inflammasome and its downstream genes, mRNA, using their specific primers, as indicated. Data represent the mean ± SD values calculated from four independent experiments ( n = 4). Significant values: 0 vs. 100 µM H 2 O 2 in LV ShControl, 0 vs. 100 µM H 2 O 2 in LV ShPrdx6, and LV ShControl vs. LV ShPrdx6 treated with H 2 O 2 ; 0 vs. 200 J/m 2 UVB in LV ShControl, 0 vs. 200 J/m 2 in LV ShPrdx6 , and LV ShControl vs. LV ShPrdx6 exposed to UVB; # p < 0.05, * p < 0.001 as indicated in figures.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection, Cell Culture, Isolation, Quantitative RT-PCR, Expressing

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 knockdown in mLECs led to the increased oxidative load, which was directly linked to increased expression of Nlrp3, ASC, Caspase-1, pro-inflammatory cytokines, and GSDMD: ( A ) photomicrograph showing stably infected mLECs expressing either LV ShControl or LV ShPrdx6 ; ( B ) Prdx6 -depleted mLECs demonstrated increased levels of ROS. ROS intensity was observed using CellROX deep red reagent. Data represent the mean ± SD values from three independent experiments ( n = 3). LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C ) Prdx6 -depleted mLECs showed elevated levels of Caspase-1. LV ShControl and LV Sh Prdx6-infected mLECs were cultured, and 48 h later, cellular extract was prepared. Caspase-1 activity was measured and compared using an equal amount of cellular extract; ( D , E ) Prdx6 -depleted mLECs displayed increased secretion of mature IL-1β and IL-18. LV ShControl and LV ShPrdx6 -infected mLECs were cultured, and 48 h later, supernatant was collected to measure IL-1β ( D ) and IL-18 ( E ) levels; and ( F , G ) total protein and RNA were isolated from mLECs stably infected with LV ShControl and LV ShPrdx6 and subjected to Western blot and mRNA analyses to examine the expression of Nlrp3, Caspase-1, ASC, inflammatory cytokines IL-1β and IL-18, and pyroptosis executor GSDMD, using their specific probes, as indicated. The data represent the mean ± SD values derived from four independent experiments ( n = 4). LV ShControl vs. LV ShPrdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Expressing, Stable Transfection, Infection, Cell Culture, Activity Assay, Isolation, Western Blot, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 delivery subsided the aberrant expression and activation of Nlrp3, bioactive Caspase-1, IL-1β, IL-18, and GSDMD in Prdx6 −/− mLECs: ( A ) total protein was isolated from Prdx6 +/+ and Prdx6 −/− mLECs with or without TAT-HA-Prdx6 and subjected to Western blot, as shown in the figure; ( B ) TAT-HA-Prdx6 delivery to Prdx6 −/− mLECs displayed reduced levels of Caspase-1. Cellular extract was prepared and Caspase-1 status was measured; ( C , D ) increased secretion of bioactive IL-1β or IL-18 observed in Prdx6 −/− mLECs was inhibited by Prdx6 delivery. Prdx6 +/+ and Prdx6 −/− mLECs with or without TAT-HA-Prdx6 were cultured. Forty-eight hours later, supernatant(s) were collected and measured for IL-1β ( C ) and IL-18 ( D ) levels; and ( E ) total RNA was isolated from Prdx6 +/+ and Prdx6 −/− mLECs transduced with or without TAT-HA-Prdx6 and subjected to qPCR, as indicated. The data represent the mean ± SD values of four independent replicates ( n = 4). Prdx6 +/+ vs. Prdx6 −/− and Prdx6 −/− vs. Prdx6 −/− with TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Activation Assay, Isolation, Western Blot, Cell Culture, Transduction

