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$Gene-silencing <t>Reticulon</t> <t>4</t> or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting <t>RTN4</t> or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).$
Rtn4, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

Image Search Results

Dynamic SILAC reveals distinct sets of proteins affected in their turnover upon TNF-α-induced muscle atrophy in C2C12 cells. (A) Experimental workflow: C2C12 myoblasts were differentiated for seven days in light medium (K0 = Lysine 0 [ 12 C 6 , 14 N 2 ]; R0 = Arginine 0 [ 12 C 6 , 14 N 4 ]) into myotubes (= t0). Medium was then replaced with heavy medium (K8 = Lysine 8 [ 13 C 6 , 15 N 2 ]; R10 = Arginine 10 [ 13 C 6 , 15 N 4 ]) for 24 or 72 h (t24, t72), with or without TNF-α to induce atrophy. Proteolytic inhibitors (BafA1: Bafilomycin A1 inhibiting autophagy; Lac: Lactacystin inhibiting the UPS) were administered for 3 h prior to cell harvest, and cells were analyzed using LC-MS/MS. n = 3 replicates. (B) Myotube diameter was measured in differentiated C2C12 myotubes under homeostatic conditions at 24 h (H24; day 8 of differentiation) and 72 h (H72; day 10), as well as following TNF-α-induced atrophy at corresponding time points (A24 and A72). Measurements were performed using ImageJ on 209 individual myotubes per condition, quantified from 15-20 randomly selected images. Statistical significance was determined using ordinary one-way ANOVA with multiple comparisons (GraphPad Prism). Images used were acquired from OPP-stained samples from . (C) Protein clustering based on total intensity dynamics of non-inhibitor treated homeostatic and atrophying cells: Proteins were categorized into four clusters according to their normalized total intensity values (light + heavy) at t0, t24, and t72. Percentages of protein counts were rounded up. (D) Proteins of each cluster of total intensities were subjected to GO term enrichment (biological processes) analysis.

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Dynamic SILAC reveals distinct sets of proteins affected in their turnover upon TNF-α-induced muscle atrophy in C2C12 cells. (A) Experimental workflow: C2C12 myoblasts were differentiated for seven days in light medium (K0 = Lysine 0 [ 12 C 6 , 14 N 2 ]; R0 = Arginine 0 [ 12 C 6 , 14 N 4 ]) into myotubes (= t0). Medium was then replaced with heavy medium (K8 = Lysine 8 [ 13 C 6 , 15 N 2 ]; R10 = Arginine 10 [ 13 C 6 , 15 N 4 ]) for 24 or 72 h (t24, t72), with or without TNF-α to induce atrophy. Proteolytic inhibitors (BafA1: Bafilomycin A1 inhibiting autophagy; Lac: Lactacystin inhibiting the UPS) were administered for 3 h prior to cell harvest, and cells were analyzed using LC-MS/MS. n = 3 replicates. (B) Myotube diameter was measured in differentiated C2C12 myotubes under homeostatic conditions at 24 h (H24; day 8 of differentiation) and 72 h (H72; day 10), as well as following TNF-α-induced atrophy at corresponding time points (A24 and A72). Measurements were performed using ImageJ on 209 individual myotubes per condition, quantified from 15-20 randomly selected images. Statistical significance was determined using ordinary one-way ANOVA with multiple comparisons (GraphPad Prism). Images used were acquired from OPP-stained samples from . (C) Protein clustering based on total intensity dynamics of non-inhibitor treated homeostatic and atrophying cells: Proteins were categorized into four clusters according to their normalized total intensity values (light + heavy) at t0, t24, and t72. Percentages of protein counts were rounded up. (D) Proteins of each cluster of total intensities were subjected to GO term enrichment (biological processes) analysis.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Staining

TNF-α-induced atrophy leads to acute translation inhibition. (A) OPP (O-propargyl-puromcyin) labeling of differentiated myotubes (homeostatic at 24 h (H24) and 72 h (H27)) and TNF-α-induced atrophying C2C12 cells (24 h (A24) and 72 h (A72)) was used to measure protein synthesis rates at time points t24 and t72. Arrows indicate puncta representing newly translated proteins. Asterisks indicate nuclei in differentiated, multinucleated myotubes. (B) Cycloheximide (CHX) was employed for 90 min as a control to inhibit protein synthesis. Puncta were manually counted across 20 images per condition, each containing over 200 cells. (C) Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 60 µm.

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: TNF-α-induced atrophy leads to acute translation inhibition. (A) OPP (O-propargyl-puromcyin) labeling of differentiated myotubes (homeostatic at 24 h (H24) and 72 h (H27)) and TNF-α-induced atrophying C2C12 cells (24 h (A24) and 72 h (A72)) was used to measure protein synthesis rates at time points t24 and t72. Arrows indicate puncta representing newly translated proteins. Asterisks indicate nuclei in differentiated, multinucleated myotubes. (B) Cycloheximide (CHX) was employed for 90 min as a control to inhibit protein synthesis. Puncta were manually counted across 20 images per condition, each containing over 200 cells. (C) Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 60 µm.

