rabbit anti cc1 Search Results


mouse anti cc1 antibody  (Thermo Fisher)
99
Thermo Fisher mouse anti cc1 antibody
Mouse Anti Cc1 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/pmc11351764-35-41-50?v=Thermo+Fisher
Average 99 stars, based on 1 article reviews
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resource source identifier antibodies rat monoclonal anti brdu abd serotec obt0030g mouse monoclonal anti apc  (Bio-Rad)
93
Bio-Rad resource source identifier antibodies rat monoclonal anti brdu abd serotec obt0030g mouse monoclonal anti apc
Resource Source Identifier Antibodies Rat Monoclonal Anti Brdu Abd Serotec Obt0030g Mouse Monoclonal Anti Apc, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti cc1  (Novus Biologicals)
92
Novus Biologicals mouse anti cc1
Mouse Anti Cc1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 1 article reviews
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mouse monoclonal  (Danaher Inc)
99
Danaher Inc mouse monoclonal
Mouse Monoclonal, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/pmc10230304-122-25-33?v=Danaher+Inc
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resource source identifier antibodies rabbit polyclonal anti fip200 proteintech  (Proteintech)
96
Proteintech resource source identifier antibodies rabbit polyclonal anti fip200 proteintech
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Resource Source Identifier Antibodies Rabbit Polyclonal Anti Fip200 Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/pm36198318-515-2-9?v=Proteintech
Average 96 stars, based on 1 article reviews
resource source identifier antibodies rabbit polyclonal anti fip200 proteintech - by Bioz Stars, 2026-08
96/100 stars
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rabbit polyclonal anti mbp antibody  (Proteintech)
96
Proteintech rabbit polyclonal anti mbp antibody
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Rabbit Polyclonal Anti Mbp Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/ppr0451521-65-10-16?v=Proteintech
Average 96 stars, based on 1 article reviews
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rabbit anti cc1  (Danaher Inc)
99
Danaher Inc rabbit anti cc1
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Rabbit Anti Cc1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/pmc08771106-96-31-36?v=Danaher+Inc
Average 99 stars, based on 1 article reviews
rabbit anti cc1 - by Bioz Stars, 2026-08
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mouse anti cc 1  (Danaher Inc)
99
Danaher Inc mouse anti cc 1
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Mouse Anti Cc 1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti cc 1 - by Bioz Stars, 2026-08
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anti apc  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology anti apc
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Anti Apc, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/10__1523_slash_jneurosci__0211___16__2016-121-18-14?v=Santa+Cruz+Biotechnology
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rabbit polyclonal anti ki 67  (Novus Biologicals)
94
Novus Biologicals rabbit polyclonal anti ki 67
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Rabbit Polyclonal Anti Ki 67, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+cc1/pmc03919648-174-13-18?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
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mouse anti cc1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology mouse anti cc1
Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of <t>FIP200</t> puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.
Mouse Anti Cc1, supplied by Santa Cruz Biotechnology, 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/product/rabbit+anti+cc1/pmc03888371-83-42-45?v=Santa+Cruz+Biotechnology
Average 93 stars, based on 1 article reviews
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anti adenomatous polyposis coli  (R&D Systems)
96
R&D Systems anti adenomatous polyposis coli
Neuronal activity-dependent Ca 2+ activities in oligodendrocytes (OCs). (A,B) Representative images (A) and quantification (B) of GCaMP expression in the motor cortex of PLP-GCaMP mice and co-localization with markers for OC + oligodendrocyte precursor cell (OPC) (Olig2), OC <t>(CC1),</t> OPC (PDGFRα, NG2), neuron (NeuN), astrocyte (S100β) (Olig2 [83.41 ± 3.899%], CC1 [72.65 ± 3.464%], PDGFRα+ [9.979 ± 0.9322%], NG2 [6.736 ± 0.2401%], NeuN [1.481 ± 0.7246%], and S100β [8.156 ± 0.5504%]). Scale bar, 30 μm. (C,D) Representative images of MBP immunostaining in the motor cortex of PLP-GCaMP6 mice. Scale bars: in (C) , 100 μm; in (D) , 30 μm. (E) Quantitative analysis of the co-localization areas of MBP and GCaMP’ + ve processes based on immunostaining data. The proportion of MBP’ + ve processes in the processes of GCaMP’ + ve cells was about 15%. (F) Experimental protocol of Ca 2+ imaging in OCs. The first craniotomy was performed one week before Pre-imaging (Pre) using two-photon microscopy. After Pre-imaging, a second craniotomy was performed and TTX was applied for 30 min, followed by a second imaging (TTX) of the same cells. (G,H) Representative image of GCaMP’ + ve cells of the motor cortex in PLP-GCaMP6 mice before and after TTX application. Red spots indicate the Ca 2+ activated areas (spot). Scale bar = 30 μm. Representative Ca 2+ traces from spots of typical GCaMP’ + ve cells are shown. (I) Changes in Ca 2+ spots, Ca 2+ events and total area under the curve (AUC) of GCaMP’ + ve cells between before and after TTX application. The number of Ca 2+ spots and Ca 2+ events and total AUC were significantly decreased after TTX application. Pre: n = 6 mice, 13 imaging fields (cells); TTX: n = 6 mice, 13 imaging fields (cells). * p < 0.05, ** p < 0.01, Mann–Whitney U test. Data are presented as mean ± standard error of mean. For detailed data, check the source data file. (J) Changes in AUC, Amplitude, and Latency of GCaMP’ + ve cells between before and after TTX application. AUC was not significantly changed, Amplitude was significantly decreased, and Latency was significantly increased after TTX application. Pre: n = 6 mice, 426 events; TTX: n = 6 mice, 189 events. N.S., not significant, * p < 0.05, *** p < 0.001, Mann–Whitney U test. Violin plots show median (black line) and distribution of the data. For detailed data, check the source data file. (K) Proportions of AUC and Latency were not significantly changed between before and after TTX application. The proportion of lower Amplitude was significantly increased after TTX application. N.S., not significant, ** p < 0.01, Kolmogorov–Smirnov test. For detailed data, check the source data file.
Anti Adenomatous Polyposis Coli, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of FIP200 puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 1. The Ca2+ transient on the ER outer surface is essential for autophagosome initiation (A–D) Pre-treatment with BAPTA-AM, but not EGTA-AM, inhibits starvation (strv)-induced formation of FIP200 puncta and WIPI2 puncta. Quantification is shown in (D) (n = 27, 36, and 29 cells for control, BAPTA-AM and EGTA-AM). COS7 cells were used unless otherwise noted. (E) Illustration of ER-localized Ca2+ release channels and pumps, and the indicators for measuring cytosolic and ER store Ca2+ levels. (F) Representative 3D surface plots of summed Ca2+ signals detected by multi-SIM under the indicated conditions. The GCaMP6f-CYB5 signal was imaged for 300 s. The bar indicates the fold increase. aa strv, amino acid starvation. (G) In situ calibration of GCaMP6f-CYB5 (n = 19 cells; see STAR Methods). (H) Number of ER Ca2+ transients detected by multi-SIM during the 300 s observation period (n = 15, 32, 20, and 18 cells for control, HBSS, aa starvation, and Torin1 treatment, respectively). (I–M) Representative patterns of Ca2+ dynamic changes on the ER outer surface. (N and O) The GCaMP6f-CYB5 signal shows no evident change under normal conditions (N) but exhibits periodic transients upon starvation (O). (P) Representative traces of GCaMP6f-CYB5 signal under normal conditions (black line) or immediately after HBSS starvation (green). Transients were detected in 65.5% of starved cells (n = 143) during 2,500 s observation. (Q) Quantification of the number of ER Ca2+ transients per cell during the first 2,500 s for each treatment (n = 18, 46, and 27 cells for bars from left to right). (R–T) Frequency distribution histograms of the amplitude, peak time (the time at which Ca2+ transient occurs), and FDHM of ER Ca2+ transients. 97 regional transients were analyzed in (R)–(T). n.s., no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM in (D), (H), and (Q). Scale bars, 20 mm in (N) and (O); 5 mm in (A)–(C) and (I)–(M). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques: Control, In Situ