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 delivery to Prdx6 -deficient mLECs suppressed elevated ROS levels and enhanced cell viability in response to UVB- or H 2 O 2 -induced cell death: ( A , C ) TAT-HA-Prdx6 protein delivery resisted against UVB- or H 2 O 2 -induced cell death in Prdx6 −/− mLECs. Prdx6 +/+ and Prdx6 −/− mLECs cells were cultured in 96-well plates. Prdx6 −/− mLECs were transduced with TAT-HA-Prdx6 (10 μg/mL) protein. 3 h later, these cells were exposed to different concentrations of UVB or H 2 O 2 . Twenty-four hours later, cell viability was measured, and ( B , D ) TAT-HA-Prdx6 protein delivery inhibited the elevated ROS level induced by UVB or H 2 O 2 exposure in Prdx6 −/− mLECs. Prdx6 +/+ and Prdx6 −/− mLECs were cultured in 96-well plates. Prdx6 −/− mLECs were transduced with TAT-HA-Prdx6 (10 μg/mL) protein. After 3 h, these cells were exposed to different concentrations of UVB or H 2 O 2 and subjected to ROS measurement at 6 h. The data reflect mean ± SD values derived from four independent experiments ( n = 4). 0 vs. 80, 160 and 320 J/m 2 UVB treatment, and Prdx6 +/+ vs. Prdx6 −/− and Prdx6 −/− vs. Prdx6 −/− supplemented with TAT-HA-Prdx6 exposed to UVB; 0 vs. 25, 50 and 100 µM H 2 O 2 treatment, and Prdx6 −/− vs. Prdx6 −/− with TAT-HA-Prdx6 under H 2 O 2 treatment; # p < 0.05, * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Cell Culture, Transduction, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 attenuated Nlrp3 inflammasome and its inflammatory genes ASC, Caspase-1, IL-1β, and IL-18 secretion by inhibiting ROS accumulation in Prdx6 −/− mLECs: ( A ) photomicrograph representing stably infected Prdx6 −/− mLECs with LV GFP-Control or GFP-tagged Prdx6 activation linked with lentiviral (LV GFP-Prdx6); ( B ) total protein was isolated from LV-GFP-Control or LV GFP-Prdx6 infected Prdx6 −/− mLECs and subjected to Western blot, as indicated in the figure; ( C ) elevated levels of Caspase-1 were inhibited by Prdx6 overexpression in Prdx6 −/− mLECs. LV GFP-Control and LV GFP-Prdx6-infected Prdx6 −/− mLECs were cultured and 48 h later, cellular extract was prepared. Caspase-1 levels were measured using an equal amount of cellular extract; ( D , E ) Prdx6 overexpression normalized the increased secretion of mature IL-1β and IL-18 in Prdx6 −/− mLECs. LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs were cultured, and supernatant was collected after 48 h to evaluate IL-1β ( D ) and IL-18 ( E ) levels; and ( F ) total RNA was isolated from LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs and subjected to q-PCR to assess the expression of Nlrp3, Caspase-1, ASC, cytokines, such as IL-1β and IL-18, and pyroptosis executor GSDMD mRNA expression using their specific probes, as indicated. Results represent the mean ± SD values from three independent replicates ( n = 3). LV GFP-Control vs. LV GFP-Prdx6 in Prdx6 −/− mLECs; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Stable Transfection, Infection, Control, Activation Assay, Isolation, Western Blot, Over Expression, Cell Culture, Expressing

