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rtn4  (Novus Biologicals)


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    Novus Biologicals rtn4
    Gene-silencing <t>Reticulon</t> <t>4</t> or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting <t>RTN4</t> or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).
    Rtn4, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 32 article reviews
    rtn4 - by Bioz Stars, 2026-05
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    1) Product Images from "Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese"

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkag233

    Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).
    Figure Legend Snippet: Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).

    Techniques Used: Transfection, Isolation, Fractionation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Derivative Assay, Membrane, BrdU Incorporation Assay, Synthesized

    ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.
    Figure Legend Snippet: ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

    Techniques Used:

    Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.
    Figure Legend Snippet: Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

    Techniques Used: Inhibition, Disruption, Control, Staining, Western Blot

    Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.
    Figure Legend Snippet: Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

    Techniques Used: Inhibition, Cell Culture

    Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).
    Figure Legend Snippet: Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).

    Techniques Used: Isolation, Fractionation, SDS Page, Control, Pulse Chase, Transfection

    ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.
    Figure Legend Snippet: ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.

    Techniques Used: Inhibition



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    93
    Proteintech antibodies against rtn4
    BMM (treated with siRNA to either CLIMP-63 or <t>RTN4</t> or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.
    Antibodies Against Rtn4, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against rtn4/product/Proteintech
    Average 93 stars, based on 1 article reviews
    antibodies against rtn4 - by Bioz Stars, 2026-05
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    rabbit anti rtn4  (Proteintech)
    93
    Proteintech rabbit anti rtn4
    BMM (treated with siRNA to either CLIMP-63 or <t>RTN4</t> or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.
    Rabbit Anti Rtn4, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rtn4/product/Proteintech
    Average 93 stars, based on 1 article reviews
    rabbit anti rtn4 - by Bioz Stars, 2026-05
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    Image Search Results


    Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques: Transfection, Isolation, Fractionation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Derivative Assay, Membrane, BrdU Incorporation Assay, Synthesized

    ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques:

    Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques: Inhibition, Disruption, Control, Staining, Western Blot

    Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques: Inhibition, Cell Culture

    Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques: Isolation, Fractionation, SDS Page, Control, Pulse Chase, Transfection

    ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

    doi: 10.1093/nar/gkag233

    Figure Lengend Snippet: ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.

    Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

    Techniques: Inhibition

    BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

    Journal: bioRxiv

    Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

    doi: 10.64898/2026.03.19.712868

    Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

    Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

    Techniques: Control, Infection, Comparison

    BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

    Journal: bioRxiv

    Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

    doi: 10.64898/2026.03.19.712868

    Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

    Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

    Techniques: Control, Infection, Comparison