Figure 3. EI24 controls the amplitude, frequency, and duration of Ca2+ transients/oscillations on the ER surface (A) Periodic Ca2+ transients in starved EI24 KO cells. (B) Trace of Ca2+ transients on the ER surface in EI24 KO cells. (C–E) The level of p62 and ratio of LC3-II/I in control and EI24 KO cells (C). Quantification is shown in (D) (n = 5) and (E) (n = 3). (F) Quantification of the number of ER Ca2+ transients per cell in control starved cells (n = 143) and EI24 KO starved cells (n = 101). (G–I) Frequency distribution histograms of the amplitude, peak time, and FDHM of global ER Ca2+ transients (n = 1,107) over 2,500 s in EI24 KO cells. (J–L) 3D surface plots of summed ER Ca2+ transients detected by multi-SIM in aa-starved EI24 KO cells. Most EI24 KO cells show large Ca2+ transients that propagate to the entire or most of the ER network. (K) and (L) show the number and amplitude of ER Ca2+ transients (n = 8 for EI24 KO cells). Data for aa-starved control cells are from Figures 1H and S1M. (M–S) More puncta are formed by FIP200, WIPI2, and GFP-ATG14 in EI24 KO cells than in controls. (M) shows quantification (n = 31, 32, 34, 35, 24, 25, 37, 33, 34, 26, 35, and 35 cells for bars from left to right). (T and U) Compared with control cells, fewer red-only (RFP+GFP) LC3 puncta are formed in EI24 KO cells after starvation. (V) Multi-SIM images showing association of GFP-LC3 structures with the ER in EI24 KO cells. (W) Percentage of red-only LC3 puncta in control (n = 21) and EI24 KO (n = 22) cells.