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A , B ) Prdx6 delivery abated the H 2 O 2 - or UVB-driven oxidative load and increased expression of Nlrp3 inflammasome-related inflammatory components in SRA-hLECs. SRA-hLECs were pretreated with TAT-HA-Prdx6 for 3 h and followed by H 2 O 2 ( A ) or UVB ( B ) exposure for 24 h in serum-free DMEM medium and thereafter treated for 3 days. Total RNA was isolated to assess the expression status of Nlrp3, ASC, Caspase-1, IL-1β, IL-18, and GSDMD mRNA expression using qPCR with their specific probes, as indicated in the figure. β-actin was used as a control. The data represent the mean ± SD values calculated from three independent experiments ( n = 3). Statistical analyses: 0 vs. 100µM H 2 O 2 treatment, and H 2 O 2 vs. H 2 O 2 plus TAT-HA-Prdx6; 0 vs. 200 J/m 2 UVB treatment, and UVB vs. UVB plus TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Isolation, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 regulation of the transcriptional activity of Nlrp3: ( A , B ) Prdx6 -depleted LECs displayed increased Nlrp3 transcription activity. Redox-active Prdx6 -depleted SRA-hLECs ( A ) or mLECs ( B ) were transiently transfected with pGL4-mNlrp3 (−1866/+166). Forty-eight hours later, luciferase activity was measured. The data are shown as mean ± SD from three different experiments. LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C ) UVB-exposed mLECs showed a significant increase in Nlrp3 transcription. mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166); 24 h later, these cells were exposed to different concentrations of UVB. Luciferase activity was measured after 24 h of UVB exposure. The results are shown as mean ± SD from four independent experiments ( n = 4). 0 vs. 80, 160, 320 J/m 2 UVB treatment; # p < 0.05, * p < 0.001; ( D – F ) Prdx6 delivery suppressed the elevated Nlrp3 transcription in redox-active Prdx6 −/− mLECs and mLECs facing oxidative stress. Prdx6 −/− mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166). Twenty-four hours later, these transfectants were transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h and luciferase activity was measured ( D ). Control vs. TAT-HA-Prdx6; * p < 0.001. mLECs were transiently transfected with pGL4-mNlrp3 (−1866/+166), and 24 h later, these cells were pretreated with TAT-HA-Prdx6 protein for 3 h, followed by H 2 O 2 or LPS ( E ) or UVB ( F ) exposure, as indicated in the figure; then, Luciferase activity was measured after 24 h. Results reflect mean ± SD values derived from four separate experiments ( n = 4). 0 vs. H 2 O 2 , 0 vs. LPS, H 2 O 2 vs. H 2 O 2 + TAT-HA-Prdx6, LPS vs. LPS + TAT-HA-Prdx6; 0 vs. UVB and UVB vs. UVB + TAT-HA-Prdx6; * p < 0.001; ( G – I ) elevated Nlrp3 transcription was inhibited by Prdx6 overexpression in Prdx6 −/− mLECs and mLECs under oxidative stress conditions. Prdx6 −/− mLECs and mLECs infected with LV GFP-Control and/or LV GFP-Prdx6 were transfected with pCAT-mNlrp3 (−1866/+166). 24 h later, these transfected mLECs were exposed to H 2 O 2 or LPS ( H ) or UVB ( I ). CAT activity was measured at 48 h. Prdx6 −/− mLECs ( G ), and mLECs exposed to 24 h of H 2 O 2 or LPS ( H ), or UVB ( I ). All measurements are shown as mean ± SD from four separate experiments ( n = 4). LV-GFP-Vector vs. LV-GFP-Prdx6, Prdx6 +/+ vs. Prdx6 −/− mLECs, 0 vs. H 2 O 2 or LPS and/or UVB, LV-GFP-Vector (H 2 O 2 or LPS or UVB) vs. LV-GFP-Prdx6 (H 2 O 2 or LPS or UVB); * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Transfection, Luciferase, Transduction, Control, Derivative Assay, Over Expression, Infection, Plasmid Preparation

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6 overexpression resulted in a reduction in NF- ĸ B and phosphorylated I ĸ Bα expression in Prdx6 -deficient LECs: ( A , B , D , E ) Prdx6 −/− mLECs and/or Prdx6 -depleted SRA-hLECs showed increased expression of NF- ĸ B and phosphorylated I ĸ Bα. Total RNA and nuclear and cytosol protein extracts were isolated and subjected to RT-qPCR ( A , B ) and Western analysis ( D , E ) of NF- ĸ B, I ĸ Bα and phospho-I ĸ Bα with specific probes, respectively. Tubulin was used as an internal loading control. Values are expressed as mean ± SD from four separate experiments ( n = 4). Prdx6 +/+ vs. Prdx6 −/− mLECs, LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( C , F ) increased NF- ĸ B and phospho-I ĸ Bα in Prdx6 −/− mLECs were suppressed by Prdx6 overexpression. Total RNA, as well as nuclear and cytosol protein extracts, were isolated and subjected to RT-qPCR ( C ) and Western analysis ( F ) of NF- ĸ B and phospho-I ĸ Bα with specific probes, respectively. Tubulin was used as an internal control. Values are expressed as mean ± SD from four independent experiments ( n = 4). LV GFP-control vs. LV-GFP-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Over Expression, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Control