Techniques: Inhibition, Labeling, Control

Increased autophagic turnover in TNF-α-induced atrophying C2C12 cells. (A) Western blot analysis of LC3B-II and p62 in C2C12 myotubes under homeostatic (H24, H72) and atrophic (A24, A72) conditions, with and without Bafilomycin (BafA1) treatment at time points 24 h and 72 h. Vehicle-treated (DMSO) cells serve as control. (B) LC3B-II and p62 intensities were normalized against Vinculin. n = 3 replicates. ns = not significant. (C-E) Flow cytometry-based assay measuring autophagic turnover by staining membrane-bound LC3-II with a fluorescently labeled antibody (C) Representative histograms display the shift in LC3-II signal following 3 h BafilomycinA1 (BafA1) treatment (D) Elevated LC3-II signal intensity (gMFI, geometric mean fluorescence intensity) in atrophying (A24, A72) cells compared to homeostatic cells (H24, H72) in dimethylsulfoxide (DMSO)-treated samples. n = 4 replicates. ns = not significant. (E) Autophagic turnover was calculated by determining the difference in LC3-II signal intensity (gMFI) between BafA1-treated and DMSO-treated cells over a 3 h incubation period. n = 4 replicates. (F) Accumulated proteins upon BafilomycinA1 (BafA1) treatment in ANOVA comparisons of atrophic (A24, A72) vs. homeostatic conditions (H24, H72) in the light channel (log 2 FC > 0 adj. p -value < 0.05). Left: Venn diagram numbers depict significantly regulated proteins per comparison. Right: Red dots in volcano plot depict autophagy machinery components.

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Increased autophagic turnover in TNF-α-induced atrophying C2C12 cells. (A) Western blot analysis of LC3B-II and p62 in C2C12 myotubes under homeostatic (H24, H72) and atrophic (A24, A72) conditions, with and without Bafilomycin (BafA1) treatment at time points 24 h and 72 h. Vehicle-treated (DMSO) cells serve as control. (B) LC3B-II and p62 intensities were normalized against Vinculin. n = 3 replicates. ns = not significant. (C-E) Flow cytometry-based assay measuring autophagic turnover by staining membrane-bound LC3-II with a fluorescently labeled antibody (C) Representative histograms display the shift in LC3-II signal following 3 h BafilomycinA1 (BafA1) treatment (D) Elevated LC3-II signal intensity (gMFI, geometric mean fluorescence intensity) in atrophying (A24, A72) cells compared to homeostatic cells (H24, H72) in dimethylsulfoxide (DMSO)-treated samples. n = 4 replicates. ns = not significant. (E) Autophagic turnover was calculated by determining the difference in LC3-II signal intensity (gMFI) between BafA1-treated and DMSO-treated cells over a 3 h incubation period. n = 4 replicates. (F) Accumulated proteins upon BafilomycinA1 (BafA1) treatment in ANOVA comparisons of atrophic (A24, A72) vs. homeostatic conditions (H24, H72) in the light channel (log 2 FC > 0 adj. p -value < 0.05). Left: Venn diagram numbers depict significantly regulated proteins per comparison. Right: Red dots in volcano plot depict autophagy machinery components.

Techniques: Western Blot, Control, Flow Cytometry, Staining, Membrane, Labeling, Fluorescence, Incubation, Comparison

During TNF-a-induced atrophy, canonical autophagy does not mediate myofibrillar protein degradation, whereas ER-phagy emerges as the predominant form of selective autophagy. (A) Immunocytochemical staining of C2C12 (atrophying) myotubes for p62/Sqstm1 and myosin heavy chain (MyHC) highlighting the M band region of the sarcomere with corresponding line plots displaying fluorescence intensity profiles for each channel along the length of the sarcomere. Scale bar 20 µm. (B) Accumulated myofibrillar proteins upon BafilomycinA1 (+B) and Lactacystin (+L) treatment in ANOVA comparisons of homeostatic (H) and atrophying (A) C2C12 cells (light channel; log 2 FC > 0, adj. p -value < 0.05). White: not significantly accumulated; grey: not significant. (C) ANOVA of differentially regulated proteins (log 2 FC > 0; adj. p -value < 0.05). Left: Newly synthesized proteins in atrophy (A24 vs. H24 and A72 vs. H72) and accumulating (Light channel) selective autophagy receptors ± BafilomycinA1 (+B) treatment in atrophy (A24 vs. H24 and A72 vs. H72). Right: Accumulating ER components ± BafilomycinA1 (+B in atrophy (A24 vs. H24 and A72 vs. H24). White: not significantly accumulated; gray: not significant.