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 3. EI24 controls the amplitude, frequency, and duration of Ca2+ transients/oscillations on the ER surface (A) Periodic Ca2+ transients in starved EI24 KO cells. (B) Trace of Ca2+ transients on the ER surface in EI24 KO cells. (C–E) The level of p62 and ratio of LC3-II/I in control and EI24 KO cells (C). Quantification is shown in (D) (n = 5) and (E) (n = 3). (F) Quantification of the number of ER Ca2+ transients per cell in control starved cells (n = 143) and EI24 KO starved cells (n = 101). (G–I) Frequency distribution histograms of the amplitude, peak time, and FDHM of global ER Ca2+ transients (n = 1,107) over 2,500 s in EI24 KO cells. (J–L) 3D surface plots of summed ER Ca2+ transients detected by multi-SIM in aa-starved EI24 KO cells. Most EI24 KO cells show large Ca2+ transients that propagate to the entire or most of the ER network. (K) and (L) show the number and amplitude of ER Ca2+ transients (n = 8 for EI24 KO cells). Data for aa-starved control cells are from Figures 1H and S1M. (M–S) More puncta are formed by FIP200, WIPI2, and GFP-ATG14 in EI24 KO cells than in controls. (M) shows quantification (n = 31, 32, 34, 35, 24, 25, 37, 33, 34, 26, 35, and 35 cells for bars from left to right). (T and U) Compared with control cells, fewer red-only (RFP+GFP) LC3 puncta are formed in EI24 KO cells after starvation. (V) Multi-SIM images showing association of GFP-LC3 structures with the ER in EI24 KO cells. (W) Percentage of red-only LC3 puncta in control (n = 21) and EI24 KO (n = 22) cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques: Control

Figure 4. Reducing Ca2+ oscillation on the ER surface suppresses the autophagy defect in EI24 KO cells (A) 3D surface plots of summed ER Ca2+ transients detected by multi-SIM in EI24 KO cells with the indicated treatment under aa starvation. (B and C) Quantification of the number and amplitude (DF/F0) of ER Ca2+ transients in the indicated cells (n = 8, 6, and 14 cells for bars from left to right). Data for EI24 KO cells are from Figures 3K and 3L. (D and E) RCI treatment reduces the number of FIP200 puncta and WIPI2 puncta in EI24 KO cells.

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 4. Reducing Ca2+ oscillation on the ER surface suppresses the autophagy defect in EI24 KO cells (A) 3D surface plots of summed ER Ca2+ transients detected by multi-SIM in EI24 KO cells with the indicated treatment under aa starvation. (B and C) Quantification of the number and amplitude (DF/F0) of ER Ca2+ transients in the indicated cells (n = 8, 6, and 14 cells for bars from left to right). Data for EI24 KO cells are from Figures 3K and 3L. (D and E) RCI treatment reduces the number of FIP200 puncta and WIPI2 puncta in EI24 KO cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques:

Figure 5. Ca2+ transients trigger the formation of liquid-like FIP200 puncta (A) Time-series imaging of starved cells showing formation of mCherry-FIP200 puncta (arrows) near or on the ER in response to the starvation-induced ER Ca2+