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Redox-active transcription factor NF- ĸ B was activated in Prdx6 -deficient LECs and LECs facing oxidative stress but its activity was normalized by Prdx6 overexpression: ( A ) HIV-1 LTR-CAT and its NF- ĸ B mutant construct; ( B , C ) Prdx6 deficiency displayed an active form of redox-active transcription factor NF- ĸ B. Prdx6 +/+ , Prdx6 −/− , and Prdx6 -depleted SRA-hLECs were transiently transfected with HIV-1 LTR-CAT; 48 h later, NF- ĸ B transcription activity was measured. The data represent the mean ± SD, from four independent repeats ( n = 4). Prdx6 +/+ vs. Prdx6 −/− and LV ShControl vs. LV ShPrdx6 ; * p < 0.001; ( D ) SRA-hLECs were transfected with HIV-1 LTR-CAT and its NF- ĸ B mutant construct; 24 h after post-transfection, these cells were exposed to H 2 O 2 or LPS or UVB, as indicated in the figure. The transactivation assay was performed after 24 h of oxidative stress exposure and NF- ĸ B activity was shown; and ( E ) LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs were transfected with HIV-1 LTR-CAT, and NF- ĸ B transcription activity was measured at 48 h. Each value represents the mean ± SD from four independent repeats. Untreated vs. H 2 O 2 /LPS/UVB treatment and LV-GFP-Control vs. LV-GFP-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Over Expression, Mutagenesis, Construct, Transfection, Transactivation Assay, Control, Infection

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A – C ) In silico analysis revealed the presence of NF- ĸ B binding sites in Klf9 promoter: aged mLECs and Prdx6 −/− mLECs, a model for aging, displayed increased Klf9 promoter activity; ( A ) schematic diagram showing the 5′-construct of mKlf9 promoter, spanning −5856/+71 bps linked to CAT reporter gene, with the position of NF- ĸ B binding sequence; ( B , C ) mLECs were transiently transfected with the Klf9 promoter, along with the GFP vector. Forty-eight hours post transfection, CAT activity was examined. The data show the mean ± SD from four independent experiments ( n = 4). 2M vs. 17M-old and Prdx6 +/+ vs. Prdx6 −/− mLECs; * p < 0.001; ( D ) Prdx6 −/− mLECs infected with LV GFP-Control or LV GFP-Prdx6 were transfected with Klf9 promoter, and CAT activity was measured at 48 h. The results represent the mean ± SD from four separate replicates ( n = 4). LV GFP-Control vs. LV-GFP-Prdx6; * p < 0.001; and ( E , F ) elevated Klf9 transcriptional activity in aging mLECs or mLECs facing oxidative stress was inhibited by TAT-HA-Prdx6. Aging mLECs and mLECs were transiently transfected with Klf9 promoter construct along with GFP plasmid; 24 h later, aging mLECs were transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h, and mLECs were transduced with TAT-HA-Prdx6 (10 µg/mL) for 3 h, followed by H 2 O 2 and/or UVB exposure for 24 h. CAT activity was measured after 24 h. Transfection efficiency was normalized using GFP O.D, measured at Ex485/Em530 nm. The results represent the mean ± SD from four independent experiments ( n = 4). 2M vs. 8M-old and 19M-old, control vs. TAT-HA-Prdx6, untreated control vs. H 2 O 2 vs. H 2 O 2 + TAT-HA-Prdx6, and untreated control vs. UVB vs. UVB + TAT-HA-Prdx6; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: In Silico, Binding Assay, Activity Assay, Construct, Sequencing, Transfection, Plasmid Preparation, Infection, Control, Transduction

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: ( A – D ) Prdx6 −/− (a model for aging) and aging mLECs showed increased levels of TXNIP with reduced antioxidant gene TRX. Elevated TXNIP levels in Prdx6 −/− and aging mLECs were related to TRX repression. Total protein and RNA were isolated from Prdx6 +/+ , Prdx6 −/− mLECs and aging mLECs and subjected to Western blot and RT-qPCR analysis. Data represent the mean ± SD values of six independent experiments ( n = 6). Prdx6 +/+ vs. Prdx6 −/− mLECs samples; young (2-month-old) vs. Old (19-month-old) mLECs samples; * p < 0.001; ( E , F ) Prdx6 delivery inhibited the elevated TXNIP expression and enhanced the TRX expression in Prdx6 −/− mLECs. Total RNA was isolated from LV GFP-Control and LV GFP-Prdx6 infected Prdx6 −/− mLECs (E) and/or Prdx6 −/− mLECs transduced with TAT-HA-Prdx6 (10 µg/mL) for 24 h. TXNIP and TRX mRNA expression levels were examined by qPCR. The data show the mean ± SD values derived from six separate experiments ( n = 6). Prdx6 +/+ vs. Prdx6 −/− mLECs and Prdx6 −/− control vs. Prdx6 −/− with TAT-HA-Prdx6 samples; * p < 0.001.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Isolation, Western Blot, Quantitative RT-PCR, Expressing, Control, Infection, Transduction, Derivative Assay