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: During TNF-a-induced atrophy, canonical autophagy does not mediate myofibrillar protein degradation, whereas ER-phagy emerges as the predominant form of selective autophagy. (A) Immunocytochemical staining of C2C12 (atrophying) myotubes for p62/Sqstm1 and myosin heavy chain (MyHC) highlighting the M band region of the sarcomere with corresponding line plots displaying fluorescence intensity profiles for each channel along the length of the sarcomere. Scale bar 20 µm. (B) Accumulated myofibrillar proteins upon BafilomycinA1 (+B) and Lactacystin (+L) treatment in ANOVA comparisons of homeostatic (H) and atrophying (A) C2C12 cells (light channel; log 2 FC > 0, adj. p -value < 0.05). White: not significantly accumulated; grey: not significant. (C) ANOVA of differentially regulated proteins (log 2 FC > 0; adj. p -value < 0.05). Left: Newly synthesized proteins in atrophy (A24 vs. H24 and A72 vs. H72) and accumulating (Light channel) selective autophagy receptors ± BafilomycinA1 (+B) treatment in atrophy (A24 vs. H24 and A72 vs. H72). Right: Accumulating ER components ± BafilomycinA1 (+B in atrophy (A24 vs. H24 and A72 vs. H24). White: not significantly accumulated; gray: not significant.

Techniques: Staining, Fluorescence, Synthesized

Schematic model of proteostatic remodeling in TNF-α-induced muscle atrophy over time (24 h to 72 h): In C2C12 myotubes, TNF-α triggers selective remodeling of proteostasis, translation (Ribosomal and mitochondrial ribosomal proteins), ER stress, autophagic flux and autophagic cargo. Arrows represent changes observed under TNF-α-induced atrophy relative to the corresponding baseline control.

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Schematic model of proteostatic remodeling in TNF-α-induced muscle atrophy over time (24 h to 72 h): In C2C12 myotubes, TNF-α triggers selective remodeling of proteostasis, translation (Ribosomal and mitochondrial ribosomal proteins), ER stress, autophagic flux and autophagic cargo. Arrows represent changes observed under TNF-α-induced atrophy relative to the corresponding baseline control.

Techniques: Control

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

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.

Techniques:

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.

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

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.

Techniques: Inhibition, Cell Culture

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).

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

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.

Techniques: Inhibition

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

Techniques:

ER association with the rootlet and mitochondria . ER associations demonstrated by immunofluorescence and immunoelectron microscopy staining in 6-month-old control mouse retina. ( A ) Schematic diagram showing the inner segment region imaged in ( B , C ) between the outer segments (OS) and outer nuclear layer (ONL). B Immunoreactivity for the ER marker protein disulfide-isomerase (PDI) was observed running through the inner segment between the mitochondria stained with mitofusin 1 (Mfn1). C ER stained with anti-KDEL shows a close association with the rootlet marker rootletin. Green arrowheads point to the rootletin staining running through the photoreceptor inner segments. ( D , E ) The association of KDEL and the rootlet (Root) was also shown by immuno-EM labeling of KDEL. D Longitudinal and E transverse orientated inner segments show KDEL labeling on the membrane surrounding the rootlet. ( F ) The concentration of KDEL labeling is much higher within 100 nm of the rootlet compared with the entire cross-sectionally orientated inner segments. Plot shows the mean and statistical significance determine using a paired, two-tailed Student's t -tests: **** P ≤ 0.0001. Scale bars = B and C = 2.5 µm and D and E = 100 nm.

Journal: Investigative Ophthalmology & Visual Science

Article Title: The P23H Rhodopsin Mouse Model Reveals a Novel Interaction Between the Endoplasmic Reticulum and Connecting Cilium Rootlet Within Photoreceptors

doi: 10.1167/iovs.67.3.57

Figure Lengend Snippet: ER association with the rootlet and mitochondria . ER associations demonstrated by immunofluorescence and immunoelectron microscopy staining in 6-month-old control mouse retina. ( A ) Schematic diagram showing the inner segment region imaged in ( B , C ) between the outer segments (OS) and outer nuclear layer (ONL). B Immunoreactivity for the ER marker protein disulfide-isomerase (PDI) was observed running through the inner segment between the mitochondria stained with mitofusin 1 (Mfn1). C ER stained with anti-KDEL shows a close association with the rootlet marker rootletin. Green arrowheads point to the rootletin staining running through the photoreceptor inner segments. ( D , E ) The association of KDEL and the rootlet (Root) was also shown by immuno-EM labeling of KDEL. D Longitudinal and E transverse orientated inner segments show KDEL labeling on the membrane surrounding the rootlet. ( F ) The concentration of KDEL labeling is much higher within 100 nm of the rootlet compared with the entire cross-sectionally orientated inner segments. Plot shows the mean and statistical significance determine using a paired, two-tailed Student's t -tests: **** P ≤ 0.0001. Scale bars = B and C = 2.5 µm and D and E = 100 nm.

Article Snippet: Single labeling with anti-KDEL (1:10, NBP1-97469, Novus Biologicals) and double labeling using two anti-Rhodopsin antibodies RET-P1 (1:100, Abcam) and 1D4 (1:250) was performed using methods previously described.

Techniques: Immunofluorescence, Immuno-Electron Microscopy, Staining, Control, Marker, Labeling, Membrane, Concentration Assay, Two Tailed Test

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