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 5. Ca2+ transients trigger the formation of liquid-like FIP200 puncta (A) Time-series imaging of starved cells showing formation of mCherry-FIP200 puncta (arrows) near or on the ER in response to the starvation-induced ER Ca2+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques: Imaging

Figure 6. Phase separation of FIP200 requires multiple FIP200 domains (A) Illustration of FIP200 domains. (B) Live-cell imaging showing formation of puncta by full-length (FL) FIP200 and its deletion mutants (D) upon exogenous Ca2+ stimulation. The medium was changed back to normal growth medium (right panels) and further images were acquired to monitor dissolution of puncta. (C and D) FRAP analysis of a GFP-FIP200 punctum formed by FL and mutant FIP200 upon exogenous Ca2+ stimulation (C). Quantitative data in (D) are shown as mean ± SEM (n = 23, 21, and 20 for FIP200 FL, DIDR, and DCC1, respectively). (E and F) Multi-SIM images showing that formation of FIP200 puncta is abolished in ATG13 KO cells upon HBSS starvation (E) or exogenous Ca2+ stimulation (F). (G) Upon exogenous Ca2+ stimulation, ATG13 and ULK1 co-partition into FIP200 puncta. (B) and (G) show spinning disk confocal images. (H and I) Multi-SIM images showing that numerous puncta containing both ATG13 and FIP200 are formed upon exogenous Ca2+ stimulation. (J) Compared with control cells, the level of endogenous FIP200 co-precipitated by GFP-VAPA is enhanced in EI24 KO cells. The relative FIP200 level (normalized by GFP-VAPA) is also shown. Scale bars: 2 mm in (E), (F), (H), and (I); 1 mm in (C) and inserts in (I); and 0.5 mm in (B) and (G). See also Figures S6 and S7.

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 6. Phase separation of FIP200 requires multiple FIP200 domains (A) Illustration of FIP200 domains. (B) Live-cell imaging showing formation of puncta by full-length (FL) FIP200 and its deletion mutants (D) upon exogenous Ca2+ stimulation. The medium was changed back to normal growth medium (right panels) and further images were acquired to monitor dissolution of puncta. (C and D) FRAP analysis of a GFP-FIP200 punctum formed by FL and mutant FIP200 upon exogenous Ca2+ stimulation (C). Quantitative data in (D) are shown as mean ± SEM (n = 23, 21, and 20 for FIP200 FL, DIDR, and DCC1, respectively). (E and F) Multi-SIM images showing that formation of FIP200 puncta is abolished in ATG13 KO cells upon HBSS starvation (E) or exogenous Ca2+ stimulation (F). (G) Upon exogenous Ca2+ stimulation, ATG13 and ULK1 co-partition into FIP200 puncta. (B) and (G) show spinning disk confocal images. (H and I) Multi-SIM images showing that numerous puncta containing both ATG13 and FIP200 are formed upon exogenous Ca2+ stimulation. (J) Compared with control cells, the level of endogenous FIP200 co-precipitated by GFP-VAPA is enhanced in EI24 KO cells. The relative FIP200 level (normalized by GFP-VAPA) is also shown. Scale bars: 2 mm in (E), (F), (H), and (I); 1 mm in (C) and inserts in (I); and 0.5 mm in (B) and (G). See also Figures S6 and S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques: Live Cell Imaging, Dissolution, Mutagenesis, Control

Figure 7. ATG9 vesicles regulate spatial organization of FIP200 puncta (A–C) Multi-SIM analysis showing the relationship of ATG9 vesicles with FIP200 puncta in the indicated cells. ATG9 refers to ATG9A in this study. (D–F) PLA assay showing that interaction of FIP200 (GFP-FIP200 knockin) with ATG9 is enhanced in EI24 KD cells. (F) shows quantification of PLA puncta (n = 32 and 33 and n = 34 cells for bars from left to right). (G–I) In ATG9 KO cells, endogenous FIP200 forms a few enlarged puncta, whose number is not further increased after 1 h aa starvation. (I) shows quantification of FIP200 puncta before (n = 30) and after (n = 29) starvation. (J and K) Exogenous Ca2+ fails to trigger GFP-FIP200 puncta in ATG9 KO cells. (L and M) Compared with control cells, the level of Myc-ATG9 co-precipitated by GFP-FIP200 is enhanced in EI24 KO cells. (M) shows quantification of ATG9 level (normalized by GFP-FIP200 level) (n = 4). (N) In ATG9 KO cells, GFP-FIP200 forms enlarged puncta. (O and P) In ATG9 KO cells, LC3 forms clusters that associate with FIP200 puncta (O). (P) shows Imaris 3D reconstruction of LC3 (green) and FIP200 (red). Multi- SIM images are shown in (J), (K), (N), and (O). (Q) Model for the role of Ca2+ transients on the ER outer surface in the assembly of autophagosome initiation sites. Data are shown as mean ± SEM in (F), (I), and (M). Scale bars: 5 mm in (D), (E), (G), and (H); 2 mm in (A)–(C), (J), (K), (N), and (O); and 1 mm in inserts in (A)–(C), (G), (H), (N), and (P).