Journal: Antioxidants

Article Title: Loss of Peroxiredoxin 6 Drives Age-Related Klf9/NF- κ B/Nlrp3 Inflammasome Activation and Pyroptosis: Therapeutic Rescue by Prdx6

doi: 10.3390/antiox15050532

Figure Lengend Snippet: Prdx6’s role in the protection and survival of lens epithelial cells in aging and oxidant-induced oxidative stress. During aging and oxidative stress, the loss of antioxidant defense in LECs leads to excessive ROS accumulation, triggering Nlrp3 inflammasome activation through NF- κ B and Klf9 signaling. Activated Klf9 further amplifies oxidative stress by increasing ROS levels and suppressing antioxidant gene expression, while NF- κ B directly enhances Klf9 transcription, together promoting sustained inflammasome activation. Aberrantly activated Nlrp3 assembles with ASC and proCaspase-1, resulting in the activation of Caspase-1, maturation of IL-1β/IL-18, cleavage of GSDMD, and induction of pyroptotic cell death. The delivery of Prdx6 suppresses NF- κ B and Klf9 activation, reduces ROS accumulation, restores antioxidant defense, and inhibits Nlrp3 inflammasome activation, thereby promoting healthy ocular aging and delaying cataract formation.

Article Snippet: For Prdx6 overexpression, Prdx6 ( NM_004905 ) human tagged ORF clone lentiviral particles linked to pLenti-C-mGFP-P2A-Puro vector (LV-GFP-Prdx6, RC207780L4V) and corresponding lentiviral Control particles (LV-GFP-Control, PS100093V) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activation Assay, Gene Expression

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: Genome wide CRISPRa high throughput screen identifies PRDX6 as a largazole target. (A) Structures of the prodrug largazole and its active form, largazole thiol, after hydrolysis. (B) Schematic of the genome wide lentiviral pooled screen for largazole. HCT116 cells expressing CRISPRa (dCas9-VP64-GFP) were infected with a lentiviral sgRNA library. After selection with 2 µg/mL puromycin, cells were split into two groups: one treated with DMSO and the other with 100 nM largazole. Following treatment, cells were lysed and genomic DNA was isolated, processed, and sequenced. (C) Differential sgRNA abundance analysis comparing largazole treated versus DMSO treated groups. A significance threshold of p -value < 2×10 -7 was used to call hits. (D) Gene level enrichment from the secondary screen comparing DMSO and largazole conditions. Genes were ranked by the summed read counts of their sgRNAs. (E) Validation of screening results using cells overexpressing individual genes. HCT116 wild type cells with the indicated cDNA overexpression were treated with 100 nM largazole for 12 days, fixed with 4% formaldehyde in PBS, and stained with 0.05% crystal violet. A 3-day DMSO treatment of HCT116 wild type cells served as a natural growth control. Images were inverted to grayscale to enhance visibility. (F) Quantification of the crystal violet assays shown in panel E. Stain was solubilized and absorbance measured at 570 nm. Data are mean ± SD. Welch one way ANOVA with Games Howell multiple comparisons test was used. *** p < 0.001, **** p < 0.0001.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Genome Wide, High Throughput Screening Assay, Expressing, Infection, Selection, Isolation, Biomarker Discovery, Over Expression, Staining, Control

