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(a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through <t>RIG-I</t> innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down <t>RIG-I-FLAG</t> <t>transfected</t> lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.
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(a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through <t>RIG-I</t> innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down <t>RIG-I-FLAG</t> <t>transfected</t> lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.
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(a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through <t>RIG-I</t> innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down <t>RIG-I-FLAG</t> <t>transfected</t> lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.
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(a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through <t>RIG-I</t> innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down <t>RIG-I-FLAG</t> <t>transfected</t> lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.
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(a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through RIG-I innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down RIG-I-FLAG transfected lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.

Journal: bioRxiv

Article Title: CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality

doi: 10.1101/2023.10.03.560559

Figure Lengend Snippet: (a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through RIG-I innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down RIG-I-FLAG transfected lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.

Article Snippet: HCT116 TP53 (-/-) cells were transfected with a FLAG-tagged RIG-I expression vector purchased from Origene (RC217615) using Lipofectamine 3000 in six 150 mm dishes plated to 90% confluency the day before.

Techniques: Functional Assay, Activation Assay, Extraction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Magnetic Beads, Transfection, Staining, Knock-Out