Journal: Cell

Article Title: Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites.

doi: 10.1016/j.cell.2022.09.001

Figure Lengend Snippet: Figure 7. ATG9 vesicles regulate spatial organization of FIP200 puncta (A–C) Multi-SIM analysis showing the relationship of ATG9 vesicles with FIP200 puncta in the indicated cells. ATG9 refers to ATG9A in this study. (D–F) PLA assay showing that interaction of FIP200 (GFP-FIP200 knockin) with ATG9 is enhanced in EI24 KD cells. (F) shows quantification of PLA puncta (n = 32 and 33 and n = 34 cells for bars from left to right). (G–I) In ATG9 KO cells, endogenous FIP200 forms a few enlarged puncta, whose number is not further increased after 1 h aa starvation. (I) shows quantification of FIP200 puncta before (n = 30) and after (n = 29) starvation. (J and K) Exogenous Ca2+ fails to trigger GFP-FIP200 puncta in ATG9 KO cells. (L and M) Compared with control cells, the level of Myc-ATG9 co-precipitated by GFP-FIP200 is enhanced in EI24 KO cells. (M) shows quantification of ATG9 level (normalized by GFP-FIP200 level) (n = 4). (N) In ATG9 KO cells, GFP-FIP200 forms enlarged puncta. (O and P) In ATG9 KO cells, LC3 forms clusters that associate with FIP200 puncta (O). (P) shows Imaris 3D reconstruction of LC3 (green) and FIP200 (red). Multi- SIM images are shown in (J), (K), (N), and (O). (Q) Model for the role of Ca2+ transients on the ER outer surface in the assembly of autophagosome initiation sites. Data are shown as mean ± SEM in (F), (I), and (M). Scale bars: 5 mm in (D), (E), (G), and (H); 2 mm in (A)–(C), (J), (K), (N), and (O); and 1 mm in inserts in (A)–(C), (G), (H), (N), and (P).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-FIP200 Proteintech Cat# 17250-1-AP; RRID:AB_1066642 8 Rabbit anti-FIP200 Sigma-Aldrich Cat# SAB4200135; RRID:AB_10621935 Mouse anti-ATG13 Merck Cat# MABC46; RRID:AB_11211663 Rabbit anti-ATG13 Cell Signaling Technology Cat# 13468S; RRID:AB_2797419 Mouse monoclonal anti-WIPI2 Abcam Cat# ab105459; RRID:AB_10860881 Rabbit anti-ATG9 MBL Cat# PD042; RRID:AB_2714019 Rabbit anti-EI24 Sigma Cat# HPA047165; RRID:AB_2679965 Rabbit anti-SERCA2 Cell Signaling Technology Cat# 4388S; RRID:AB_2227684 Mouse monoclonal anti-LC3 (clone 4E12) MBL Cat# M152-3; RRID: AB_1279144 Rabbit polyclonal anti-LC3 Cell Signaling Technology Cat# 2775S; RRID:AB_915 950 Rabbit polyclonal anti-p62 MBL Cat# PM045; RRID:AB_1279301 Rabbit anti-VAPA Proteintech Cat# 15275-1-AP; RRID:AB_2256991 Mouse anti-VAPB Proteintech Cat# 66191-1-IG; RRID:AB_2881586 Rabbit anti-S6K Cell Signaling Technology Cat# 2708S; RRID:AB_390722 Rabbit anti-p-S6K Cell Signaling Technology Cat# 9234L; RRID:AB_2269803 Mouse anti-Myc Sigma-Aldrich Cat# M5546; RRID:AB_260581 Mouse anti-His Sigma-Aldrich Cat# 70796-M; RRID:AB_10807496 Mouse monoclonal anti-GFP (clone 7.1 and 13.1) Roche Cat# 11814460001; RRID:AB_390913 Mouse monoclonal anti-Actin (clone 7D2C10) Proteintech Cat# 60008-1-Ig; RRID:AB_2289225 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID:AB_2107436 Bacterial and virus strains E. coli BL21-CodonPlus (DE3) Agilent Cat# 280230 Chemicals, peptides, and recombinant proteins Dulbecco’s Modified Eagle’s Medium, high glucose HyClone Cat# SH30022.01B Fetal Bovine Serum ThermoFisher Scientific Cat# 10099-141C Hank’s balanced salt solution (HBSS) Gibco Cat# 14025-092 DMEM without amino acids HyClone Cat# SH4007.01 Thapsigargin (TG) Sigma Cat# T9033 BAPTA-AM Abcam Cat# AB120503 EGTA-AM ThermoFisher Scientific Cat# E1219 Isoprenaline (ISO) Thermo Scientific Cat# 11683009 Caffeine Thermo Scientific Cat# 11364569 Ryanodine Merck Cat# 559276 Anisomycin EMD Millipore Cat# 176880 Bafilomycin A1 Sigma Aldrich Cat# B1793 KB-R7943 Sigma Aldrich Cat# K4144 Torin 1 Cell Signaling Technology Cat# 14379 Chloroquine (CQ) Sigma Aldrich Cat# C6628 Poly-L-Lysine Sigma Aldrich Cat# P4832 Triton X-100 ThermoFisher Scientific Cat# HFH10 digitonin Sigma Cat# D141 protease inhibitor cocktail Roche Cat# 11836170001 (Continued on next page) Cell 185, 4082–4098.e1–e8, October 27, 2022 e1