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 alters cell sensitivity to largazole without comprising HDAC inhibition. (A) Largazole sensitivity assay in cells with PRDX6 knockdown and the indicated rescues. HCT116 cells with PRDX6 knockdown and PRDX6 knockdown rescued with WT, S32A, C47S, or S32A+C47S were treated with 100 nM largazole for 12 days, fixed with 4% formaldehyde in PBS, and stained with 0.05% crystal violet. A 3-day DMSO treatment of HCT116 parental cells served as a natural growth control. Images were inverted to grayscale to enhance visibility. (B) Quantification of panel A. Crystal violet stain was solubilized and absorbance measured at 570 nm. Data are mean ± SD. One way ANOVA with Tukey multiple comparisons test was used. **** p < 0.0001. (C) Western blot of pan H4ac levels in HCT116 parental, PRDX6 knockdown, and PRDX6 knockdown cells with the indicated rescues. Cells were treated with 100 nM largazole for 2 hours. GAPDH was used as a loading control. (D) Schematic of the combination treatment assay. 3×10 5 HCT116 parental cells were seeded per well in a 6 well plate. All wells were treated with 100 nM largazole for 9 days, then washed with PBS and replaced with 100 nM largazole, 10 µM MJ33, or 100 nM largazole plus 10 µM MJ33 for 3 additional days. (E-F) Results and quantification of panel D. 3-day DMSO and MJ33 treatments of HCT116 parental cells were used to indicate natural growth rate and any growth effect of MJ33 alone. Images were inverted to grayscale to enhance visibility. Crystal violet stain was solubilized and absorbance measured at 570 nm. Data are mean ± SD. One way ANOVA with Tukey multiple comparisons test was used. **** p < 0.0001. (G-H) IC 50 values for largazole and paragazole in HCT116 parental versus PRDX6 knockdown cells. HCT116 parental and PRDX6 knockdown cells were seeded into a 96 well plate in 4 lanes × 12 wells with 4,000 cells per well. Twelve hours after seeding, medium was replaced with fresh DMEM containing a serial dilution of largazole, and cells were treated for 3 days. Cells were then washed with PBS, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized, and absorbance was read at 530 nm. Wells without largazole were used as controls for normalization. Data are mean ± SD. A four-parameter logistic regression was used to calculate IC 50 . Student t -test was used for statistical analysis. **** p <0.0001.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Inhibition, Sensitive Assay, Knockdown, Staining, Control, Western Blot, Serial Dilution

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 knockdown increases vulnerability to largazole induced lipid peroxidation stress. (A) Largazole induces lipid peroxidation in HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate at 10,000 cells per well. After 24 hours, medium was replaced with fresh DMEM containing the indicated treatments for the indicated durations. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde in PBS, and stained with 10 µM Image iT Lipid Peroxidation Sensor and 10 µg/mL Hoechst 33342 for 3 days at 4°C in the dark. After incubation, cells were washed three times with PBS and imaged using DAPI, FITC, and TRITC channels. Scale bar, 100 µm. (B) Quantification of panel A. Fifty cells per condition were randomly selected and the mean FITC and TRITC fluorescence intensities per cell were measured. The FITC/TRITC ratio was used as a readout of lipid peroxidation stress. Two-way ANOVA with Tukey multiple comparisons test was used. ** p < 0.01, **** p < 0.0001, n.s.= not significant. (C) PRDX6 knockdown induces iron accumulation in HCT116 cells upon largazole treatments. After indicated treatments, live cells were stained with 1LµM FerroOrange (Dojindo) and costained with 10Lµg/mL Hoechst 33342 for 30 minutes prior to imaging, without washout. Imaging was performed on the Revvity Opera Phenix Confocal System using a 20X water objective and standard settings. Nuclei were segmented using Hoechst signal, and perinuclear cytoplasmic regions were defined to quantify FerroOrange fluorescence on a single-cell basis. Border cells were excluded, and integrated fluorescence intensity per cell was calculated. “High” Fe²⁺ cells were defined using Otsu’s threshold from control wells, and the percentage of high-signal cells was quantified per well. Bars represent mean ± SEM from biological replicates. Statistical significance was determined by two-way ANOVA with Tukey’s post-hoc test. *** p < 0.001, **** p < 0.0001, n.s.= not significant. (D) Largazole increases GSSG in PRDX6 knockdown HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate at 10,000 cells per well. After 24 hours, medium was replaced with fresh DMEM containing 100 nM largazole for 18 hours. GSH and GSSG were measured using the GSH/GSSG Glo assay, with readings normalized using the CellTiter Glo luminescent viability assay. Two way ANOVA with Tukey multiple comparisons test was used. * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. = not significant. ( E-F ) Largazole shows increased synergy with erastin in PRDX6 knockdown HCT116 cells. HCT116 parental and PRDX6 knockdown cells were seeded in a 96 well plate in 8 lanes × 8 wells at 8,000 cells per well. Twelve hours after seeding, medium was replaced with fresh DMEM containing serial dilutions of largazole and erastin2, and cells were treated for 3 days. Cells were then washed twice with PBS, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized, and absorbance was read at 530 nm. Wells without treatment served as normalization controls. Synergy scores were computed and visualized using SynergyFinder+.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Knockdown, Staining, Incubation, Fluorescence, Imaging, Control, Glo Assay, Viability Assay

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 is critical to maintain GPX4 level in head and neck squamous cell carcinoma. (A) Overall pan-cancer survival analysis stratified by PRDX6 expression. Patients were grouped by significantly upregulated versus significantly downregulated PRDX6 in tumor relative to matched normal tissue. Shaded bands indicate 95% confidence intervals. Log-rank test p -value < 0.05. (B) PRDX6 expression level is critical for survival probability for patients with head and neck squamous cell carcinoma (HNSCC). Five year-Kaplan-Meier overall survival from TCGA data stratified by median PRDX6 expression (High PRDX6, red; Low PRDX6, blue). Shaded bands represent 95% confidence intervals. Log-rank test p -value < 0.05. (C-D) PRDX6 knockdown slows proliferation in squamous carcinoma cell lines. For all indicated cell lines, 12 wells of 500 cells were seeded into 7 plates of 96-well plate (1 plate/day). After indicated duration of incubation, plates were fixed and crystal violet staining was performed. The plates were solubilized, and absorbance was measured at 570 nm. Data are mean± SD. (E-F) Largazole shows synergistic effect with RSL3 and with erastin2 in A223 and FaDu PRDX6 knockdown cells. For both FaDu and A223, shNT and PRDX6 knockdown cells were seeded into 8 lanes x 8 wells of 96-well plate with 8,000 cells in each well. 12 hours after seeding, DMEM was replaced by fresh DMEM with largazole and RSL3 or and erastin2 in serial-diluted manner, and cells were treated for 3 days. After 3 days treatment, cells were washed with PBS twice, fixed with 4% formaldehyde in PBS, stained with 0.05% crystal violet, solubilized and quantified by reading absorbance for each well at 570 nm. Wells with no treatment were used as control to normalize the data and synergy scores were calculated and visualized using SynergyFinder+. (G-H) Largazole downregulates PRDX6 and GPX4 mRNA in FaDu and A223 cells. FaDu and A223 cells with either shNT or PRDX6 knockdown were treated with 100LnM largazole for the indicated times. Total RNA was extracted at each time point and subjected to RT-qPCR to assess PRDX6 and GPX4 mRNA expression. β-actin was used as the housekeeping gene for normalization. mRNA levels were normalized to the respective 0Lhr WT control group for both cell types. Data represent the relative expression of PRDX6 and GPX4 over time in response to largazole treatment. A regular one-way ANOVA was performed for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. = not significant. (I) PRDX6 regulates GPX4 protein level. Western blot for GPX4 expression levels in A223 or FaDu WT, PRDX6 KD and PRDX6 overexpression (OE) cell lines. Actin was used as a loading control. (J) PRDX6 and GPX4 co-expression and survival in HNSCC. Five year Kaplan Meier overall survival from TCGA stratified by combined PRDX6 and GPX4 expression (Both high, red; Both low, blue) using median cutoffs. Shaded bands indicate 95 percent confidence intervals. Log rank test p < 0.05.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Expressing, Knockdown, Incubation, Staining, Control, Quantitative RT-PCR, Western Blot, Over Expression