Techniques: Knock-In, Control

Neuronal activity-dependent Ca 2+ activities in oligodendrocytes (OCs). (A,B) Representative images (A) and quantification (B) of GCaMP expression in the motor cortex of PLP-GCaMP mice and co-localization with markers for OC + oligodendrocyte precursor cell (OPC) (Olig2), OC (CC1), OPC (PDGFRα, NG2), neuron (NeuN), astrocyte (S100β) (Olig2 [83.41 ± 3.899%], CC1 [72.65 ± 3.464%], PDGFRα+ [9.979 ± 0.9322%], NG2 [6.736 ± 0.2401%], NeuN [1.481 ± 0.7246%], and S100β [8.156 ± 0.5504%]). Scale bar, 30 μm. (C,D) Representative images of MBP immunostaining in the motor cortex of PLP-GCaMP6 mice. Scale bars: in (C) , 100 μm; in (D) , 30 μm. (E) Quantitative analysis of the co-localization areas of MBP and GCaMP’ + ve processes based on immunostaining data. The proportion of MBP’ + ve processes in the processes of GCaMP’ + ve cells was about 15%. (F) Experimental protocol of Ca 2+ imaging in OCs. The first craniotomy was performed one week before Pre-imaging (Pre) using two-photon microscopy. After Pre-imaging, a second craniotomy was performed and TTX was applied for 30 min, followed by a second imaging (TTX) of the same cells. (G,H) Representative image of GCaMP’ + ve cells of the motor cortex in PLP-GCaMP6 mice before and after TTX application. Red spots indicate the Ca 2+ activated areas (spot). Scale bar = 30 μm. Representative Ca 2+ traces from spots of typical GCaMP’ + ve cells are shown. (I) Changes in Ca 2+ spots, Ca 2+ events and total area under the curve (AUC) of GCaMP’ + ve cells between before and after TTX application. The number of Ca 2+ spots and Ca 2+ events and total AUC were significantly decreased after TTX application. Pre: n = 6 mice, 13 imaging fields (cells); TTX: n = 6 mice, 13 imaging fields (cells). * p < 0.05, ** p < 0.01, Mann–Whitney U test. Data are presented as mean ± standard error of mean. For detailed data, check the source data file. (J) Changes in AUC, Amplitude, and Latency of GCaMP’ + ve cells between before and after TTX application. AUC was not significantly changed, Amplitude was significantly decreased, and Latency was significantly increased after TTX application. Pre: n = 6 mice, 426 events; TTX: n = 6 mice, 189 events. N.S., not significant, * p < 0.05, *** p < 0.001, Mann–Whitney U test. Violin plots show median (black line) and distribution of the data. For detailed data, check the source data file. (K) Proportions of AUC and Latency were not significantly changed between before and after TTX application. The proportion of lower Amplitude was significantly increased after TTX application. N.S., not significant, ** p < 0.01, Kolmogorov–Smirnov test. For detailed data, check the source data file.