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 restrains largazole induced inflammatory and ferroptotic responses in squamous carcinoma cells. (A) GSEA shows that PRDX6 knockdown in A223 cells enhances largazole induced inflammatory responses. Bubble plot summarizing GSEA results of Hallmark gene sets enrichment across three conditions: KD_DMSO vs WT_DMSO, WT_LAR vs WT_DMSO and KD_LAR vs KD_DMSO. Each point represents a significantly enriched pathway (FDR < 0.25). Color indicates the normalized enrichment score (NES), reflecting the direction and magnitude of enrichment. Bubble size reflects the negative log10 of the adjusted p -value. (B-C) Largazole induces inflammatory signaling and ferroptosis in a PRDX6 knockdown dependent manner. Heatmaps display expression of genes in inflammation and ferroptosis pathways across the indicated conditions. Gene values were taken from DESeq2 normalized counts, log2 transformed, and row scaled (z score). The color scale represents relative expression (blue, low; red, high). Only genes detected in the RNA seq dataset and present in the normalized matrix are shown. (D) Cytokine array reveals elevated inflammatory responses induced by largazole treatment in A223 PRDX6 KD cells. A223 WT and PRDX6 KD cells were seeded into a 6-well plate with 2×10 6 cells in each well. After 24 hours, medium was replaced with 1 mL fresh DMEM containing DMSO or 100 nM largazole for 18 hours. Cells were collected and cytokine levels were measured using the Proteome Profiler Array Mouse XL Cytokine kit. (E) Quantification of panel D. Spot intensities were measured and analyzed by two way ANOVA with Tukey multiple comparisons. * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. = not significant.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Knockdown, Expressing, Transformation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 knockdown sensitizes largazole treatment in vivo . (A) PRDX6 KD enhances tumor growth inhibition by OKI-179 in A223 tumors. A223 parental or PRDX6 KD cells were implanted in the flank of WTB6 mice. After tumor volume reached ∼180 mm (A223 WT recipient mice were treated from day 12 onwards and PRDX6 KD recipient mice were treated from day 16 onwards) animals were treated with 60mg/kg OKI-179 or vehicle via oral gavage at 2 days interval for 9 doses. Tumor volume was measured and groups were compared on day 34. A223 parental + Ctrl, n=13; A223 parental + OKI-179, n=14; A223 PRDX6 KD + Ctrl, n=19; A223 PRDX6 KD + OKI-179, n=23. Different groups were compared using two-way ANOVA followed by Tukey’s multiple comparisons test. ** p <0.01, **** p <0.0001. (B) Individual tumor growth curves of recipients in different treatment groups. (C) Kaplan-Meier survival curve of recipient mice in different treatment groups. Groups were compared using log-rank tests. * p <0.05. (D) Representative immunohistochemical staining (DAPI, CD4, CD8 and CD3) of tumor tissues from A223 WT and PRDX6 KD mice with and without OKI-179 treatment. (E) Representative immunohistochemical staining (DAPI, PRDX6, CD11b, CD4, CD8 and CD3) of tumor tissues from A223 WT and PRDX6 KD mice with and without OKI-179 treatment.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Knockdown, In Vivo, Inhibition, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

doi: 10.64898/2026.01.21.700636

Figure Lengend Snippet: PRDX6 modulates immune checkpoint inhibitor response. Combination of anti-PD-L1/OKI-179 led to better outcomes in A223 PRDX6 KD cell line. (A) A223 parental cells were implanted in the flank of WTB6 mice and on day 12 tumor bearing mice were randomized into 4 groups, Control (n=6), OKI-179 (60 mg/kg/dose/2 days via oral gavage for 9 doses, n=5), anti-PD-L1 (200 µg/mouse/2days, i.p., for 3 doses, n=9), and OKI-179+anti-PD-L1 combination (n=8). Tumor volume was measured and groups were compared on day 28. (C) A223 PRDX6 KD cells were implanted in the flank of WTB6 mice and on day 16 tumor bearing mice were randomized into 4 groups, Control (n=8), OKI-179 (60 mg/kg/dose/2 days via oral gavage for 9 doses, n=9), anti-PD-L1 (200 µg/mouse/2days, i.p., for 3 doses, n=10), and OKI-179+anti-PD-L1 combination (n=10). Tumor volume was measured and groups were compared on day 40. Different groups were compared using two-way ANOVA followed by Tukey’s multiple comparisons test. ** p <0.01, *** p <0.001, **** p <0.0001. (B-D) Individual tumor growth curves of recipients in different treatment groups of A223 parental and A223 PRDX6 KD cells. (E, F) Kaplan-Meier survival curves of recipient mice in different treatment groups A223 parental and A223 PRDX6 KD cells. Groups were compared using log-rank tests. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, n.s. = not significant. (G-H) Combination of anti-PD-L1/OKI-179 led to a higher percentage of responders. A223 WT or PRDX6-KD cells were implanted at the flank of WT B6 mice and treated as described above. RCTV was calculated and compared for different treatment groups. Based on RCTV values, treatment recipients were divided into Responders (R, RCTV<0), Slow progressors (SP, 01.5). (G) Percentage of each response group (R, SP or NR) in different treatment groups. Different groups were compared using one-way ANOVA followed by Tukey’s multiple comparisons test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. (H) Summary of responses to different treatment regimens. For each treatment regimen, multiple cohorts of mice were used for independent experiments.

Article Snippet: Wild type and mutant human PRDX6 coding sequences were codon optimized and synthesized by Twist Biosciences.

Techniques: Control