Journal: Frontiers in Cellular Neuroscience

Article Title: Activity-dependent oligodendrocyte calcium dynamics and their changes in Alzheimer’s disease

doi: 10.3389/fncel.2023.1154196

Figure Lengend Snippet: Neuronal activity-dependent Ca 2+ activities in oligodendrocytes (OCs). (A,B) Representative images (A) and quantification (B) of GCaMP expression in the motor cortex of PLP-GCaMP mice and co-localization with markers for OC + oligodendrocyte precursor cell (OPC) (Olig2), OC (CC1), OPC (PDGFRα, NG2), neuron (NeuN), astrocyte (S100β) (Olig2 [83.41 ± 3.899%], CC1 [72.65 ± 3.464%], PDGFRα+ [9.979 ± 0.9322%], NG2 [6.736 ± 0.2401%], NeuN [1.481 ± 0.7246%], and S100β [8.156 ± 0.5504%]). Scale bar, 30 μm. (C,D) Representative images of MBP immunostaining in the motor cortex of PLP-GCaMP6 mice. Scale bars: in (C) , 100 μm; in (D) , 30 μm. (E) Quantitative analysis of the co-localization areas of MBP and GCaMP’ + ve processes based on immunostaining data. The proportion of MBP’ + ve processes in the processes of GCaMP’ + ve cells was about 15%. (F) Experimental protocol of Ca 2+ imaging in OCs. The first craniotomy was performed one week before Pre-imaging (Pre) using two-photon microscopy. After Pre-imaging, a second craniotomy was performed and TTX was applied for 30 min, followed by a second imaging (TTX) of the same cells. (G,H) Representative image of GCaMP’ + ve cells of the motor cortex in PLP-GCaMP6 mice before and after TTX application. Red spots indicate the Ca 2+ activated areas (spot). Scale bar = 30 μm. Representative Ca 2+ traces from spots of typical GCaMP’ + ve cells are shown. (I) Changes in Ca 2+ spots, Ca 2+ events and total area under the curve (AUC) of GCaMP’ + ve cells between before and after TTX application. The number of Ca 2+ spots and Ca 2+ events and total AUC were significantly decreased after TTX application. Pre: n = 6 mice, 13 imaging fields (cells); TTX: n = 6 mice, 13 imaging fields (cells). * p < 0.05, ** p < 0.01, Mann–Whitney U test. Data are presented as mean ± standard error of mean. For detailed data, check the source data file. (J) Changes in AUC, Amplitude, and Latency of GCaMP’ + ve cells between before and after TTX application. AUC was not significantly changed, Amplitude was significantly decreased, and Latency was significantly increased after TTX application. Pre: n = 6 mice, 426 events; TTX: n = 6 mice, 189 events. N.S., not significant, * p < 0.05, *** p < 0.001, Mann–Whitney U test. Violin plots show median (black line) and distribution of the data. For detailed data, check the source data file. (K) Proportions of AUC and Latency were not significantly changed between before and after TTX application. The proportion of lower Amplitude was significantly increased after TTX application. N.S., not significant, ** p < 0.01, Kolmogorov–Smirnov test. For detailed data, check the source data file.

Article Snippet: The primary antibodies used in this study were as follows: anti-Olig2 (rabbit; Millipore, AB9610; 1:1000), anti-adenomatous polyposis coli (clone CC1) (mouse; Calbiochem, OP80; 1:500), anti-PDGFRα (goat; R&D systems, AF1062; 1:200), anti-NG2 (rabbit; Millipore, AB5320; 1:500), anti-NeuN (mouse; Millipore, MAB377; 1:500), anti-S100β (rabbit; Abcam, ab52642; 1:1000), anti-MBP (mouse; BioLegend, clone SMI 99; 1:100), and anti-GFP (chicken; Novus biologicals, NB100-1614; 1:2000).

Techniques: Activity Assay, Expressing, Immunostaining, Imaging, Microscopy, MANN-WHITNEY